Advances in Cancer Research Volume 70

-~ ~

Advances in

CANCER RESEARCH Volume 70

This Page Intentionally Left Blank

Advances in

CANCER RESEARCH Volume 70 Edited by

George F. Vande Wude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center ( M E ) Karolinska Znstitutet Stockholm. Sweden

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1996 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NWI 7DX, UK

http:lfwww.hbuk.co.ukfapf International Standard Serial Number: 0065-230X International Standard Book Number: 0- 12-006670-X

PRINTED IN THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 01 BB 9 8 7 6

5

4

3 2

1

Contents

Contributors to Volume 70 ix

FOUNDATIONS IN CANCER RESEARCH Fundamentals of Cancer Cell Biology Michael S t o k e r 1. Introduction 1 11. Antecedents 2 111. Autobiographical Note 3 1V. Foundations of Tissue Culture 4 V. Foundations of Cell Culture 5

VI. VII. VIII. IX. X. XI.

Clones 5 The Immortals: Stable Cell Lines 8 The Mortals: Cell Strains 8 Food for Cells in Culture 9 Growth Factors 9 Short-Range Cell Interactions 10 XU. Junctional Communication 11 XIII. Cell and Substrate Adhesion 12 XIV. The Tumor Viruses 12 XV. Cell Fusion 15 XVI. Conclusion 16 References 17

FOUNDATIONS IN CANCER RESEARCH The Stepby-Step Development of Epithelial Cancer: From Phenotype to Genotype Ernrnanuel Farber I. Cancer Development as Basic to Cancer Research 22 11. Patterns of Development of Epithelial Cancers 23 111. A Working Hypothesis 27

V

Contents

vl

IV. The Phenotypes 27 V. The Genotypes 40 VI. The Challenge 41 References 44

Genetics of the Nevoid Basal Cell Carcinoma Syndrome Abirami Chidambaram a n d Michael Dean I. Introduction 49 11. Clinicopathological Features of NBCCS 50 111. Genetics of NBCCS 52 IV. Strategies for Isolation of Candidate Genes 54 V. Discussion 57 References 59

Transforming Growth Factor-f! System and I t s Regulation by Members of the Steroid-Thyroid Hormone Superfamily Katri Koli a n d jorma Keski-Oja I. Introduction 63 11. Transforming Growth Factor-p 64 111. Dual Effects of TGF-f! on Cell Proliferation 71

IV. V. VI. VII. VIII. 1X.

Regulation of Cell Differentiation by TGF-f! 73 TGF-P in the Regulation of the Immune System 73 The Steroid-Thyroid Hormone Superfamily 75 Steroid Hormone Regulation of TGF-p Isoform Expression 79 Regulation of Plasminogen Activation by Steroids 84 Summary 86 References 87

c-Myc in the Control of Cell Proliferation and Embryonic Development Jean-Marc Lemaitre, Robin S. Buckle, a n d Marcel Mechali I. Introduction 96 11. The c-myc Gene 96 111. Structural and Functional Features of the c-Myc Protein 101 IV. c-Myc as a Transcription Factor 108 V. c-Myc and Cell Proliferation 116 VI. c-Myc in Embryonic Development 125 VII. c-Myc and Differentiation 128 VIII. c-Myc and Apoptosis 130

References 134

Contents

vii

Identification of the Genes Encoding Cancer Antigens: Implications for Cancer lmmunotherapy Steven A. Rosenberg, Yutaka Kawakami, Paul F. Robbins, a n d Rongfu Wang I. 11. 111. IV.

Introduction 145 Methodology 147 Human Melanoma Antigens Recognized by T Cells 149 Cancer Therapies Based on the Molecular Identification of Cancer Antigens 169 References 172

The MEN I1 Syndromes and the Role of the ret Proto-oncogene Bruce A. J. Ponder a n d Darrin Smith I. Introduction 180 11. The MEN I1 Syndromes 181 111. The ret Proto-oncogene 192 IV. Development of the Tissues Involved in MEN 11, and Patterns of ret Expression 207 V. Speculations on How Different ret Mutations Result in the Associated Phenotypes and in Tumor Formation 21 1 V1. Other Events in Tumor Progression 213 VII. Animal Models of MEN I1 213 VIII. Clinical Implications of the Identification of ret Mutations in MEN I1 214 IX. Future Prospects 215 References 216

Index 223

This Page Intentionally Left Blank

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Robin S . Buckle The Randall Institute, King’s College London, London WC2 SBRL, England (95) Abirami Chidambaram Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 (49) Michael Dean Human Genetics Section, Laboratory of Viral Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 (49) Emmanuel Farber Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 (21) Yutaka Kawakami Surgery Branch, Division of Clinical Science, National Cancer Institute, Bethesda, Maryland 20892 (145) Jorma Keski-Oja Department of Virology, the Haartman Institute, and Department of Dermatology and Venereology, University of Helsinki, SF-00014 Helsinki, Finland (63) Katri Koli Department of Virology, the Haartman Institute, University of Helsinki, SF-00014 Helsinki, Finland (63) Jean-Marc Lemaitre Institut J. Monod CNRS, 75251 Paris cedex 05, France (95) Marcel MCchali Institut J. Monod CNRS, 75251 Paris cedex 05, France (95) Bruce A. J. Ponder CRC Human Cancer Genetics Research Group, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, England (179) Paul F. Robbins Surgery Branch, Division of Clinical Science, National Cancer Institute, Bethesda, Maryland 20892 (145) Steven A. Rosenberg Surgery Branch, Division of Clinical Science, National Cancer Institute, Bethesda, Maryland 20892 (145) Darrin Smith CRC Human Cancer Genetics Research Group, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, England (179) Michael Stoker Cambridge University, Cambridge, England (1) Rong-fu Wang Surgery Branch, Division of Clinical Science, National Cancer Institute, Bethesda, Maryland 20892 (195)

ix

This Page Intentionally Left Blank

FOUNDATIONS IN CANCER RESEARCH Fundamentals of Cancer Cell Biology Michael Stoker Cambridge University, Cambridge, England

1. Introduction

II. 111. IV. V. VI. V11. VIII.

IX. X. XI. XII. XIII. XIV.

XV.

XVI.

Antecedents Autobiographical Note Foundations of Tissue Culture Foundations of Cell Culture Clones The Immortals: Stable Cell Lines The Mortals: Cell Strains Food for Cells in Culture Growth Factors Short-Range Cell Interactions Junctional Communication Cell and Substrate Adhesion The Tumor Viruses A. Transformation of Cultured Cells B. Integration of Viral and Cell Genomes Cell Fusion Conclusion References

I. INTRODUCTION When I was asked by the editors of Advances in Cancer Research to contribute a personal account of the early stages in the development of cancer cell biology, I turned to the earlier articles in the Fundamentals series to see if there was an overlap that would make any such attempt superfluous. There is great variety in these excellent articles, not only in subject matter but in the approach of the authors. Some are mostly, and justifiably, accounts of the important contributions made by the author’s own group, almost autobiographies. One is a very interesting biographical account of the contributions of a deceased colleague. Others are broadly based and wide-ranging historical accounts of the foundations of a subject (often with only modest reference to the author’s own contributions). Several, quite Advances in CANCER RESEARCH, Vol. 70 Copyright 8 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

Michael Stoker

rightly, include a good deal of cell biology, the subject now allocated to me. This is inevitable, and 1 cannot grumble because the topic is so pervasive; anyway, I assume that the editors want the contributions of the authors to reflect their own view of events. This article is mostly about the work of others, and, because it is a personal choice, it is selective and incomplete. However, I also include a short autobiographical note to indicate the influences that led me personally into cell biology. The reader may skip this with impunity.

II. ANTECEDENTS Cell biology can be described as the study of individual cells, in contrast to tissues, individual molecules, and individual genes. It is reductionist, but it includes frequent and anxious glances over the shoulder at the complexity from which it is derived. Much of cell biology has been cancer cell biology because, in the early days, cancer cells were easier to study than normal cells. However, to identify the unique features of cancer cells, a comparison with normal cells became an obvious necessity. One of the most important advances in an understanding of cancer has been the realization that cancer does not arise as an abnormality of an individual or even a tissue. Nearly all cancers originate from one single abnormal cell-that is, they are clonal. This is true even in individuals carrying an abnormal gene predisposing to cancer in all their cells, and it applies during progression to greater malignancy through sequential selection of a series of clones. The clonal origin of cancer was realized in experimental cancers through cell cloning in culture and single-hit initiation of virus-induced tumors, but it was not until the discovery of X chromosome inactivation, and of chromosome translocation in myeloid leukemia, that the clonal nature of naturally occurring human cancers was confirmed. It might have been expected that the field of cell biology would emerge, in reductionist fashion, from the study of larger and more complex structures such as organs and tissues, via histology and pathology. This was indeed true during the long and halting development of tissue culture, during the first half of this century, from which contemporary cell biology emerged after the end of World War 11. I shall deal with this emergence later, but it followed the development of techniques for the isolation and long-term culture of individual eukaryotic cells in large numbers, in cell culture as opposed to tissue culture. At this stage there was not much attention to the tissues of origin, often a cancer. The expansion of virology also played a part because of the demand for vaccines produced in cell cultures, and later from the study of the tumor viruses and their effect on cells (see Levine, 1994).

Fundamentals of Cancer Cell Biology

3

And we must not ignore the fortunate coincidence of the expanding use of the electron microscope and of cell fractionation, which began to reveal the functional microanatomy of individual cells at about the same time, thanks to George Palade and Christian de Duve (1971).

111.

AUTOBIOGRAPHICAL NOTE

My own slow path toward cell biology was influenced both by local events in Cambridge and distant developments overseas. After a medical education and army service during World War 11, my early research, up to 1953, had been mainly on rickettsiae, which were then considered to be large viruses. I also had to teach virology, but with one exception this field, and particularly animal virology, was still in the dark ages and, though important, the real understanding of the nature of viruses came later. The notable exception lay with the bacterial viruses, and my colleague and mentor, John Miles, drew my attention to the momentous pioneering discoveries that were then taking place, led by Max Delbruck in the United States and by Andre Lwoff and his colleagues in France. The explanation of the growth and assembly of the lytic bacteriophages, the crucial role of phage DNA shown by the elegant experiment of Hershey and Chase, and the integration of lambda phage in lysogenic bacteria surely pointed the way for future work on animal viruses and cells. It certainly influenced me. At the same time, I could not fail to be affected by local events in Cambridge. Some of the very early work on cultured animal cells had been carried out by Strangeways and his colleague Honor Fell long before my time, but I shall return to this later in the history of tissue culture. In 1946, one of the first Siemans electron microscopes was.brought from Germany and installed in the Cavendish Laboratory under V. E. Cosslet, and, of particular importance, an electrical engineer, Bob Horne (later Professor Horne), who took over the maintenance and operation of the machine for assorted biologists who wanted to try out their materials in this strange object. It attracted not only the animal virologists but a diverse group, including bacteriologists, cell biologists, plant virologists, and, because of their proximity in the Cavendish Laboratory, the first molecular biologists. Through this very informal assembly at the microscope, and in neighboring pubs, I got to know in particular the plant virologists Kenneth Smith and Roy Markham, who were interested in virus structure, as well as Max Perutz and John Kendrew, and not least Francis Crick and Jim Watson (the latter also through his membership of my college). It was the wide-ranging discussions with these individuals and others in related laboratories, and the first pictures of viruses and cell structures pro-

4

Michael Stoker

duced by Bob Horne, that attracted me more and more to research at the cellular and molecular level, rather than other approaches to the medical problems of that period. In 1954, with lambda phage in mind, I began to study the growth of herpesvirus in cultured cells, then a few years later turned to tumor viruses and cell transformation, and from this to normal and cancer cell biology.

IV. FOUNDATIONS OF TISSUE CULTURE Attempts to maintain living tissue outside the body began haltingly about 100 years ago, but it was the work of two American groups that put it on a firm footing. First, in Baltimore, Harrison (1907) reported nerve fiber growth in cultures of chick embryo tissue, and this was followed in similar studies by Lewis and his wife (Lewis and Lewis, 1911), which were continued for many years. But it was the report from Carrel (1912) at the Rockfeller Institute in New York that is generally agreed to be the classic origin of tissue culture. By adding embryo extract to fragments of chick tissue in plasma clot cultures, Carrel obtained a continuous outgrowth of cells, due to mitosis as well as migration. Upon splitting the cultures, the growth could be maintained, and the progeny cells were still alive long after the expected life span of a chicken. Carrel’s success is thought to have been due to his expertise as a surgeon in maintaining sterility, when in the absence of antibiotics so many cultures were ruined by contamination. It may also be noted that, among the many types of normal chick tissue with which he worked, he cultured cells from Rous sarcomas, thus initiating cancer cell biology. Carrel and his group at the Rockefeller Institute continued their investigations and published many papers over the next few years, but tissue culture research in general was hindered throughout the period of World War I. During the 1 9 2 0 however, ~~ research in the field developed considerably in Europe. In particular, Albert Fischer, who had studied in New York with Carrel and then moved to Copenhagen, investigated the behavior of various tissues in culture, especially epithelia, as well as a variety of tumors. This led to his massive textbook on the subject, published in Munich (Fischer, 1930). In England, Thomas Strangeways led the way in Cambridge, using Carrel’s system to help his investigation of rheumatoid arthritis. Tissue culture became Strangeways’ main interest, and he made the first detailed study of the morphological changes during mitosis. He went on to investigate the effect of various types of irradiation on cultured cells, and showed the special sensitivity of cells in mitosis (Strangeways, 1922; Strangeways and Oakley, 1923). Later he was joined by Honor Fell (Strangeways and Fell, 1926), and it was Honor Fell who, after the early death of Strangeways, succeeded him

Fundamentals of Cancer Cell Biology

5

as head of what became the Strangeways Laboratory. She became a pioneer in the use of organ cultures of intact tissues, especially bone, in which, for example, she revealed the effects of vitamins A and C. Strangeways also cooperated with Canti, working in London (Strangeways and Canti, 1927), and provided him with cultures for the first cinematograph film of cultured cells, including the famous film first shown in 1928. Another pioneer of tissue culture in Cambridge in the interwar period was Willmer, who developed improved methods for measuring growth in cultures (Willmer and Jacoby, 1936) and, with Pomerat, investigated the role of carbohydrate metabolism (Pomerat and Willmer, 1939).

V. FOUNDATIONS OF CELL CULTURE In the first 30 years after Harrison and Carrel first showed that animal cells would survive and grow in vitro, tissue culture became well established. Progress was slow, however, and limited to few laboratories. It was generally restricted to tissues, short-term cultures of cells migrating from small fragments of intact tissue, and it was handicapped by the need for strict aseptic conditions to avoid bacterial contamination. For investigation of isolated cells to progress, there was a need for continuous growth of large homogeneous, preferably clonal, populations, suitable for biochemical and eventually genetic analysis. Paradoxically, it was methods used for study of the enemies of early tissue culture, namely bacteria and the bacterial viruses, which provided models for the further investigation of animal cells in culture. Progress was made remarkably quickly in the first decade or so after the end of World War 11. This was due largely to a detailed analysis of the nutritional requirements of cells in culture, to the use of trypsin and chelating agents for cell suspension, and to the introduction of antibiotics to control contamination. The improved culture media that became widely available then allowed continuous culture and subculture of a variety of cell types, to yield very large populations. At the same time the techniques for isolation and propagation of single cells to provide pure clones also became available.

VI. CLONES After several unsuccessful attempts in previous years, the first clear demonstration that a single isolated cell would divide and give rise to a clonal

6

Michael Stoker

population was reported by Earle and his colleagues at Bethesda, Maryland, in an important paper in 1948 (Sandford et al., 1948). Already, many years before, Earle and Thompson (1931) had suggested that the usual culture medium of serum and embryo extract was only adequate for growth in a dense population of cells that could release additional metabolites and so enrich the surrounding fluid medium. Single cells, however, were probably unable to enrich the relatively large volume of surrounding fluid sufficiently. The successful solution in 1948 was to use medium that had been used for, and then separated from, a mass culture of cells-so-called conditioned medium. Separated cells were then cultured in very small volumes of this medium, each one in a section of fine-bore glass tubing. In this way the division of single cells was observed, and ultimately yielded a large population that migrated out of the original tube and was subcultured as a pure clone. (Figure 1 is from the original paper and shows the capillary tube culture [Sanford et al., 19481). This was a most important advance, but the method was tricky and demanded skillful manipulation. Cloning of cells in culture was not commonly practiced until the discovery of “feeder layers” 7 years later in Denver by Puck and Marcus (1955). Instead of exposing single cells to used culture medium in a small volume, they added them to glass slides suspended over a preexisting layer of cells cultured on the base of a Petri dish and then exposed to x-rays. The irradiated cells would not themselves divide and obscure the growth of the added, nonirradiated cells, but they could act as “feeders” and release metabolites into the microenvironment of the added cells. The result was dramatic. After 8-19 days, large colonies of cells had appeared on the glass slides in numbers approximating the number of single cells plated. Thus a mixed population of animal cells could now be grown into colonies for subculture and cloned in the same way as bacteria. Soon afterward, Puck and Fisher (1956) showed how mutants with different growth requirements could be isolated in this way. Despite the relative ease of cloning by plating for colonies, and because it was impossible to exclude colony development from more than one cell, it was still sometimes necessary to select identified single cells. Various techniques were used, such as manipulation in microdrops of medium under oil, used by Wildy and Stoker (1958) in Cambridge, or identification after serial dilution of suspensions in microwells. Much later this became less important because of the clear identification of clonal populations by insertion of retroviral markers. In time, with improvements in culture medium, some types of cells could be grown and isolated in individual colonies, without the need for a feeder layer of nondividing cells. Even with feeder cells, however, the probability of growth from isolated cells, or plating efficiency, varied a good deal, being

Fundamentals of Cancer Cell Biology

7

Fig. I (A) A single cell in a capillary segment embedded in a conditioned medium plasma on the floor of a Carrel D 3.5 flask. X200. (B) Same preparation as in (A) taken 65 hours later, 87 hours after planting and 45 hours after a fluid change. The cell had proliferated to yield 6 cells, 1 of which cannot be seen due to curvature of the capillary. The cell at the right is dead. ~ 2 0 0 . (C)Same preparation as in (B) taken 45 hours later, 135 hours after planting and 24 hours after the last fluid change, when there were 12 live cells and 1 dead. Several of the cells are out of focus. One cell was undergoing division. ~ 2 0 0 From . Sandford et al. (1948), courtesy of the Journal of the National Cancer Institute.

8

Michael Stoker

generally low with freshly isolated cells and high for well-established cultures known as stable cell lines.

VII. THE IMMORTALS: STABLE CELL LINES For the isolation of the first clones, Earle’s group used L cells. These were fibroblasts that had originally been isolated from mouse connective tissue by Earle (1943). After treatment with a carcinogen, they were found to be transplantable as sarcomas and, in the laboratory at least, were immortal, capable of growing in culture indefinitely with undiminished vigor. They were the first “stable line” and the forerunner of many others, which are relatively easy to grow in culture and to store at low temperature. Most of these stable cell lines are derived from cancers or become neoplastic, and selection in culture usually involves chromosomal abnormalities. Nevertheless, stable lines are favorites in the study of cell biology. One of these early favorites was the first human line, isolated by George Gey and his colleagues (1952) in Baltimore from a cervical carcinoma and named Hela after abbreviation of the deceased patient’s name. Subsequently other stable lines were isolated from tumors of humans and experimental animals. Later, as we shall see, cell lines from normal tissues, without obvious characteristics of cancer cells, were needed to study neoplastic transformation in vitro.

VIII. THE MORTALS: CELL STRAINS Cells freshly obtained from normal animal tissues were also found to grow well at first, and they have been used very successfully in large numbers-for example, to produce poliovirus vaccine in monkey kidney cells. Such cells, compared to cells from stable lines, were more reluctant to grow at low density, and it was difficult to isolate clones from single cells. Moreover, they could not be propagated indefinitely. After a sequence of subcultures from the original primary cultures, further growth ceased, and they had to be replaced with freshly isolated cells. This was at first assumed to be due to inadequate nutrition, and did not attract much attention until the important studies of Hayflick and Moorhead (1961) at the Wistar Institute in Philadelphia. Working with a series of cultures from human embryos and a strict regimen of subculture, they found that cells continued to multiply logarithmically, maintaining their diploid karyotype and other characteristics, for about SO generations. Then inevitably, and independently of cell source or medium used, the cells ceased to grow and eventually died. This could not have been due to gradual dilution and loss of an essential metabolite present

Fundamentals of Cancer Cell Biology

9

in the original tissue cells, and the authors concluded that normal human diploid cells were endowed with a built-in mechanism leading to inevitable “cell senescence.’’ Though there may be variation between species, this important conclusion has become generally accepted. It was a forerunner of an equally important, and much later, discovery in Edinburgh of programmed cell death, or apoptosis (Kerr et al., 1972). When I was shown the first evidence for apoptosis in Alistair Currie’s laboratory, I completely failed to recognize its significance. How wrong can one be? Cultures of diploid and mortal cells are generally referred to as “strains” to distinguish them from the mutant aneuploid cells that arise from preexisting cancers or appear as variants in propagation of cell strains. These, as we have seen earlier, are the stable cell “lines.”

IX. FOOD FOR CELLS IN CULTURE The advances described previously, and the resulting expansion of research in the 1950s and 1960s, could not have taken place without the identification of the detailed nutritional requirements of, and consequently the provision of suitable medium for, cultured cells. Much had been achieved with serum, plasma, embryo extract, and simple buffered salt solutions, but these complex media could not be standardized and were inadequate for many purposes. They were gradually replaced by partially synthetic media, for example, by Charity Weymouth (1956). However, Eagle (1955), at the National Institutes of Health in Bethesda, Maryland, in a painstaking study, analyzed in detail the essential requirements of HeLa cells and mouse fibroblasts for amino acids and vitamins, glucose, and salts. It allowed him to identify the necessary constituents of a minimal synthetic medium that, together with proteins from dialyzed serum, was sufficient to promote growth of the cultured cells. Eagle’s medium, subsequently modified by Dulbecco and Vogt (1960), and named DMEM (Dulbecco modified Eagle’s medium) is still, 40 years later, the medium most commonly used in the study of normal and tumor cells, and it would be difficult to find a better example of a fundamental contribution to cell biology.

X. GROWTH FACTORS It was many years before the proteins required for growth and other activities of cultured cells were identified. Most of them, including growth factors, are now known as cytokines. These act as intercellular signals by

10

Michael Stoker

binding to, and activating, specific receptors on the cell surface. Although they include insulin, a long-established agent, the role of growth factors and similar cytokines at the cell level was not at first recognized. The first growth factor to be identified as such was epidermal growth factor (EGF), originally found in the salivary glands of mice by Stanley Cohen (1962) in Nashville, after the pioneer studies in Italy with Rita Levi Montalcini that had led to nerve growth factor (see Levi Montalcini, 1966). Using extraordinary assays such as eyelid opening in chicks, Cohen purified EGF and sequenced the amino acids, before going on to show EGF’s mitogenic activity in epithelial cells. Subsequently, a range of other cultured cells, including fibroblasts, was shown to respond to EGF. The discovery of EGF eventually led to the identification of a whole range of signaling cytokines, and to their receptors, which were eventually found to be the products of oncogenes. A little later, Plutznik and Sachs (1965,1966)in Rehovot, and Bradley and Metcalf (1966) in Melbourne, quite independently discovered two growth factors, or colony-stimulating factors, for macrophages and granulocytes of the hematopoietic system. This story is fully documented by Metcalf (1994). It also led to the discovery of the numerous cytokines, or lymphokines, affecting T and B lymphocytes and macrophages in the immune system. Untangling the complex autocrine and paracrine interplay of all the cytokines and their receptors, and changes found in cancer cells, remains a major research activity to this day.

XI. SHORT-RANGECELL INTERACTIONS Regulation of cell behavior by direct contact between neighboring cells and with the supporting substrate has also become a major research topic. The first pioneering observation that contact between cells could affect their behavior was made in London by Abercrombie and Heaysman (1954), who showed that the motility of fibroblasts was paralyzed by contact with a neighbor. Cells in confluent sheets of cells were subject to “contact inhibition” of movement. Abercrombie and Heaysman also showed that cancer cells were insensitive to this inhibition, and continued to move actively even in dense cultures. It should be stressed that this important observation, and subsequent studies by Abercrombie, were confined to cell motility and not growth. Nevertheless, it drew attention to another phenomenon, the inhibition of growth of most normal cells in close contact at high density, in contrast to tumor cells, which continued to grow as well as move. It was at first assumed that contact inhibition of growth and movement were closely linked. But there was already evidence that growth at high density was affected by other factors, such as medium depletion, so to avoid

Fundamentals of Cancer Cell Biology

11

confusion Rubin and 1 proposed the term “density-dependent inhibition” of growth, to distinguish it from “contact inhibition” of movement (Stoker and Rubin, 1967). Holley and Kiernan (1968), in La Jolla, then reported that growth was indeed affected by serum requirement more than cell density. Later I was able to show how changes in the diffusion boundary layer in the medium close to the cell surface could have a short-range effect that might simulate cell contact (Stoker, 1973). Nevertheless, regulation by direct cell contact cannot be excluded, and Dulbecco (1970) has suggested the term “topoinhibition” for any effect that may be identified. Contact inhibition and density-dependent inhibition, though still unexplained, are important because of the loss of sensitivity of cancer cells. This characteristic is recessive in most cancer cell lines that have been tested, because it was shown early on that the tumor cells temporarily regain the normal phenotype when in contact with normal cells in dense cultures (Stoker, 1964). Later it was found that the tumor phenotype is suppressed in a hybrid with a normal cell (see later). In recent years the attempts to explain regulation at short range between neighboring cells have been carried out at the molecular level and focused on cell surface and substrate molecules that promote adhesion. Before turning to this major field, however, I wish to deal with the pioneer work in another aspect of cell contact, namely gap junction communication.

XII. JUNCTIONAL COMMUNICATION Although it was known that electrical connections allowed ionic exchange between nerve and muscle cells, it was Lowenstein and his associates (1965) in Miami who first showed similar connections between epithelial cells. Ionic coupling was also shown between a variety of normal and transformed cells in culture by Potter and colleagues (1966). Independently, Subak Sharpe and coworkers (1966) in Glasgow, using autoradiography, reported metabolic cooperation due to direct contact and showed that hypoxanthine was transferred between fibroblasts. Subsequent work by these groups confirmed that molecules up to a molecular mass of about 1000 Da would pass freely between like and unlike cells, but some tumor and transformed cells would not communicate with each other. Meanwhile, gap junctions were being identified and characterized by electron microscopists, and these junctions, finally shown to be composed of connexin molecules, were identified as the pathway of communication (Revel and Karnovsky, 1967). Lowenstein has from his earliest contributions put forward the view that cell communication is implicated in the regulation of growth in confluent cultures of normal cells, and that this control is lost when communication is deficient,

12

Michael Stoker

as in many tumor cells. Although originally an attractive hypothesis, it is gratifying to see that in recent years increasing evidence in favor of this proposal has been obtained, especially by Mehta, a colleague of Lowenstein (Mehta et al., 1986).

XIII. CELL AND SUBSTRATE ADHESION In recent years, one of the most active and exciting fields of research in cell biology has been concerned with the adhesion of cells to one another and to the extracellular substrate. This is now known to be due to families of cell adhesion molecules (CAMs) and substrate adhesion molecules (SAMs) that bind specifically, either homotypically to one another or hetereotypically to a receptor molecule. CAMs and SAMs are now thought to be responsible for the arrangement of different cell types in tissues during development and, by their association with the cytoskeleton, to play a role in cell movement, either normal or abnormal as seen in dissemination of tumor cells. Many distinguished scientists have been leaders in these developments, notably Hynes (1973), who discovered fibronectin, the first SAM, in London, and more recently Edelman (1985) in New York, who showed the importance of the CAMs. In this article, however, I should like to draw attention to the earlier foundations of this field, beginning over 40 years ago. Following some earlier work by Holfreter, it was the reaggregation experiments of Moscona (1957, 1961) in Chicago that showed clearly that separated embryo cells not only reaggregated but sorted themselves into associations of different cell types. Moscona and his colleagues extended their studies of reassortment to other animal tissues, including sponges, and he postulated that the arrangement might be due to molecules on the cell surface with specific binding sites. In the absence of direct evidence of such molecules at the time, other views were put forward, such as Steinberg’s model based on differential adhesiveness. It was many years before the first cell-specific adhesion molecules, the integrins and cadherins, were eventually identified and Moscona’s idea was shown to be substantially correct.

XIV. THE TUMOR VIRUSES A great deal of cancer research is at present centered on the protooncogenes and tumor suppressor genes, and the way in which their perturbation as oncogenes, for example, gives rise to cancer itself. Oncogenes take us all the way back to the discovery of tumor viruses, and the later research that

Fundamentals of Cancer Cell Biology

13

revealed the similarity to lysogeny in bacteria. The Rous sarcoma virus [an RNA virus], discovered in New York over 80 years ago (Rous, 1911) has been particularly important in the discovery of oncogenes, while the papillomavirus, first studied in the 1930s by Shope (1932), was the forerunner of the DNA viruses. Additional tools of special significance for tumor cell biology were two viruses discovered in the 1950s-polyomavirus of mice (Stewart et al., 1957), and simian virus 40 (SV40) of monkeys, which came to light as a result of the poliovirus vaccine program in the United States. A little later, the first human tumor virus was found in Burkitt’s lymphoma by Epstein and his colleagues working in London and Africa (Epstein et al., 1964). Fortunately, however, the whole story of the DNA-containing tumor viruses has been dealt with very thoroughly already by Levine (1994), and I therefore confine myself to the role of tumor viruses in expanding our knowledge of normal and tumor cell biology, and ask the editors (and Dr. Levine) to excuse any overlap.

A. Transformation of Cultured Cells A turning point in tumor cell biology was the observation by Temin and Rubin (1958), working in Dulbecco’s laboratory in Pasadena, that cultured chick fibroblasts exposed to Rous sarcoma virus alter in morphology and grow into recognizable dense foci, which were easily distinguished in the culture. The virus could therefore induce a genetic change affecting the morphology and growth of normal cells; in addition, the technique permitted an assay of virus particles as “focus-forming units.” In turn, it showed that single cells could be transformed by single virus particles. (At this stage it was not known if the transformed cells were tumor cells because of the difficulty of transplantation in chickens.) Rous sarcoma virus particles were shown to contain RNA and not DNA, but a few years later Dulbecco and Vogt (1960) in Pasadena, and Medina and Sachs (1961) in Rehovot, as well as Macpherson and I in Glasgow (Stoker and Macpherson, 1961), showed that the recently isolated DNAcontaining polyomavirus would transform cultured hamster cells. When transplanted into hamsters, these transformed cells gave rise to sarcomas. The discovery of neoplastic transformation in vitro by tumor viruses opened the way to many of the major developments in tumor cell genetics that followed in later years. It was now possible to study the characteristics of pure, clonal populations of newborn tumor cells, but since they arose in a mixed population of normal cells, the predecessor of a single transformed cell giving rise to a clone could not be identified for comparison. The isolation of two stable cell lines, which had many of the characteris-

14

Michael Stoker

tics of normal fibroblasts, solved this problem and greatly improved the quantitative analysis of transformation. First in Glasgow, Macpherson and I isolated a baby hamster kidney cell line (BHK21), which resembled the normal fibroblast in morphology, arrangement, and growth. It could be passaged indefinitely like other stable lines, and clones could easily be isolated from single cells. These pure populations could then be transformed by polyomavirus (Macpherson and Stoker, 1962), and this for the first time allowed a comparison of the transformed cell with its cloned precursor (Stoker, 1962; Stoker and Abel, 1962). Soon afterward, using a clonal population of the same BHK21 cells, Macpherson and Montagnier (1964) developed a selective assay for transformation using agar suspension, which inhibits the growth of the anchorage-dependent normal cells but not transformed cells. This assay system allowed us to transform the cells with isolated viral DNA (Crawford et al., 1964). Meanwhile, another important cell line had been isolated from mouse fibroblasts by Todaro and Green (1963) in New York. This was a specially selected mouse fibroblast line, named 3T3, which had a high colony-forming efficiency and strongly arrested growth in confluent cultures. These cells could be transformed in vitro by SV40, and colonies of transformed cells that escaped growth arrest were easily distinguished on the thin monolayer of normal cells (Todaro and Green, 1964). The 3T3 cell system, and the SV40 transformed derivative developed by Green and his colleagues, subsequently became very important and were used widely for the study of both normal growth regulation and its perturbation by a tumor virus. This research on neoplastic transformation by tumor viruses was followed by reports of similar transformation of cultured cells by chemical carcinogens, first by Lasnitski (1963) in preliminary research with organ cultures at the Strangeways Laboratory in Cambridge, and then in cell cultures by Berwald and Sachs (1965) in Rehovot, and by Chen and Heidelberger (1969) in Madison.

B. Integration of Viral and Cell Genomes The viruses did not multiply in cells transformed by polyomavirus or SV40, or the equivalent tumor cells from animals. The transforming virus apparently disappeared, and at first its continued presence seemed unnecessary for persistence of the transformed phenotype and transmission to progeny, a sort of hit and run. However Habel (1961) had observed that the transformed cells still expressed a viral antigen, suggesting the presence of at least one virus gene. Then came the reports from Koprowski and colleagues (1967) in Philadelphia, and from Watkins and Dulbecco (1967) in La Jolla, that fusion of a virus-transformed cell with a second “permissive” cell,

Fundamentals of Cancer Cell Biology

15

which allowed virus multiplication, yielded a harvest of virus particles and revealed the presence of hidden virus in the transformed cells after all. Soon afterward, Westfal and Dulbecco (1968) used DNA hybridization to detect the presence of the whole virus genome integrated in the chromosomal DNA of the transformed cell. These important discoveries showed that DNAcontaining viruses could behave like temperate bacteriophages such as lambda, and introduce new genes into animal cells. It was difficult to see how this could apply to RNA-containing viruses such as the Rous sarcoma virus. However, in 1963, Temin, the codiscoverer of in vitro transformation, had made the outrageous proposal that the Rous virus RNA might persist in a tumor cell as a DNA copy. In the absence of a suitable enzyme this seemed impossible, and there was much sad shaking of heads. Nevertheless, Temin (1968) showed a requirement for DNA synthesis in the early stages of infection, and there had already been suggestive but not conclusive evidence of DNA sequences matching the viral RNA (Temin, 1964). But there was still a requirement for an unlikely enzyme that could achieve reverse transcription from RNA into DNA. Its discovery did not take long; the isolation of reverse transcriptase independently by Baltimore (1970) and by Temin and Mizutani (1970) burst upon us a few years later. This was a real foundation pillar, which not only led to new concepts in cancer but revolutionized techniques available to geneticists for manipulating genes.

XV. CELL FUSION I wish to discuss one more topic that I have already mentioned briefly, namely cell fusion and the formation of artificial heterokaryons. The foundation of this important research cannot be ignored, since it has led to such a rich harvest of applications in many fields, including cancer cell biology. Okada (1962) in Osaka originally noticed that infection with Sendai virus would lead to the formation of giant polynuclear cells. Then, in 1965 in Oxford, Harris and Watkins used inactivated Sendai virus to deliberately fuse two dissimilar cells, HeLa cells and Erlich ascites tumor cells. Harris (1965) followed up this advance by reporting that heterokaryons with two nuclei could be made with differentiated cells from a variety of species, and that a multiplying partner could initiate DNA synthesis in cells such as lymphocytes and erythrocytes, in which DNA synthesis is normally suppressed. Cells with mixed karyotype also arise occasionally in mixed cultures, as observed earlier by Barski and his colleagues (1960). This was extended and analyzed by Sorieul and Ephrussi (1961) at Gif using cells from two mouse

16

Michael Stoker

strains with different marker chromosomes. Further improvement in selection of the rare spontaneous hybrids was obtained by Littlefield (1964) in Boston, by using mutant cell lines and selective medium. Finally, the discovery of Sendai virus-mediated cell fusion allowed Puck and his colleagues in Denver to study hybrids between their well-characterized set of mutants (Kao et al., 1969). Meanwhile, Weiss with Ephrussi in Gif (1966) and with Green (1967) in New York had made the important observation that human-mouse hybrids retained the mouse chromosomes but human chromosomes were lost. This allowed mapping of certain human genes to individual chromosomes, as shown, for example, by Ruddle and his colleagues in New Haven, and others. The analysis of malignancy in hybrids derived from fused cells was reported in 1971 by Wiener and Klein in Stockholm, working with Harris in Oxford (Weiner et af., 1971). After fusion of normal mouse fibroblasts with various malignant cell lines, most surviving hybrids had lost significant numbers of chromosomes, but some carried the full or almost full chromosome complement of both parents. When tested for malignancy by transplantation, however, it was found that with chromosome loss the hybrid was still malignant, but a full complement of chromosomes suppressed the malignancy. This important result showed that cancer could be recessive in character, and so forecast the tumor suppressor genes.

XVI. CONCUISION We have now reached 1971, and it is time I stopped. This does not mean that pioneering research has been absent in the following 25 years; quite the contrary. I have already indicated a few later dates, but have not even reached the antioncogenes, or signaling cascades, or many other exciting topics. Some discoveries being made today will be the foundations of entirely new, at present unidentified, fields. However, 1 stop at 1971 because 1 can identify the crucial advances before and during the first 25 years of my life in research with greater clarity than those that are closer to the present day. Judgment of the relative importance of critical discoveries in this later period would be hazardous and perhaps contentious. I leave it to another reviewer, 20 years hence. This article may be criticized for lack of attention to the word “cancer.” However, the wealth of information that is now being reported on the biology of cancer cells is largely based on earlier work described here simply on “cells.” As I have already indicated, much early research on cells in culture was carried out on tumor cells, not because of their special properties but because they were easier to handle than normal cells. It was only later, when

Fundamentals of Cancer Cell Biology

17

valid comparisons with normal cells were available, that cancer cells could be characterized. I request forgiveness for the large areas of omission. Most of these were deliberate because of overlap with excellent articles by others in this series on Advances in Cancer Research. It may be noted that I have frequently referred to the geography of discoveries, rather than particular institutions. I did this to draw attention to the fact that important research is not concentrated in one or a few centers but comes from widely distributed laboratories around the world.

ACKNOWLEDGMENT I owe a debt to a n old friend, Dr. Bob Pollack, whose collection of important papers on cell biology, published as a book from Cold Spring Harbor Laboratory in 1975, made my task much easier.

REFERENCES Abercrombie, M., and Heaysman, J. E. (1954). Exp. Cell Res. 6, 293-306. Baltimore, D. (1970). Nature (London) 226, 1209-121 1. Barski, G., Sorieul, S., and Cornefert, F. (1960). Comptes Rendes Seances (Paris) 251, 1825. Berwald, Y., and Sachs, L. (1965).1.Natl. Cancer Inst. 35, 641-661. Bradley, T. R., and Metcalf, D. (1966). Aust. J. Exp. Biol. Med. 44, 287-300. Carrel, A. (1912).J. Exp. Med. 15, 516-528. Chen, T. T., and Heidelberger, C . (1969). Int. J. Cancer 4, 166-178. Cohen, S. (1962).J. Biol. Cbem. 237, 1555-1562. Crawford, L. V., Dulbecco, R., Fried, M., Montagnier, L., and Stoker, M. G. P. (1964). Proc. Natl. Acad. Sci. U.S.A. 52, 148-152. Dulbecco, R. (1970). Nature (London) 227, 802-806. Dulbecco, R., and Vogt, M. (1960). Proc. Natl. Acad. Sci. U.S.A. 46, 365-370. Eagle, H. (1955). Science 122,501-504. Earle, W. R. (1943).1. Natl. Cancer Inst. 4, 165-212. Earle, W. R., and Thompson, J. W. (1931). Public Health Rep. 45, 2672-2698. Edelman, G. E. (1985). Am. Rev. Biochem. 54, 135-139. Epstein, M. A., Achong, B. G., and Barr, Y. M. (1964). Lancet 1, 702-703. Fischer, A. (1930). “Gewebeguchtung.” Verlag Rudolph Muller Steinicke, Munchen. Gey, G. O., Coffman, W. D., and Kubicek, M. T. (1952). Cancer Res. 12, 264-265. Habel, K. (1961). Proc. SOC. Exp. Biol. ( N e w York) 106, 722-725. Harris, H. (1965). Nature (London) 206, 583-588. Harris, H., and Watkins, J. F. (1965). Nature (London) 205, 640-646. Harrison, R. G. (1907). Proc. SOC. Exp. Biol. Med. 4, 140-143. Hayflick, L., and Moorhead, P. S. (1961). Exp. Cell Res. 25, 585-621. Holley, R. W., and Kiernan, J. A. (1968). Proc. Natl. Acad. Sci. U.S.A. 60, 300-301. Hynes, R. 0. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 3170-3174.

18

Michael Stoker

Kao, F. T., Johnson, R. T., and Puck, T. T. (1969). Science 164, 312-314. Kerr, J. F. R., Wylie, A. H., and Currie, A. R. (1972). Br. J. Cancer 26, 239-257. Koprowski, H., Jensen, F. C., and Steplewski, Z. (1967). Proc. Natl. Acad. Sci. U.S.A. 58,127133. Lasnitski, 1. (1963). In "Biology of the Prostate and Related Tissues" (Cancer Institute Monograph 12), pp. 381-403. Levi Montalcini, R. (1966). Harvey Lect. 60, 217-259. Levine, A. (1994). Adv. Cancer Res. 65, 141-168. Lewis, M. R., and Lewis, W. H. (1911). John Hopkins Hosp. Bull. 22, 126-127. Littlefield, J. (1964). Science 145, 709-710. Lowenstein, W. R., Socolar, S. J., Higashino, S., Kanno, Y.,and Davidson, N. (1965). Science 149,295-298. Macpherson, I., and Montagnier, L. (1964). Virology 23, 291-294. Macpherson, I., and Stoker, M. (1962). Virology 16, 147-151. Medina, D., and Sachs, L. (1961). Br. /. Cancer 15, 885-904. Mehta, P. P., Bertram, J. S., and Loewenstein, W. R. (1986). Cell 44, 187-196. Metcalf, D. (1994). Adv. Cancer Res. 63, 41-91. Moscona, A. (1957). Proc. Natl. Acad. Sci. U.S.A. 43, 184-193. Moscona, A. (1961). Exp. Cell Res. 22, 455-475. Okada, Y. (1962). Exp. Cell Res. 26, 98-107. Palade, G. E., and de Duve, C. (1971). /. Cell. Biol. 50, 5D-56D. Pluenik, D. H., and Sachs, L. (1965). J. Cell. Comp. Physiol. 66, 319-324. Plutznik, D. H., and Sachs, L. (1966). Exp. Cell Res. 43, 553-563. Pomerat, C. M., and Willmer, E. N. (1939). J. Exp. Biol. 16,232-249. Potter, D. D., Furshpan, E. J., and Lennox, E. S. (1966). Proc. Natl. Acad. Sci. U.S.A. 55,328336. Puck, T. T., and Fisher, H. W. (1956). J. Exp. Med. 104, 427-434. Puck, T. T., and Marcus, P. 1. (1955). Proc. Natl. Acad. Sci. U.S.A. 41,432-437. Revel, J. P., and Karnovski, M. J. (1967). J. Cell. Biol. 33, C7-Cl2. Rous, P. J. (1911). /. Exp. Med. 13, 397-411. Sandford, K. K., Earle, W. R., and Likely, G. D. (1948). J. Natl. Cancer Inst. 9, 229-246. Shope, R. E. (1932). J. Exp. Med. 56, 803-810. Sorieul, S., and Ephrussi, B. (1960). Nature (London) 190, 653-654. Stewart, S. E., Eddy, B. E., Gochenour, A. M., Borchese, N. G., and Grubbs, G. E. (1957). Virology 3, 380-400. Stoker, M. (1962). Virology 18, 649-651. Stoker, M. (1964). Virology 24, 165-174. Stoker, M. (1973). Nature (London) 246,200-203. Stoker, M., and Abel, P. (1962). Cold Spring Harbor Symp. Quant. Biol. 27, 375-385. Stoker, M., and Macpherson, I. (1961). Virology 14, 359-370. Stoker, M., and Rubin, H. (1967). Nature (London) 215, 171-172. Strangeways, T. S. P. (1922). Proc. R. SOC. B (London) 94, 137-141. Strangeways, T. S. P., and Canti, R. G. (1927). Q. J. Microsc. Sci. 71, 1-14. Strangeways, T. S. P., and Fell, H. B. (1926). Proc. R. SOC. B (London) 99, 240-366. Strangeways, T. S. P., and Oakley, H. E. H. (1923). Proc. R. SOC. B (London) 95, 373-381. Subak Sharpe, J., Burke, R., and Pitts, J. (1966). Heredity 21, 342-343. Temin, H. M. (1963). Virology 20, 577-582. Temin, H. M. (1964). Proc. Natl. Acad. Sci. U.S.A. 52, 323-329. Temin, H. M. (1968). Cancer Res. 28, 1835-1838. Temin, H. M., and Mizutani, S. (1970). Nature (London) 226, 1211-1213. Temin, H., and Rubin, H. (1958). Virology 6, 669-688.

Fundamentals of Cancer Cell Biology Todaro, G. I., and Green, H. (1963).J. Cell Biol. 17, 299-313. Todaro, G. I., and Green, H. (1964). Virology 23, 117-119. Watkins, J. F., and Dulbecco, R. (1967).Proc. Natl. Acad. Sci. U.S.A. 58, 1396-1403. Weiss, M. C., and Green, H. (1967). Proc. Natl. Acad. Sci. U.S.A. 58, 1104-1111. Weiss, M. C., and Ephrussi, B. (1966).Genetics 54, 1095-1109. Westfal, H., and Dulbecco, R. (1968).Proc. Nutl. Acad. Sci. U.S.A. 59, 1158-1165. Weymouth, C. (1956).J. Nutl. Cancer Inst. 17, 305-311. Wiener, F., Klein, G., and Harris, H. (1971).J. Cell Sci. 8, 681-692. Wildy, P., and Stoker, M. (1958).Nature (London) 181, 1407-1408. Willmer, E. N., and Jacoby, F. (1936).J. Exp. Biol. 13,237-248.

19

This Page Intentionally Left Blank

FOUNDATIONS IN CANCER RESEARCH The Step-by6tep Development of Epithelial Cancer: From Phenotype to Genotype Emmanuel Farber Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University,Philadelphia, Pennsylvania 19107

Pathology is the science of the physiological reactions of the living organism to an abnormal environment. And since this is much wider and more varied than its more usual and narrower form, known as the normal or physiological environment, 1 would define pathology as the Greater Physiology. Just as health is the sum o f the physiological reactions of the organism to usual or normal stimuli, disease is the sum of the physiological reactions to unusual or abnormal stimuli. G . W. de P. Nicholson (1950)

1. Cancer Development as Basic to Cancer Research 11. Patterns of Development of Epithelial Cancers Ill. A Working Hypothesis IV. The Phenotypes A. Liver B. Other Solid Organs C. Skin D. Colon E. Other Surfaces V. The Genotypes VI. The Challenge A. Cancer Development as an Evolutionary Adaptive Process B. From Phenotype to Genotype: A Desirable Conceptual Approach References

Advances in CANCER RESEARCH, Vol. 70 Copyright Q 1996 by Academic Press, Inc. All righis of reproduction in any form reserved.

22

Emmanuel Farber

I. CANCER DEVELOPMENT AS BASIC TO CANCER RESEARCH Cancer research has at least two major objectives: (a) the understanding of the cancer phenotype and how it develops and evolves as the immediate basis for the diagnosis and treatment of cancer, and (b) the elucidation of the fundamental molecular-biochemical-biological modulations that underlie the development of the cancer phenotype and its continual evolution to increasingly malignant behavior. The second objective is to add an important facet to our current concepts of basic biology, both “normal” and in disease. In many research enterprises that fall under the rubric of “cancer research,” both of these major objectives are important and relevant, even though the therapeutic aspects may dominate. An important phase of cancer research that is receiving ever-increasing attention is the prevention of cancer in several sites. This is the major longterm medical objective in my studies. Despite our limited knowledge in this important area of cancer research, the past several decades have seen large changes in the occurrence of some cancers, including large unexplained decreases (e.g., stomach), in which cancer research has contributed little to their control or to understanding the rational basis for these phenomena. However, it is likely that a knowledge of the etiology of the cancer development and/or the steps through which cancer develops may be necessary for the further occurrence of decreases in the incidence of many other cancers. Ideally, the identification of major etiological factors, such as cigarette smoking, ultraviolet radiation, and hepatitis due to hepatitis B virus, and their removal or their neutralization are the easiest and most efficient approaches to the prevention of some major cancers. The prospects for the prevention of bronchogenic carcinoma, some skin cancers, including melanomas, and hepatocellular carcinoma are real, even though cultural-economic considerations may retard the rate of progress. Unfortunately, for many other epithelial cancers, this prospect is not currently evident. With these carcinomas, such as in breast, colon-rectum, pancreas, kidney, and prostate, and in some lung cancers, a realistic identification of a modulatable etiology remains a goal for the future. For such cancers, our only approach to date is to interrupt the long step-by-step processes that precede the cancer and to do so in a scientifically rational and safe manner. Since epithelial cancers and their preceding and precursor epithelial changes are known only by their phenotypes, it is important to delineate these in a scientifically meaningful way. This is a rational basis for the study of the genomic and molecular backgrounds that, in composite, determine in part the phenotype of each precursor lesion for each type of cancer and how

Development of Epithelial Cancer

23

these various components relate to the phenotypes of the cancers that occur later. Thus, the major objective of this area of cancer research is to delineate the phenotype of the cells of each major step on the path to cancer and to so change the phenotype as to prevent or even delay its evolution to carcinoma. The evidence already available clearly indicates that cancer development can be either delayed in a major way or even prevented by modulation of the phenotype of the precursor cells. Unfortunately, the currently known approaches in this area are indeed limited and are by no means established as safe. Agents such as the retinoids and tamoxifen, which have preventive effects on the phenotype of some cancer-precursor lesions, have potent cellular physiological effects and are themselves carcinogens in experimental animals. Their uses are fraught with potential hazards when employed more or less continuously for long periods of time. Although the knowledge of altered genotypes may ultimately prove to be necessary for us to understand how cancer develops, it is axiomatic that a knowledge of altered phenotypes is a necessary prerequisite. Currently, the relevance to cancer development of many genomic alterations remains unclear, since they do not offer, in any known system, a testable hypothesis for understanding the altered phenotype at the particular step in the process. For example, virtually every oncogene, including suppressor-altered genes, has as its major focus loss of control of cell proliferation. As discussed shortly, this phenotype is not an early change in the development of cancer and cannot be the basis for the several earlier steps that precede the appearance of malignant neoplasia in epithelial tissues.

11. PATTERNS OF DEVELOPMENT

OF EPITHELIAL CANCERS As outlined in Table I, there are at least three major patterns for epithelial cancer development in humans and animals. Patterns I and I1 are commonly seen and studied in humans. However, model systems for molecular-biochemical and biological analyses are mainly available for pattern I. Pattern I1 is largely studied in humans by pathologists and clinicians from a descriptive vantage point. Although some genes and immediate gene products (mRNA) are being studied in pattern 11, such studies have been largely descriptive and casual, mainly because of the problems of availability and access of precursor lesions and their lack of synchrony. These complexities are commonly seen in studies of pattern I in humans as well. In fact, since pattern I1 is seen mainly on surfaces, generally readily seen in humans, it is easier to study than is pattern I in humans in some sites. The

24

Emmanuel Farber

Table I Biological Patterns of Epithelial Cancer Development 1. With discrete focal proliferations as putative precursor steps-largely

monoclonal

Examples: Skin Papilloma Urinary bladder Papilloma Larynx Papilloma Cervix Papilloma Liver Nodule Gastrointestinal tract Polyp (adenomatous) 11. Without obvious focal discrete proliferations-largely monoclonal Examples: Cervix Dysplasia, carcinoma-in-situ Skin Dysplasia, carcinoma-in-situ Bronchi Dysplasia, carcinoma-in-situ Urinary bladder Dysplasia, carcinoma-in-situ (“intraepithelial neoplasia ”) Ill. Without any evident precursor or precancerous lesions-polyclonal Neoplasms induced with retroviruses with oncogenes

skin, larynx, cervix, and urinary bladder are amenable to some study, while the gastrointestinal liver and tract are less so. However, as emphasized repeatedly by Foulds (1969,1975) “the statistical time relationship alone is not sufficient evidence of sequential development which requires demonstration of material continuity of the suspected sequential lesions” (1975). Foulds stresses that “it is important to distinguish between merely temporal precursors of a particular kind of tumour and the material or morphological precursors within whose substance the sequential tumour actually develops” (1975).If more than one precursor lesion is seen at any time point before a single cancer is evident, a highly synchronized occurrence or appearance of the lesions is a necessity if one is to propose a step-by-step sequence as a working hypothesis that can be subjected to test. As discussed elsewhere (Farber and Sarma, 1987), such analyzable model systems that show a reasonably high degree of synchrony are not generally available except for very few models, among which is the resistant hepatocyte model in the liver of the rat. Both pattern 1 and pattern I1 processes, despite their apparent differences, are slow, long-term, and multistep and involve a small minority of the altered epithelial cells at each of the several steps that are seen during the evolution to cancer. In humans, these processes may last from three to five decades from beginning to cancer. In experimental animals, one-third to two-thirds of the average life span may be required. During these long processes, new phenotypes can be recognized in the different steps as some of the precursor lesions evolve to cancer.

Development of Epithelial Cancer

25

The time frame for neoplasms of pattern 111 is quite different. In experimental animals, the appearance of malignant neoplasia after exposure to the agent is very rapid, occurring within one cell cycle or less. So far, of the neoplasms that have been studied in intact animals, only an occasional epithelial neoplasm, such as a carcinoma in the mammary gland, has been observed (Reddy et al., 1988). Pattern 111 appears to be rare, if it occurs at all, in humans. These neoplasms are almost always polyclonal. The process of cancer development with retroviruses with oncogenes resembles viral diseases, with the virus spreading from cell to cell. No known examples of this type have been established in humans, at least in epithelial tissues. Since the liver in the rat has been the most amenable to a step-by-step analysis of carcinoma development with chemical carcinogens, it receives the major attention in this article. However, analysis for each step will compare our knowledge in the liver with that in the skin, urinary bladder, colon, cervix, and larynx, in so far as this is possible. Given that the classical separation of the carcinogenic process in the liver and other organs into three different phases or sequences is still somewhat useful and is well known generally, each step is related here to one of the three phases: initiation, promotion (selection), and progression (Fig. 1). As with every other site in patterns I and 11, carcinogenesis begins in the liver with the appearance of scattered newly altered hepatocytes in a seemingly random fashion (Gindi et al., 1994). These altered hepatocytes with a new phenotype show clonal expansion only when appropriately and differentially stimulated to undergo cell proliferation. The expanded clones, hepatocyte nodules, in turn become sites for the appearance of another new altered hepatocyte that again undergoes clonal expansion by cell proliferation. Thus, carcinogenesis in the liver and in most other organs and tissues is not a continuum, but rather a discontinuous process involving a small number of altered cells at several steps over a long period of time (Foulds, 1975). When a malignant neoplasm does finally appear, the cancer cells are now much less restricted in their growth patterns, can grow to a large size, and, of course, can metastasize. In any single individual, one or very few of the original hepatocyte nodules (or the expanded clones in other sites) show this evolution in any single life span. Before beginning to discuss the individual steps in any detail, I must say a word about nomenclature. For pragmatic reasons in clinical medicine, the focal proliferations that precede the appearance of cancer are often designated as benign neoplasms (adenoma, papilloma, etc.). While justified as a practical way to communicate between pathologists and clinicians, this custom is often scientifically inappropriate. The terms denote a judgment or conclusion that can seriously prejudice the scientist. It has become my policy to use descriptive terms unless 1 am certain that the designation in question

Initiation Carcinogm

I

Initiated Metabolic Cell Target cell L L Hepatocyte Activation Prolif. Inhibition

Promotion

Progression

+ Metastasis

Fig. I Schematic representation of several key steps in the sequence of steps between exposure to a chemical carcinogen and the appearance of hepatocellular carcinoma. Modified from Farber, E. (1984). Cancer Res. 44, 5463.

D e v e l o p m e n t of Epithelial Cancer

27

is valid. In carcinogenesis, most of the focal proliferations, including nodules, are clearly not neoplastic, and the focal proliferations with evolution to cancer represent only a quantitatively minor option. The term “preneoplastic” also may have biological implications that are quite unjustified, as discussed critically in detail by Foulds (1969). Because of these serious reservations, I have used descriptive terms for the most part based on the physiological behavior (biological phenotype) until unequivocal malignant behavior appears.

111. A

WORKING HYPOTHESIS

Based upon our knowledge of the early steps in liver carcinogenesis and upon the remarkable similarity in the patterns of development of epithelial cancers in several other sites, such as the skin, the pancreas, and probably the colon, 1 consider the development of epithelial cancers in the adult to be composed of two different sequences as follows: Sequence A-an initial sequence of several steps that are predominantly part of an adaptive physiological process of long duration that I have called “clonal adaptation” (Farber, 1990). Sequence B-a subsequent sequence of shorter duration involving progression from advanced steps in sequence A to advanced malignant neoplasia. Sequence B might well involve progressive mutations and selection as proposed by Nowell (1976), involving oncogenes, suppressor genes, and other genomic components. Underlying this sequence could well be an increasing degree of genomic instability favoring progressive disturbance in gene expression and control. The following discussion of phenotypes and genotypes relates as far as possible to this working hypothesis. Included is an analysis of the degrees to which the current information base and understanding supports or refutes this attempted synthesis.

IV. THE PHENOTYPES A. Liver I . INITIATION

The essence of the initiation process is the production or appearance of widely scattered isolated hepatocytes in which a new resistant phenotype

Emmanuel Farber

28

(active Protein

Polysaccharide

SH Carcinogen

ETC.

Fig. 2 Interaction of chemical carcinogens or their active derivates with various cellular molecules. From Farber, E. (1973). Cancer Res. 33, 2537.

has been induced constitutively, not transiently. A key physiological phenotype as a consequence of the new biochemical phenotype is resistance to the inhibitory effects of many carcinogens and other xenobiottcs. This new phenotype is induced rapidly by genotoxic carcinogens-that is, carcinogens that by themselves, or more commonly after suitable metabolic activation, interact with DNA as well as with RNA and protein (Fig. 2). When suitably assayed, at least one nongenotoxic (“epigenetic” (Weisburger and Williams, 1981)) carcinogen, clofibrate, can also induce a similar phenotype but much more slowly, taking several weeks of exposure rather than several minutes or hours (Nagai, 1993; Nagai et al., 1993). Since genotoxic carcinogens, by definition, induce alterations in the genome, and since the first identifiable altered cell is a rare cell in the liver (1 per 105-106 hepatocytes), as well as in the skin and probably in other sites as well, it is readily assumed that this first step is a mutagenic one and that the induced rare altered cell is functionally mutated. This is a presumption that still remains to be scientifically and carefully documented. Rubin (1993, 1995; Rubin et al., 1995a,b), in an extensive series of studies in cells in culture, has shown that rare altered cells can appear without apparent mutation and that these can become transformed to malignant neoplasms. Also, we know that many “new” cells in small numbers appear frequently at many steps during normal development from the fertilized ovum to the adult organism. Magee (1995) also has critically questioned the validity of this supposition and speculation.

Development of Epithelial Cancer

29

a. Phenotype I The rare altered hepatocytes that appear during initiation do not show any spontaneous cell proliferation either in vivo in the intact organism or in vitro in culture. This is the case in every system studied in which a small but effective dose of a chemical carcinogen can initiate the carcinogenic process but cannot enable further steps to occur without exposure to additional chemicals (promoting or selecting agents), including carcinogens. Since no ultimate cancer has been observed with an “initiating dose” of a carcinogen in any site without expansion by cell proliferation of the rare altered cell appearing during initiation, we must conclude tentatively that such expansion is necessary for the ultimate appearance of cancer. This expansion, appropriately called clonal expansion, follows initiation and is designated as promotion or, better, “selection.” Clonal expansion by selection can occur by one or more of at least four different processes: (a) differential inhibition, (b) differential stimulation, (c) differential recovery, or (d) differential cytotoxicity (Farber, 1982a,b). The only one for which there is considerable evidence in its support is differential inhibition. This type of selection depends upon a resistance of the few altered postinitiation cells to the inhibitory effect of the selecting procedure. Based upon suggestions made by Haddow in 1938, which in turn reflected previous opinions, it was hypothesized that an effect of a carcinogen was to induce a resistance to the inhibiting effects of carcinogens and other xenobiotics on cell proliferation (Farber, 1973). This resistance would allow for clonal expansion during promotion. Such a resistance was first demonstrated experimentally (and quantitatively) by Solt and Farber in 1976. This physiologically new phenotype was accompanied by several biochemical alterations that have been designated in composite as the “resistance phenotype” (Farber, 1984, 1987; Roomi et al., 1985) (Table 11). The genesis of this new constitutive phenotype, phenotype 1, in the rare hepatocyte is a two-step process. Some change is induced by the activated form of carcinogen, possibly via an interaction with DNA. The designation of DNA as the major target is attractive but is as yet unproven. This first step must be followed by a single round of cell proliferation within about 96 h (Cayama et al., 1978). The new phenotype then appears. The cell proliferation can be triggered by induction of cell death by the carcinogen, by a hepatonecrogenic agent, or by partial hepatectomy (Pitot and Sirica, 1980; Columbano et al., 1981; Ying et al., 1982). Cell proliferation triggered by a primary hyperplastic agent, such as phenobarbital or lead nitrate, is ineffective (Columbano et al., 1983, 1984, 1987a,b). Phenotype 1 can be induced by any one of many different chemical carcinogens of widely differing chemical structures. The phenotype appearing in different hepatocytes with the same or different agents shows some quantitative variations in any single biochemical parameter but has a measurable

30

Emmanuel Farber

-\able II Phenotype 1: Biochemical Pattern Associated with Resistance Phenotype A. Decrease in xenobiotic metabolizing and activating components: Cytochrome P450 Mixed-function oxygenases Glucose-6-phosphatase P-glucuronidase Glutathione peroxidase Nucleotide polyphosphatase (ATPase) Ribonudeases and deoxyribonucleases Serine dehydratase Sulfotransferase Superoxide dismutase Lipid peroxidation B. Increase in detoxification and related components: Aldehyde dehydrogenase isozyme DT-diaphorase (quinone reductase) Epoxide hydrase (epoxide hydrolase) (microsomal) Glucose-6-phosphate dehydrogenase Gamma-glutamyl transpeptidase Glutathione Glutathione-S-transferase, particularly rGSTP1-1 N-Acetylglucosaminyltransferase P-glycoprotein (rnultidrug resistance, mdr) UDP G-transferase I

degree of resistance in each. The resistance with any single carcinogen shows no relation to the carcinogen used, since it is seen with a wide range of quite different xenobiotics (Tsuda et al., 1980). Whether the variation in the biochemical phenotype among the altered hepatocytes appearing during initiation is significantly greater than the biochemical differences between hepatocytes in different zones of the liver acini (or lobules) or in different zones in different lobes has not been established (Pitot et al., 1978; Pitot and Sirica, 1980; Ogawa et al., 1980). The pattern of biochemical components in phenotype 1 does not appear to be “abnormal” since it can also be induced by exposure to one of a few xenobiotics. For example, as recorded in Table 111, there is a remarkable resemblance between phenotype 1 and the new phenotype induced by a single intravenous dose of lead nitrate (Columbano et al., 1983, 1984; Ledda-Columbano et al., 1989; Roomi et al., 1986). A similar phenotype can also be induced by the antioxidants butyl hydroxyanisole (BHA) and butyl hydroxytoluene (BHT) (Cha and Bueding, 1979; Cha and Heine, 1982). Unlike the constitutive phenotype 1 appearing during initiation, the phenotype associated with these agents is of a transient nature, lasting only a few days, and appears regionally in the liver, not in isolated, seemingly random hepatocytes (Koo et al., 1994).

Development of Epithelial Cancer

31

Table 111 Lead-Inducible Phenotype vs Phenotype 1 in Nodulesa Nodules

__

Cholesterol synthesis Glucose-6-phosphate dehydrogenase Glycogen phosphorylase Phase I Cytochrome P450 Cytochrorne b, Various mixed-function oxygenases Total microsomal heme Phase I1 GSH GHS-S-transferase Blutathione-S-transferase-7-7(P) DT-diaphorase UDPG-transferase I Epoxide hydrolase a

t

Lead

t

-

-

.1

.1

-

-

, increase; -, no change; 1, decrease.

Phenotype 1 can persist for a long time in the rare altered hepatocytes but can be reversed with appropriate environmental manipulation. For example, the population of initiated hepatocytes can show almost complete reversion of phenotype 1 to the control hepatocyte phenotype if the animal is exposed to a peroxisome proliferator (clofibrate) for just a brief period of time (1-2 weeks) (Boult, 1994; Boult et af., 1994, 1995). S-adenosylmethionine can also induce a similar reversion (Pascale et af., 1992). In addition to chemical carcinogens, it has been suggested by Blumburg and London (1982; London and Blumburg, 1982) that hepatitis B may also have resistant hepatocytes (“R cells”) as early cells from which hepatocellular carcinoma may evolve. Initiation in skin, urinary bladder, and some other sites is also known to induce rare epithelial cells that are altered and that persist for long periods of time. However, the nature of the phenotypes induced and the mechanisms for their subsequent selection to form focal proliferations have not been explored mechanistically. Whether phenotype 1, as seen in the liver after initiation, is also associated with initiation in other sites would be a very important area for exploration. It must be emphasized that in no known site does initiation show any spontaneous cell proliferation of the altered cells. This is observed even when examined months after the time of initiation with an initiating dose of a carcinogen. The resistance phenotype in the initiated hepatocytes has several similarities to the phenotype in some human cancer cells that are resistant to some chemotherapeutic agents (Wolfe et al., 1986; Fairchild et al., 1987;

32

Emmanuel Farber

Mantle et al., 1987; Endicott and Ling, 1989; Farber and Sarma, 1987). The “natural” resistance of some cancers to chemotherapy could well be acquired very early in the carcinogenic process, as occurs in some hepatocellular carcinomas, and not necessarily be acquired much later in response to treatment (Farber, 1982a,b, 1990; Sikic, 1993).

2. PROMOTION AND PROGRESSION As already mentioned, the physiological resistance in phenotype 1 is used as the basis for clonal selection during promotion in some model systems. When the liver is stimulated to proliferate by partial hepatectomy or by a necrogetic agent, such as CCI,, and is exposed at the same time to an agent that can inhibit cell proliferation after suitable metabolic activation, such as 2-acetylaminofluorene or other carcinogens, only the rare hepatocytes with the resistance phenotype can respond (Solt and Farber 1976; Soh et al., 1977a,b; Eriksson et al., 1983). This “differential inhibition” is a major basis for promotion or selection in liver cancer development in the rat. Phenobarbital (Peraino et al., 1971, 1973, 1975; Eckl et al., 1988) and orotic acid (Sarma et al., 1986; Laconi et al., 1988) may well promote by a similar mechanism. It has been claimed that promotion by phenobarbital may occur by differential stimulation (Schulte-Hermann et al., 1984). Unfortunately, the results in an essential control, the effect of phenobarbital on cell proliferation of the hepatocytes in the surrounding liver relative to that in livers of control animals not exposed to the carcinogen, were not reported. a. Phenotype 2 The clonal expansion of the rare altered postinitiation hepatocytes with the resistant phenotype 1 leads to the formation of focal proliferations (nodules) in a seemingly random fashion throughout the liver (Solt et al., 1977b; Gindi et al., 1994). The nodules composed of phenotype 1 cells have a very distinctive organizational pattern of their hepatocytes. The cells are arranged in two-cell-thick or more than two-cell-thick plates and various other patterns, such as tubules (Reuber, 1965; Bannasch, 1968, Teebor and Becker, 1973; Farber, 1973, 1976; Ogawa et al., 1979a,b; Tatematsu et al., 1979). This hepatocyte pattern is similar to that in some nodules in human cirrhosis (Sasaki and Yoshida, 1935; Kinosita, 1937; Phillips and Steiner, 1966; Rubin and Popper, 1967). The nodules also show a distinctive alteration in blood supply, with a decrease in the supply from the portal vein and a relative increase in the arterial supply (Solt et al., 1977a; Conway et al., 1983, 1984). The nodules may appear rapidly and synchronously, as in the resistant hepatocyte model (Farber et al., 1978, 1989; Farber and Cameron, 1980; Farber, 1982a,b), or more slowly and asynchronously. Conceivably, the dif-

Development of Epithelial Cancer

33

ferent rates of clonal expansion may be a reflection of the intensities of the selection process and of the level of the resistance phenotype in different nodules. The biochemical phenotype is as already described under phenotype 1 (Table 11). In addition, the nodules show reproducible alterations in iron metabolism, such as decreases in iron uptake and/or concentration, total iron, total heme, heme enzymes (cytochrome P450,cytochrome b5, catalase, tryptophan-2,3-dioxygenase(“pyrolase”)), and heme-binding protein (cytosolic) (Becker et al., 1971;Stockert and Becker, 1980;Moore et al., 1983; L. C.Eriksson et al., 1986).Increases occur in heme oxygenase and transferrin receptors (60X)(Stout and Becker, 1987; Eriksson and Anderson, 1992).Alterations in glucose metabolism, including glycolytic enzymes, and in the pentose shunt enzymes have also been found (Scherer et al., 1972; Scherer and Emmelot, 1975;Emmelot and Scherer, 1980;Bannasch, 1968, 1986;Bannasch et al., 1982, 1984).In addition, a-fetoprotein is increased considerably in both phenotypes 1 and 2 (Becker and Sell, 1974;Becker et al., 1973, 1974). Thus, the hepatocyte nodules composed of hepatocytes and supporting cells show a wide spectrum of biochemical changes, both quantitative and qualitative. This pattern is unusually consistent in nodules from model to model and for many components between species, including humans. While some of the changes are also seen during liver regeneration and may reflect the hepatocyte proliferation in the clonal expansion, many of the changes are special to the nodules and may even be in the opposite direction to changes during regeneration (Farber and Cameron, 1980; Ernmelot and Scherer, 1980;Enomoto et al., 1981). Again, it must be pointed out that the cell proliferation in this clonal expansion is not spontaneous but is due to the regenerative stimulus applied. When the total hepatocyte nodule population reaches that in the control liver (i.e., when the regenerative component is complete), the majority of nodules no longer show any cell proliferation and demonstrate another phenotype, phenotype 3. Phenotype 2 is not associated with any increased degree of cell loss (socalled apoptosis). As with liver regeneration following either partial hepatectomy or liver cell necrosis, the hepatocytes proliferate until the mass of liver cell is reformed and then stop. There is virtually no cell loss o r cell death during this phase of the carcinogenic process (Rotstein et al., 1984, 1986; Farber et al., 1988).

b. Phenotype 3 When the hepatocyte proliferation returns to the control level in the nodules with the reformation of the original liver cell mass, the nodules show two major options: (a) phenotype 3-remodeling by redifferentiation to

34

Emmanuel Farber

normal-appearing liver in the majority of nodules (over 90-95 %) (so-called regression); and (b) phenotype 4-persistence in the small minority of nodules with spontaneous cell proliferation that is almost balanced by cell loss or cell death. Phenotype 3 consists of a remarkable restructuring of the nodule hepatocytes with a rearrangement to single-cell plates and an integration into the organizational pattern of the surrounding liver. With this change, the nodules “disappear” as nodules, but their cells appear as normal control liver cells without any obvious distinction from the surrounding liver (Enomoto and Farber, 1982; Tatematsu et al., 1983). During this redifferentiation, not only does the architecture become rearranged but the pattern of biochemical changes seen in phenotype 1 reverts to that of the control surrounding liver (Tatematsu et al., 1983; Kitagawa, 1971, 1976; Kitagawa and Pitot, 1995). In those areas where the nodules have remodeled, the previously nodular liver is now virtually indistinguishable from control liver. Unlike the aneuploidy in the later appearing hepatocellular carcinomas, the DNA content per nucleus in nodules is mainly euploid (Becker et al., 1971). The nodule hepatocytes respond well to the proliferative stimulus of partial hepatectomy, as do the nonnodular hepatocytes in the surrounding liver (Becker et al., 1971). They also respond to some inducing agents by increases in enzymes, as does the control liver. c. Phenotype 4 The hepatocyte nodules generated by clonal expansion have, as their minor option, persistence with slow evolution to malignant neoplasia. They acquire a new phenotype, phenotype 4 (Table IV). During this evolution, new foci of altered hepatocytes appear within the nodule. These have a more basophilic cytoplasm than do the surrounding cells and show some nuclear Tabie IV Phenotype 4 and Beyond: Properties of Persistent Nodules Leading to Cancer

I. Persistence of resistance phenotype 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 1.

12.

Spontaneous hepatocyte proliferation Balance between cell proliferation and cell loss until malignant neoplasia appears Slow progressive remodeling of increasing number of nodules “Ground glass hepatocytes” as a common hepatocyte appearance before cancer Normal diurnal rhythm Normal respose to phenobarbital New pattern of growth on transplantation to spleen with slow evolution to cancer Appearance of nodules in nodules Appearance of hepatocellular carcinoma within nodules with metastasis Imbalance between cell proliferation and cell loss “Full-blown” hepatocellular carcinoma

Development of Epithelial Cancer

35

anisocytosis and basophilia. The number of such foci of altered hepatocytes is not known. Although the persistent nodules are remarkably synchronous, the step-by-step evolution to cancer during progression has yet to be studied in great detail. However, it can be clearly and reproducively shown that the persistent nodules have a sequential phenotypic pattern that appears to be important in their evolution (Table IV). The persistence of a small number of nodules is, of course, relative. The progression sequence is by far the slowest in liver cancer development as well as for cancer development in several other sites. During this period, five phenotypic properties stand out:

1. The hepatocytes with phenotype 4, unlike those with phenotypes 1,2, and 3, show spontaneous cell proliferation, without the need for an external reproductive stimulus. However, this new property is also accompanied by the new appearance of cell loss, presumably by cell death, so that the overall growth of the nodules is very slow (Rotstein et al., 1984, 1986; Farber et al., 1988; Farber, 1990). The degree of cell loss almost balances the degree of cell proliferation (Rotstein et al., 1986). It is not known whether or not the cell loss is the expression of the well-known principle that an increase in liver cell number above a “physiological” range is the stimulus for the cell loss (Schulte-Hermann, 1974, 1979). 2. The persistent nodules continue to show slow remodeling to normalappearing liver with disappearance of the nodular arrangement. Thus, with increasing duration of progression, the number of nodules progressively decreases. Theoretically, if the development of cancer were delayed sufficiently, all the nodules would remodel. However, almost always, further phenotypic changes occur focally in the few persistent nodules. These foci of altered hepatocytes enlarge and involve whole nodules. These in turn show further focal phenotypic change that accelerates the evolution to cancer. This apparent option, remodeling versus progression with evolution to cancer, is seen in cancer development in other sites as well. However, in other sites, the nature of the “regression” or “disappearance” of nodules is not known. 3. The nodule hepatocytes that continue to persist have a striking “ground glass” appearance (Farber, 1976) very similar to the ground glass appearance of the persistent focally altered hepatocytes in hepatitis B carriers in humans (Stein et al., 1972; Huang et al., 1972; Huang and Groh, 1973; Gerber et al., 1974) and in mice that received transgenically the genes for the large surface antigen of hepatitis B virus (Babinet etal., 1985; Chisari et al., 1985, 1987, 1989). In the latter two cases, it has been shown that the large surface protein accumulates within the endoplasmic reticulum (ER) as fibers, along with the proliferation and dilation of the ER. In the ground

36

Emmanuel Farber

glass cells appearing in chemical hepatocarcinogens, unknown material is seen to accumulate inside the ER. Also, in humans with hereditary a,-antitrypsin (AAT) deficiency (S. Eiksson et al., 1986) and in mice that received the gene for AAT transgenically (Geller et al., 1994), AAT minus the appropriate addition of carbohydrate for secretion accumulates inside the ER. The humans and the mice develop a high incidence of hepatocellular carcinoma, as do the transgenic hepatitis B mice. 4. An interesting change from phenotype 3 to phenotype 4 concerns the behavior of nodular hepatocytes in the spleen. Hepatocytes from normal control rats can be transplanted to the spleen in syngeneic animals. These transplanted cells grow slowly and replace progressively more of the spleen. Hepatocytes of nodules with phenotype 3 behave like the hepatocytes from the controls, with the exception that they often express biochemical properties of phenotype 1, such as increase in gamma-glutamyl transpeptidase. Hepatocytes from nodules with phenotype 4 and subsequent, as yet uncharacterized phenotypes show a totally different behavior in the spleen. They grow focally as nodules, not diffusely, and show a high incidence of hepatocellular carcinoma with metastasis after many months (Finkelstein et al., 1983; Lee et al., 1983; Tatematsu et al., 1987). This is a striking new behavior and suggests that the nodules with phenotype 4, even early, are already programmed for ultimate malignant progression. 5. The persistent nodules show a slow, progressive appearance of new altered hepatocytes that show increasing basophilia with some altered appearance of the nuclei, suggesting dysplasia. The number of such steps during the evolution to malignancy is unknown. The progressive set of phenotypes during this evolution is also unknown. However, continued occurrence of synchrony with progression makes it feasible and possible to analyze this sequence in detail biochemically and with the newer tools of molecular biology (Farber et al., 1989).

3. THE CANCER PHENOTYPES

The main features of the cancer phenotype are well known: relatively uncontrolled growth, invasion, and metastasis. These biological functional parameters are associated with obvious alterations in nuclear structure and appearance. The nuclei show major changes in the organization and distribution of chromatin. Also, as discussed by Loeb (1994; Cheng and Loeb, 1993) and by Prehn (1994), the cancer cell shows considerable evidence of genomic instability. This fits well with older suggestions that cancer cells characteristically show inappropriate expression of genetic information (see Farber and Cameron, 1980). The inappropriate expression, for example, of isozymes and hormones has been documented many times.

Development of Epithelial Cancer

37

It must be emphasized that we do not know yet which relevant mutations precede and which follow the major disturbances in nuclear genomic organization, as emphasized by Prehn and by Loeb. It is widely appreciated that cancers of the same cell type of the same organ or tissue show large quantitative variations in their rates of growth and degrees of invasion and metastasis and in the extent of their inappropriate expression of genetic information. These variations, including the degree of alterations in gene instability, in the degree and pattern of differentiation, and in the biochemical properties in general, are probably largely responsible for the well-known uniqueness of each and every cancer of the same cell type (Heppner, 1984). This overwhelming variation makes it very difficult, if not impossible, to delineate and to understand in great detail the molecular genomic foundation for the behavior of any single cancer. Whether we can hope to obtain meaningful insight into malignant behavior by the largely random approaches now in common use without a much better understanding of the major steps in the development of cancer remains to be demonstrated effectively.

B. Other Solid Organs Major other solid organs, such as the pancreas, kidney, prostate, and brain, fall into patterns I or I1 in their development of cancers. Despite continued study to reveal cancer precursor and “premalignant” lesions, there is almost no understanding of how any of the early lesions develop and their fundamental phenotypes. Konishi and coworkers (Mizumoto et al., 1988, 1989, 1990; Scarpelli, 1995) have reported that reproducible early precursor lesions can be induced rapidly in the pancreas by the development of a model in the hamster using the same principle as used in the resistant hepatocyte model in the liver, a resistant carcinogen-induced pancreatic duct model. These observations offer a rational testable hypothesis for understanding how the first focal proliferation, focal ductular hyperplasia, might be generated. The rnolecular-biochemical nature of the pancreatic resistance phenotype will be awaited with interest as a major early step in the delineation of a possible testable mechanism for the early steps in one model for pancreatic cancer development. No such possible mechanistic analyses for kidney or prostate carcinogenesis have been proposed. For these organs, we are largely at the early morphological descriptive stage, without any insights into the possible new phenotypes that could explain how the first focal proliferative lesions develop. Without such insights into phenotypes, genotypic studies remain largely conjectural and speculative.

38

Emmanuel Farber

C. Skin The skin and the liver are the two sites that have received the lion’s share of attention over the past five to six decades. The delineation of the few major visible steps in cancer development were pioneered by Rous and coworkers and by Berenblum and coworkers in the late 1930s to early 1940s. The dominant pattern catalogued, pattern I, has been a major model for attention in chemical and other carcinogenesis. The almost exclusive emphasis during this period has been on the analysis of agents rather than processes (Yuspa and Poirier, 1988; Yuspa, 1994; Greenhalgh and Roop, 1994). It has been thought that the increasingly detailed study of what an agent does (carcinogen, promoter, modulator, etc.) will generate the major insights into the fundamentals of both phenotype and genotype of each visible step. Unfortunately, this conceptual approach to the step-by-step analysis has been disappointingly frugal. The fundamental molecular-biochemical phenotype of the papilloma and its precursor cells altered during initiation, and the phenotype used for clonal expansion to generate the papilloma, remain essentially unknown. This also applies to the subsequent steps between a persistent papilloma and an ultimate cancer and to the nature of the “regression” or “disappearance” of the majority of papillomata. A possible lead was the observation that one group of promoting agents, the phorbol esters, may act by inducing terminal differentiation in uninitiated epithelial cells, but not in initiated ones (see Yuspa and Porier, 1988). However, papillomas induced with a carcinogen plus a phorbol ester show differentiation from basal cells to keratinized surface cells just as does the surrounding skin. Thus, the suggestion regarding the action of phorbol esters remains to be established in the intact animal (in vivo). In the absence of a rational testable working hypotheses for how a papilloma develops (see Section 1II.A.l.a and III.A.2.a) and for a phenotype relevant to clonal expansion and for the subsequent steps, any genotypic analysis becomes again largely conjectural and speculative. The malignant melanoma and its genesis from precursor nonneoplastic or nonmalignant lesions have been profitably studied in humans by Clark and others (see Clark, 1994, for references). Unfortunately, again, how the first step might develop mechanistically and what the relevant phenotype might be are as yet unknown. A meaningful genotypic analysis of these and subsequent steps must await the presentation of some reasonable molecular and biochemical phenotypes if the analysis is to be relevant and useful.

D. Colon Despite the intense activity surrounding colon cancer and its development during the past few years (see Vogelstein et al., 1988; Fearon and Vogelstein,

Development of Epithelial Cancer

39

1990; Fearon and Jones, 1992; Liu et al., 1995a,b), the phenotypes of the major steps remain unclear and ill-defined. As with the early focal proliferative lesions in almost all other sites, the basis for their genesis is poorly defined. Of all the common sites for cancer in the Western world, the colon is the site with the most active cell proliferation in the normal or control individual. The colon epithelium shows vigorous cell proliferation in the lower segments of the glands followed by a highly reproducible pattern of differentiation and movement of the epithelial cells with shedding into the lumen (Lipkin, 1988; Lipkin and Higgins, 1988). Naturally, any focal increase in the number of cells in a polyp, either hyperplastic or adenomatous (Deschner and Lipkin, 1978), is insufficient to account for a focal proliferative lesion, such as the polyp, unless the balance between the rates of cell proliferation, cell differentiation, cell movement, and cell loss is disturbed. Although an increase in cell proliferation is reported to occur as a basis for a polyp, this cannot generate the lesion unless the rates of the other processes do not increase to the same extent. Although mitotic figures and DNAlabeled epithelial cells can be seen in the more superficial segments of the glands and polyps, this could fundamentally be due to a primary disturbance in cell differentiation, cell movement, and/or cell loss. A major set of gene products, the enzymes and other proteins, that go to make up the phenotype must be characterized if gene analysis and its alterations are to be meaningful biologically. So far, almost none of those that might be part of the first or early phenotype have been identified. The evidence that the early steps in colon cancer development are primarily focused on an altered control of the cell cycle and cell proliferation is lacking. This of necessity introduces a large element of speculation, uncertainty, or even doubt about the relevance and biological significance of the reported genomic changes in the development of colon cancer in humans. The increasing emphasis during the past few years on the focal alterations in the crypt epithelium (the aberrant crypt foci) as possible precursors of polyps is encouraging (Bird, 1987; Pretlow et al., 1991; Roncucci et al., 1991; Otori et al., 1995). Unfortunately, so far the emphasis (of necessity) is largely on the morphological characterization. Since most individual gene products are ultimately proteins, including enzymes, it is to be anticipated that the increasing delineation of the biochemical phenotype may give guidance to the analysis of appropriate and relevant genes during these early putative steps in colon cancer development.

E. Other Surfaces The lower urinary tract is interesting. Although lined by a stratified epithelium, the component cells are quiescent in the adult and remain so until

40

Emmanuel Farber

injury stimulates some reparative cell proliferation. As indicated in Table I, the urinary tract, especially the urinary bladder, shows two patterns, I and 11. There is considerable evidence in humans that pattern I1 may be the most common precursor for transitional cell carcinoma (Koss, 1979), even though pattern 1 is commonly seen. Unfortunately, the mechanism(s) of formation of focal hyperplasias in either pattern have no rational working phenotypic hypotheses. This situation is also present in carcinogenesis on other surfaces, such as the oral cavity, pharynx, larynx, bronchi, and esophagus.

V. THE GENOTYPES Cancer research in the past several years has been focusing increasingly on attempts to define the genotypic changes in malignant and benign neoplasia. The heavy redirection of research to the gene is in some respects gratifying and necessary. However, other aspects, such as the phenotypes, are not receiving even the small attention needed to explore this very fundamental base for cancer. Also, the gene studies are directed very much toward the late phase of cancer development, cancers themselves and their immediate precursors. The evidence in favor of sequence B in cancer development being mutational is outlined in Table V. Judging by what we know about the first few steps in cancer development in many sites, the emphasis on the late phases may not necessarily be illuminating in respect to fundamental mechanisms in the early carcinogenic sequence. By neglecting these early phases, we may very well be overlooking relatively “simple” and innovative ways to develop safe regimens for cancer prevention. The gene changes in a variety of different epithelial cancers have been reviewed in extenso in the past few years and need not receive much attenTable V Some Evidence in Favor of Sequence B Being Mutational-AdversarialConfrontational Cancer Development 1. Mutagenic, genotoxic range of many carcinogens-chemical, radiation, DNA viruses, and possibly RNA viruses 2. Wide range of genotoxic alterations by carcinogens 3. “Abnormal” behavior of cancer cells 4. Many genomic alterations in most cancers 5. Hereditary behavior of the cancer phenotype 6. Altered genomic control in cancer 7. Genomic disorganization common in most, if not all cancers 8. Diversity and heterogeneity of cancers of any single cell type

D e v e l o p m e n t of Epithelial Cancer

41

tion in this article (e.g., see Yuspa and Poirier, 1988; Balmain and Brown, 1988; Fearon and Vogelstein, 1990; Fearon and Jones, 1992; Van De Vijver, 1993; Nowell, 1993; Kovacs, 1993; Nakamura, 1993; Greenhalgh and Roop, 1994; Yuspa, 1994; Ozbun and Butel, 1995; MacPhee, 1995). Suffice it to say that almost no discussion was related to the early events in carcinogenesis, including those in the liver. What little was presented related to altered control of cell proliferation, a phenotypic change that only occurs spontaneously late in the process. Despite the major effort to develop some rational working hypothesis of the role of DNA damage in initiation (e.g., see Lawley, 1994), no testable suggestion has been presented that might explain the phenotypes seen in the liver after the exposure to one of many different carcinogens (phenotype 1, 2, and 3). It must be emphasized that, even though cell proliferation is an important component of several steps in the carcinogen process, the only obvious loss of major control of the cell cycle is seen quite late in the advanced cancer precursors and especially in malignancy. Prior to this time, in all the systems that have been studied, one sees a very highly controlled increase in cell proliferation almost balanced by cell loss (see Farber, 1991; Farber et al., 1988). The strong impression one has from the data in several systems is that the cell proliferation-cell loss is a part of a carefully programmed phenomenon that can persist for a considerable segment of sequence A and into sequence B until late in the process. If this impression is valid, one would have to entertain the suggestion that many of the sequential changes prior to bona fide neoplasia are by no means random but very carefully predetermined, probably by an appropriate genetic mechanism.

VI. THE CHALLENGE A. Cancer Development as an

Evolutionary Adaptive Process Cancer research during the past four to five decades has clearly indicated that environmental carcinogenic influences have probably been in existence for as long as living organisms have been on earth. Such influences include various types of radiation, such as ultraviolet light, and chemicals such as polycyclic aromatic hydrocarbons generated by burning organic compounds. Given this prolonged history of exposures to at least some carcinogens, intriguing questions arise: “How has nature handled carcinogens?” “How has nature allowed the development of so many species of living organisms in such a hostile environment?”

42

Emmanuel Farber

Table VI Some Evidence in Favor of Sequence A in Carcinogenesis as an Adaptive, Physiological Process 1. No immune response until late in carcinogenesis 2. Common molecular-biochemical and biological phenotype of new cell population with

many different carcinogens 3. New cell populations have different states of differentiation with options 4. Differentiation of focal new cell populations is a major option 5. Clonal expansion after initiation (promotion, selection) is protective and has survival value for the host (Harris et al., 1989) 6. Cell proliferation in expanded clones almost balanced by cell loss until late in carcinogenesis 7. The constitutive new resistance phenotype in rare altered hepatocytes after initiation is very similar to that induced transiently by lead nitrate, BHA, BHT, or interferon

For example, despite the numerous carcinogens to which humans are exposed, we are living longer today than ever before. Has the evolutionary process developed protective mechanisms (Farber, 1980; Farber and Rubin, 1991)?One such mechanism that is well documented throughout the whole system of living organisms, from prokaryotes to humans, is the development of repair processes for DNA altered by ultraviolet light and by chemicals. As indicated by the quotation from Nicholson (1950) at the beginning of this article, the cell and the living organism have developed many other adaptive processes that are physiological. Reversible induction of (a) metabolizing enzymes, (b) acute-phase reactive proteins, (c) heat shock proteins, and (d) a variety of DNA repair enzymes are some of the physiological adaptations. With genotoxic carcinogens, clonal adaptation in the liver generating resistant hepatocytes appears to be an additional response that has survival value for the host (Harris etal., 1989; Farber, 1991). This protective response involving 30 or more different enzymes and other components could well be responsible, at least in part, for the long period of carcinogenesis before cancer appears either late in the active reproductive period or in the postreproductive period of the host (Farber and Rubin, 1991). Such a delayed response to potent carcinogens would thus be protective for the reproduction of the species, with evolution having effectively performed one of its roles in biology. Some of the evidence in support of this working hypothesis is outlined briefly in Table VI.

From Phenotype to Genotype: A Desirable Conceptual Approach

B.

It is indeed regrettable that in almost no system is there a genotype that offersa testable working hypothesis as a possible basis for understanding the

Development of Epithelial Cancer

43

phenotype. Where we are beginning to obtain a rational testable hypothesis for the phenotype, such as in the early steps in liver carcinogenesis, we have very little knowledge about the appropriate genotype. The only possible exception might be the late steps in the further progression of malignant neoplasms to more malignant forms. The current research, while still early, nevertheless begins to offer possible rational hypotheses for understanding the phenotype. Clearly, this overall lack of integration of genotype with phenotype is a major challenge to cancer research. With respect to the late steps in cancer, the challenge is the development of more detailed and exact hypotheses for specific phenotypes in the vast array of cancer phenotypes. How detailed this can become remains for further research to delineate. Suggestions concerning the genotypic pattern of the very early steps in carcinogenesis would be of great importance. Given the many single components in the early phenotype as judged by the liver, it would be of particular interest to explore two reasonable hypotheses that come to mind. The first hypothesis would propose that multiplicity in the phenotypic components is due to a cascade effect, with one component being the first and the others simply triggered by the first. This hypothesis would only require that one gene, that of the first, be “altered” during the generation of this step (initiation). The second hypothesis would propose that there is a different kind of genetic program, a gene or genes that are of a controlling nature. The “alteration” of this gene would trigger a whole complex of genes simultaneously, generating the biochemical pattern that we see. Although the second hypothesis is more attractive from one point of view, it does not have, so far, an abundance of supporting data. In favor of this hypothesis is the observation that, with regard to phenotype 1 in the liver, a similar biochemical phenotype can be induced transiently by exposure to agents not carcinogenic for the liver, such as BHA, BHT, lead nitrate, or an interferon (see Section 1V.A.1). Developments of possible relevance to this problem appeared in Nature (Coupland, 1995; Weigel and Nilsson, 1995; Mandel and Yanofsky, 1995). In two different plants, seemingly single genes can trigger flower formation at times in the development of the plants at which flowering does not occur “naturally.” This flower formation involves many genes that somehow can be triggered appropriately by a single one. Such physiological responses are of course well known in many animal eukaryotes as well as in prokaryotes. Conceivably the induction by a single gene of a complex set of biochemical properties involving many proteins as individual gene products could be the basis for some of the phenotypes seen in the liver (and elsewhere) during the early steps in carcinogenesis. It is obvious that the exploration of the genetic basis for some phenotypes in cancer development could have very important practical considerations for cancer prevention. Also, such studies could contribute very significantly

44

Emmanuel Farber

to our understanding of biology generally, especially as to how cells respond to selective environmental perturbations, an aspect so fundamental to our ultimate understanding of biology.

ACKNOWLEDGMENTS The research of the author and his junior and senior colleagues was aided by research grants from the National Cancer Institute of Canada, the Medical Research Council of Canada (M-5994), and the Canadian Liver Foundation. I wish to express my sincere thanks to Ms. Carla Aldi for her most able assistance in the preparation of this manuscript.

REFERENCES Babinet, C., Farza, H., Morello, D., Hadchowel, M., and Pourcel, C. (1985). Science 230, 11 60-1 163. Balmain, A., and Brown, K. (1988).Adv. Cancer Res. 51, 147-182. Bannasch, P. (1968). “Recent Results in Cancer Research,” Vol. 19. Springer-Verlag, Berlin. Bannasch, P. (1986).Carcinogenesis 7, 689-695. Bannasch, P., Moore, M. A., Klimek, F., and Zerban, H. (1982).Toxicol. Pathol. 10, 19-34. Bannasch, P., Hacker, H. J., Klimek, F., and Meyer, 1. (1984).Adv. Enzyme Regul. 22,97-121. Becker, F. F., and Sell, S. (1974).Cancer Res. 34,2489-2494. Becker, F. F., Fox, R. A., Klein, K. M., and Wolman, S. R. (1971). J. Natl. Cancer Inst. 46, 1261-1269. Becker, F. F., Klein, K. M., Wolman, S. R., Asofsky, R., and Sell, S. (1973). Cancer Res. 33, 3330-3338. Becker, F. F., Horland, A. A., Shurgin, A., and Sell, S. (1974). Cancer Res. 35, 1510-1513. Bird, R. P. (1987). Cancer Lett. 37, 149-151. Blumberg, B. S., and London, W. T. (1982). Cancer 50, 2657-2665. Boule, C. (1994).MSc Thesis, University of Toronto. Boule, C., Nagai, M. K., and Farber, E. (1994). Proc. Am. Assoc. Cancer Res. 35, 830. Boule, C., Nagai, M. K., Montalvo, M., and Farber, E. (1995).Proc. Am. Assoc. Cancer Res. 36, 1060. Cayama, E., Tsuda, H., Sarma, D. S. R., and Farber, E. (1978).Nature (London) 275,60-61. Cha, Y. N., and Bueding, E. (1979).Biochem. Pharmacol. 28, 1917-1921. Cha, Y. N., and Heine, H. S. (1982). Cancer Res. 42, 2609-2615. Cheng, K. C., and Loeb, L. A. (1993).Adv. Cancer Res. 60, 121-156. Chisari, F. V., Pinkert, C. A., Milich, D. R., Filippi, P., McLachlan, A., Palmiter, R. D., and Brinster, R. L. (1985).Science 230, 1157-1160. Chisari, F. V., Filippi, P., Buras, J., McLachlan, A., Popper, H., Pinkert, C., Palmiter, R., and Brinster, R. L. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 6909-6913. Chisari, F. V., Klopchin, K., Moriyama, T., Pasquinalli, C., Dunsford, H., Sell, S., Pinkert, C . A., Brinster, R. L., and Palmiter, R. D. (1989).Cell 59, 1145-1156. Clark, W. H. Jr. (1994).Adv. Cancer Res. 65, 113-140. Columbano, A., Rajalakshmi, S., and Sarma, D. S. R. (1981). Cancer Res. 41, 2079-2083.

Development of Epithelial Cancer

45

Columbano, A., Ledda, G. M., Sirigu, P., Perra, T., and Pani, P. (1983). Am. J. Pathol. 110,8388.

Columbano, A., Ledda-Colurnbano, G. M., Coni, P., Vatgiu, M., Faa, G., and Pani, P. (1984). Toxicol. Pathol. 12, 89-95. Columbano, A., Ledda-Columbano, G. M., Coni, P., and Pani, P. (1987a). Carcinogenesis 8, 345-347.

Colurnbano, A., Ledda-Columbano, G. M., Lee, G., Rajalakshimi, S., and Sarma, D. s. R. (1987b). Cancer Res. 47,5557-5559. Conway, J. G., Popp, J. A., Vi, S., and Thurman, R. G. (1983). Cancer Res. 43, 3374-3378. Conway, J. G., Popp, J. A., and Thurrnan, R. G. (1984). Fed. Proc. 43,590. Coupland, G. (1995). Nature (London) 377,482-483. Deschner, E. E., and Lipkin, M. (1976). Clinics in Gastroenterology 5,543-561. Eckl, P. M., Meyer, S. F., Rive, W., Whitcomb, W. R., and Jirtle, R. L. (1988). Carcinogenesis 9, 479-483.

Emmelot, P., and Scherer, E. (1980). Biochim. Biophys. Acta 605, 247-304. Endicott, J. A., and Ling, V. (1989). Annu. Rev. Biochem. 58, 137-171. Enomoto, K., and Farber, E. (1982). Cancer Res. 42, 2330-2335. Enomoto, K., Ying, T. S., Griffin, M. J., and Farber, E. (1981). Cancer Res. 41, 3281-3287. Eriksson, L. C., and Anderson, G. N. (1992). Crit. Rev. Biochem. Mol. B i d . 27, 1-55. Eriksson, L. C., Ahluwalia, M., Spiewak, J., Lee, G., Sarma, D. S. R., Roorni, M. J., and Farber, E. (1983). Environ. Health Perspect. 49, 171-174. Eriksson, L. C., Torndal, U.-B., and Anderson, G. N. (1986). Carcinogenesis 7, 1467-1474. Eriksson, S., Carlson, J., and Velez, R. (1986). New Engl. J. Med. 314, 736-739. Fairchild, C. R., Ivy, S. P., Rushmore, T., Lee, G., Koo, P., Goldsmith, M. E., Meyers, C. E., Farber, E., and Cowan, K. H. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 7701-7705. Farber, E. (1973). Methods Cancer Res. 7, 345-375. Farber, E. (1976). In “Liver Cell Carcinoma” (H. M. Cameron, D. A. Linsell, and G. P. Warwick, Eds.), pp. 243-277. Elsevier/North-Holland Biomedical Press, Amsterdam. Farber, E. (1980). Biochim. Biophys. Acta 605, 149-166. Farber, E. (1982a). In “Cancer: A Comprehensive Treatise” (F. F. Becker, Ed.), 2nd ed., Vol. 1, pp. 485-506. Plenum Press, New York. Farber, E. (1982b). Am. J. Pathol. 108, 270-275. Farber, E. (1984). Can. J. Biochem. Cell Biol. 62, 486-494. Farber, E. (1987). Chem. Sci. 27A, 131-133. Farber, E. (1990). Biochem. Pharmucol. 39, 1837-1846. Farber, E. (1991). Dig. Dis. Scripta. 36, 973-978. Farber, E., and Cameron, R. G. (1980). Adv. Cancer Res. 35, 125-226. Farber, E., and Rubin, H. (1991). Cancer Res. 51, 2751-2761. Farber, E., and Sarma, D. S. R. (1987). Lab. Invest. 56, 4-22. Farber, E., Cameron, R. G., Laishes, B., Lin, J.-C., Medline, A., Ogawa, K., and Solt, D. B. (1978). In “Carcinogens: Identification and Mechanisms of Action” (A. C . Griffin and C. R. Shaw, Eds.), pp. 319-335. Raven Press, New York. Farber, E., Rotstein, J., Harris, L., Lee, G., and Chen, Z.-Y. (1988). In “Chemical Carcinogenesis Models and Mechanisms” (F. Feo, P. Pani, A. Columbano, and R. Garcea, Eds.), pp. 167-172. Plenum Pub]., New York. Farber, E., Chen, Z.-Y., Harris, L., Lee, G., Rinaudo, J. S., Roomi, M. W., Rotstein, J., and Semple, E. (1989). In “Liver Cell Carcinoma” (P. Bannasch, D. Keppler, and G. Weber, Eds.), pp. 273-291. Kluwer Academic Publ., Dordrecht. Fearon, E. R., and Jones, P. A. (1992). FASEB J. 6, 2783-2790. Fearon, E. R., and Vogelstein, B. (1990). Cell 61, 759-767. Finkelstein, S. D., Lee, G., Medline, A., Tatematsu, M., Makowka, L., and Farber, E. (1983). Am. J. Pathol. 110, 119-126.

46

Emmanuel Farber

Foulds, L. (1969). “Neoplastic Development,” Vol. 1, pp. 1-15. Academic Press, New York. Foulds, L. (1975). “Neoplastic Development,” Vol. 2. Academic Press, New York. Geller, S. A., Nichols, W. S., Kim, S. S., Tolmachoff, T., Lee, S., Dycaico, M. J., Felts, K., and Sorge, J. A. (1994). Hepatology 19, 389-397. Gerber, M. A., Hadziyannis, S., Vissoulis, C., Schaffner, F., Paronetta, F., and Popper, H. (1974). Am. J. Patbol. 75, 489-496. Gindi, T., Ghazarian, D. M. D., Deitch, D., and Farber, E. (1994). Cancer Lett. 83, 75-80. Greenhalgh, D. A., and Roop, D. R. (1994). Adu. Cancer Res. 64,247-296. Haddow, A. (1938). Acta Unio Intern. Contra Cancrum 3, 342-352. Harris, L., Morris, L. E., and Farber, E. (1989). Lab. Invest. 61, 467-470. Heppner, G. H. (1984). Cancer Res. 44, 2259-2265. Huang, S.-N., and Groh, V. (1973). Lab. Invest. 29, 353-366. Huang, S.-N., Millman, I., and O’Connell, A. (1972). Am. J. Pathol. 67, 453-470. Kinosita, R. (1937). Trans. Jpn. Patbol. SOC. 27, 665-729. Kitagawa, T. (1971). Gann 62, 207-216. Kitagawa, T. (1976). Cancer Res. 36, 2534-2539. Kitagawa, T., and Pitot, H. C. (1975). Cancer Res. 35, 1075-1084. Koo, P., Nagai, M. K., and Farber, E. (1994).I. Biol. Cbem. 269, 14601-14606. Koss, L. G . (1979). Human Pathol. 5, 533-548. Kovacs, G. (1993). Adu. Cancer Res. 62, 89-124. Laconi, E., Li, F., Semple, E., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. R. (1988). Carcinogenesis 9, 675-677. Lawley, P. D. (1994). Adu. Cancer Res. 65, 17-111. Ledda-Columbano, G. M., Columbano, A., Curto, M., Ennas, M. G., Coni, P.,Sarma, D. S. R., and Pani, P. (1989). Carcinogenesis 10, 847-850. Lee, G., Medline, A., Finkelstein, S. D., Tatematsu, M., Makowka, L., and Farber, E. (1983). Transplantation (Baltimore) 36, 218-221. Lipkin, M. (1988). Cancer Res. 48, 235-245. Lipkin, M., and Higgins, P. (1988). Adv. Cancer Res. 50, 1-24. Liu, B., Farrington, F. M., Petersen, G. M., Hamilton, S. R., Parsons, R., Papadopoulos, N., Fyiwata, T., Jen, J., Kinzler, K. W., Vogelstein, B., and Dunlop, M. G. (1995a). Nature Med. 1,348-352. Liu, B., Necolaides, M. C., Markowitz, S., Wilson, J.K.-V., Parsons, R. E., Jen, J., Papadopoulos, N., Peltomaki, P., de la Chapelee, A., Hamilton, s. R., Kinzler, K. w., and Vogelstein, B. (1995b). Nature Genet. 9, 48-55. Loeb, L. A. (1994). Cancer Res. 54, 5059-5063. London, W. T., and Blumberg, B. S. (1982).Hepatology 2, 10s-14s. MacPhee, D. G. (1995). Cancer Res. 55,5489-5492. Magee, P. N. (1995). In “Molecular and Cellular Mechanisms of Toxicity” (F. DeMatteis and L. L. Smith, Eds.), pp. 125-143. CRC Press, Boca Raton, FL. Mandel, M. A., and Yanofsky, M. F. (1995). Nature (London) 377, 522-524. Mantle, T. J., Pickett, C. B., and Hayes, J. D. (1987).“Glutathione S-Transferases and Carcinogenesis.” Taylor and Francis, London. Mizumoto, K., Tsutsumi, M., Denda, A., and Konishi, Y. (1988). J. Natl. Cancer Inst. 80, 1564-1 567. Mizumoto, K., Kitazawa, S., Ito, S., Takashima, Y., Tsutsumi, M., Denda, A., and Konishi, Y. (1989). Carcinogenesis 10, 1457-1459. Mizumoto, K., Tsutsumi, M., Kirazawa, S., Denda, A., and Konishi, Y. (1990).Cancer Lett. 49, 211-215. Moore, M. A., Hacker, H. J., and Bannasch, P. (1983). Carcinogenesis 4,595-603. Nagai, M. K. (1993). PhD Thesis, University of Toronto. Nagai, M. K., Armstrong, D., and Farber, E. (1993). Proc. Am. Assoc. Cancer Res. 34, 981.

Development of Epithelial Cancer

47

Nakamura, Y. (1993). Adu. Cancer Res. 62, 65-87. Nicholson, G. W. de P. (1950). “Studies on Tumor Formation,” pp. 249-261. Butterworth & Co., London. Nowell, P. C . (1976). Science 194, 23-28. Nowell, P. C. (1993). Adu. Cancer Res. 62, 1-17. Ogawa, K., Medline, A., and Farber, E. (1979a). Lab. Invest. 41, 22-35. Ogawa, K., Medline, A., and Farber, E. (1979b). Br. 1. Cancer 40, 782-790. Ogawa, K., Solt, D. B., and Farber, E. (1980). Cancer Res. 40, 725-733. Otori, K., Sugiyama, K., Hasebe, T., Fukushima, S., and Esumi, H. (1995). Cancer Res. 55, 4743-4746. Ozbun, M. A., and Butel, J. S. (1995). Adu. Cancer Res. 66, 71-141. Pascale, R. M., Marras, V., Simile, M. M., Daino, L., Pinna, G., Bennati, S., Carta, M., Seddaiu, M. A., Massarelli, G., and Feo, F. (1992). Cancer Res. 52, 4979-4986. Peraino, C., Fry, R. J. M., and Staffeldt, E. (1971). Cancer Res. 31, 1506-1512. Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisieleski, W. E. (1973). Cancer Res. 33, 27012705. Peraino, C., Fry, R. J. M., Staffeldt, E., and Christopher, J. P. (1975). Cancer Res. 35, 28842890. Phillips, M. J., and Steiner, J. W. (1966). Lab. Invest. 15, 801-817. Pitot, H. C., and Sirica, A. E. (1980). Biochim. Biophys. Acta 605, 191-215. Pitot, H. C., Barsness, I., Goldsworthy, T., and Kitagawa, T. (1978). Nature (London) 721, 456-45 8. Prehn, R. T. (1994). Cancer Res. 54, 5296-5300. Pretlow, T. P., Barrow, B. J., Ashton, W. S., O’Riordan, M. A., Pretlow, T. P., Jureisek, J. A., and Sakellato, T. A. (1991). Cancer Res. 51, 564-567. Reddy, E. P., Shalka, A. M., and Curran, T. (1988). “The Oncogene Handbook.” Elsevier/ North-Holland Biomedical Press, Amsterdam. Reuber, M. D. (1965). J. Natl. Cancer Inst. 34, 697-723. Roncucci, L., Stamp, D., Medline, A., Cullen, J., and Bruce, W. (1991). Hum. Puthol. 22,287294. Roomi, M. W., Ho, R. K., Sarma, D. S. R., and Farber, E. (1985). Cancer Res. 45,564-571. Roomi, M. W., Columbano, A., Ledda-Columbano, G. M., and Sarma, D. S. R. (1986). Carcinogenesis 7, 1643-1646. Rotstein, J., Macdonald, P. D., Rabes, H. M., and Farber, E. (1984). Cancer Res. 44, 291329 17. Rotstein, J,, Sarma, D. S. R., and Farber, E. (1986). Cancer Res. 46, 2377-2385. Rubin, E., and Popper, H. (1967). Medicine 46, 163-183. Rubin, H. (1993). Differentiation 53, 123-137. Rubin, H. (1995). Frontier Perspett. 4, 9-16. Rubin, H., Yao, A., and Chow, M. (1995a). Proc. Natl. Acad. Sci. U.S.A. 92, 7734-7738. Rubin, H., Yao, A., and Chow, M. (1995b). Proc. Natl. Acad. Sci. U.S.A. 92, 4843-4847. Sarma, D. S. R., Rao, P. M., and Rajalakshimi, S. (1986). Cancer Suru. 5, 781-798. Sasaki, T., and Yoshida, T. (1935). Virchows Arch. Patol. Anat. Physiol. Klin. Med. 295, 175200. Scarpelli, D. G. (1995). Jpn. J. Cancer 86, 706. Scherer, E., and Emmelot, P. (1975). Eur. 1. Cancer 11, 145-154. Scherer, E., Hoffmann, M., Emmelot, P.,and Friedrich-Freksa, H. (1972).1.Natl. Cancer Inst. 49,93-106. Schulte-Hermann, R. (1974). Crit. Rev. Toxicol. 3, 97-158. Schulte-Hermann, R. (1979). In “Toxic Injury of the Liver” (E. Farber and M. M. Fisher, Eds.), pp. 385-445. Marcel Dekker, New York. Schulte-Hermann, R., Timmermann-Trosiener, I., and Schuppler,J. (1984). In “Models, Mech-

48

Emmanuel Farber

anisms and Etiology of Tumour Promotion” (M. Borzsonyi, N. E. Day, K. Lapis, and H. Yamasaki, Eds.), IARC Scientific Publ. No. 56, pp. 67-75. IARC, Lyon. Sikic, B. J. (1993).J. Clin. Oncol. 11, 1629-1635. Solt, D. B., and Farber, E. (1976). Nature (London) 263, 702-703. Solt, D. B., Hay, J. B., and Farber, E. (1977a). Cancer Res. 37, 1686-1691. Solt, D. B., Medline, A., and Farber, E. (1977b). Am. 1. Pathol. 88,595-610. Stein, O., Fainaru, M., and Stein, Y. (1972). Lab. Invest. 26, 262-269. Stockert, R. J., and Becker, F. F. (1980).Cancer Res. 40,3632-3634. Stout, D. L., and Becker, F. F. (1987). Cancer Res. 47, 963-966. Tatematsu, M., Murasaki, G., Nakanishi, K., Miyoto, Y.,Shinohara, Y., and Ito, N. (1979). Cann 70,125-130. Tatematsu, M., Nagamine, Y., and Farber, E. (1983). Cancer Res. 43,5049-5058. Tatematsu, M., Lee, G., Hayes, M. A., and Farber, E. (1987). Cancer Res. 47,4699-4705. Teebor, G . W., and Becker, F. F. (1973).Cancer Res. 31, 1-3. Tsuda, H., Lee, G., and Farber, E. (1980). Cancer Res. 40, 1157-1164. Van De Vijver, M. J. (1993). Adu. Cancer Res. 61,25-56. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preisinger, A. C., Leppert, M., Nakumura, Y.,White, R., Smits, A.M.M., and Bos, J. L. (1988). New Engl. 1. Med. 319, 525-532. Weigel, D., and Nilsson, 0. (1995). Nature (London) 379, 495-500. Weisburger, J. A., and Williams, G. M. (1981). Science 214,401-407. Wolfe, C. R., Adams, D. J., Balkwill, F., Griffin, B., and Hayes, J. D. (1986).Proc. Am. Assoc. Cancer Res. 27, 10. Ying, T. S., Enomoto, K., Sarma, D. S. R., and Farber, E. (1982). Cancer Res. 42, 876-880. Yuspa, S.-H. (1994).Cancer Res. 54, 1178-1189. Yuspa, S.-H., and Pokier, M. C. (1988).Adv. Cancer Res. 50.25-70.

Genetics of the Nevoid Basal Cell Carcinoma Syndrome Abirami Chidambaram’ and Michael DeanZ ‘Intramural Research Support Program, SAIC Frederick, and 2Human Genetics Section, Laboratory of Viral Carcinogenesis, National Cancer InstituteFrederick Cancer Research and Development Center, Frederick, Maryland 21 702

I. Introduction 11. Clinicopathological Features of NBCCS 111. Genetics of NBCCS

A. Linkage and Mapping B. Molecular Aspects of NBCCS IV. Strategies for Isolation of Candidate Genes A. Cosmid Selection and Analysis B. Comparative Mapping V. Discussion References

I. INTRODUCTION The nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), odontogenic kerarocysts, pits of the palms and soles, and a spectrum of skeletal and developmental abnormalities. The condition was first described by Jarisch (1894) and White (1894). It is a complex hamartoneoplastic-malformation syndrome with over 100 signs and symptoms primarily involving the skin, central nervous system, and skeletal system. The term “basal cell nevus” was coined by Nomland (1932) because the tumors resembled pigmented moles (nevi), although, microscopically, the cells were “like dark staining cells.” Therefore, the term “nevus of basal cells” was used to describe these growths (Howell and Anderson, 1982). The report that originally described this syndrome (Howell and Caro, 1959) included only three major phenotypic features: basal cell nevi, jaw cysts, and rib defects. Since then, several reports (Ward, 1960; Gorlin and Goltz, 1960; Anderson et al., 1964; Clendenning et al., 1964; Maddox et al., 1964; Pollard and New, 1964; Anderson and Advanccs in CANCER RESEARCH. Vol. 70 Copyrighr Q 1996 by Academic Press, Inc. All rights of rcproduction in any form reserved

50

Abirami Chidambaram and Michael Dean

Cook, 1966) have described NBCCS. It is now known that this syndrome also comprises cancers such as medulloblastoma and ovarian fibromas. In addition to other benign and malignant tumors, malformations are a significant component of this syndrome. These include a wide and varying spectrum of defects such as calcification of the falx cerebri, bifid ribs and other rib anomalies, kyphoscoliosis, imperfect segmentation of cervical vertebrae, and cleft lip and/or palate. While BCC is the most common nonmelanoma skin cancer in Caucasian populations in the United States as well as in various parts of the world, the distribution of BCCs in NBCCS patients differs substantially from that observed in the general population in age of onset as well as distribution of these cancers. Gorlin syndrome is estimated to have a prevalence of 1 in 56,000, and about 0.5% of all BCCs are attributable to the syndrome (Evans et al., 1991; Springate, 1986). Statistics (Cancer Facts and Figures, 1993) indicate that BCCs account for about 80% of all nonmelanoma skin cancers and, of these, 1 in 200 patients with one or more BCCs over a lifetime has the syndrome and 1 in 5 patients who developed a BCC before 19 years of age has NBCCS. About 40% of NBCCS cases represent new cases (Gorlin, 1982, personal communication cited in Online Mendelian Inheritance in Man (OMIM) entry #109400). A study of 118 Australian NBCCS cases (Shanley et al., 1994)reports a new mutation rate of 14-81%.

11. CLINICOPATHOLOGICAL FEATURES OF NBCCS The frequencies of individual features associated with NBCCS are listed in Table I (Gorlin, 1987). The list underscores the possible extent of involvement of various organs and systems that are affected in patients with this syndrome. One of the most striking and consistent features of this syndrome is the number and distribution of BCCs in NBCCS patients in contrast to that observed in the general population. In the latter group, 87% of BCCs occur on the face, head, neck, and arms (areas exposed to sunlight), while 912% of BCCs occur on the trunk. In NBCCS patients, however, up to 38% of BCCs occur on the trunk, with about 65% occurring on the face, head, neck, and arms. Large numbers of BCCs have been observed following radiation treatment of NBCCS patients for medulloblastoma (Strong, 1977). While the age at onset for BCCs in the general population is about 50 years on average, NBCCS patients are known to develop these tumors as early as their first few years. In addition, the number of BCCs over a patient’s lifetime can range from zero to thousands. Clinical differences in the presentation of NBCCS between African American and Caucasian NBCCS patients have

Nevoid Basal Cell Carcinoma Syndrome

51

Table I

Some of the Most Commonly Observed Features Associated (50% or greater frequency) with NBCCS (Gorlin Syndrome) Multiple basal cell carcinoma Jaw cysts (odontogenic keratocysts) Palmar and plantar pits Calcified falx cerebri Rib anomalies (e.g., bifid or fused) Spina bifida Mild ocular hypertelorism Epidermal cysts Other features (15-45% frequency) include: Vertebral anomalies (e.g., kyphoscoliosis) Short metacarpals Calcified ovarian fibromas Anomalies of the sternum Hamartomas (pseudolytic bone lesions) Strabismus Non-random but rare features include: Medulloblastoma Meningioma Cardiac fibroma Cleft lip/cleft palate Sprengel deformity of scapula Eye disorders such as congenital cataract and glaucoma Minor renal anomalies Mental retardation, fetal rhabdomyoma and ovarian fibrosarcomas have also been observed in NBCCS patients Adapted from Gorlin (1987).

been reported (Goldstein et al., 1994a,b). Blacks with NBCCS have fewer skin tumors than Caucasian NBCCS patients even though it has been observed that blacks may be prone to increased numbers of BCCs in the field of irradiation if they have received radiation treatment for medulloblastoma (Kimonis et al., 1995). These features are important to consider with regard to effective diagnosis and syndrome delineation in order to identify patients as well as family members at risk for NBCCS. These observations also serve as criteria in selecting families in which the NBCCS gene may be segregating, because phenotypic features are crucial in ascertaining probands and their families for segregation and linkage analysis. The phenotypic manifestation and

52

Abirami Chidambaram and Michael Dean

spectrum of associated defects also provide valuable clues as to possible candidate loci that may be involved in the syndrome.

Ill. GENETICS OF NBCCS A. Linkage

and Mapping

An autosomal dominant mode of inheritance was suggested for NBCCS (Gorlin and Goltz, 1960). The gene was localized to 9q22.3-q31 and shown to be due to a single dominant locus; the most likely location was reported to be between the DNA markers D9S12 and D9S53 (Gailani et al., 1992; Reis et al., 1992; Farndon et al., 1992). Analysis of additional kindreds has further refined the localization of the gene to a region between the markers D9S196 and D9S180 (Chenevix-Trench et al., 1993; Compton et al., 1993; Goldstein et al., 1994a,b; Wicking et al., 1994). In addition, analyses of BCCs, squamous cell carcinomas, and medulloblastomas have confirmed the presence of deletions in these tumors in the candidate region of the NBCCS gene (Albrecht et al., 1994; Quinn et al., 1994; Shanley et al., 1995). The genes for Fanconi anemia complementation group C (FACC)and xeroderma pigmentosum complementation group A (XPAC), as well as selfhealing squamous epithelioma (ESS; Goudie et al., 1993), also map to the same region of 9q (Figs. 1 and 2).

B. Molecular Aspects of NBCCS NBCCS has features that are compatible with the two-hit model of the Knudson hypothesis (Knudson, 1971) pertaining to the mode of action of tumor suppressor genes such as retinoblastoma and Wilms tumor. Susceptibility to disease is inherited in a dominant fashion while the gene acts in a recessive fashion at the cellular level, requiring a second hit or mutation for neoplastic transformation of the cells. Gailani et al. (1992) found loss of heterozygosity of polymorphic molecular marker loci in the 9q22.1-3 1 region in 11 of 16 sporadic BCCs, 2 hereditary BCCs, and one ovarian fibroma. This lends support to the hypothesis that the gene acts as a tumor suppressor, and hemizygous inherited (germline) mutations cause developmental anomalies that are observed associated with this syndrome. The same study also speculated on the possibility of Gorlin syndrome and ESS being allelic disorders due to the fact that pitting of the soles and palms is observed in NBCCS patients. ESS patients also exhibit such pitting, though

53

Nevoid Basal Cell Carcinoma Syndrome

0 c

m rn

cu

n

I

24

5197 5196 SZ80 S180 XPAC

S173 TMOD COL15A1 S6 FACC

60C20

D9S29 32

II -

Fig. Map of NBCCS region on human chromosome 9. Schematic remesentation of the - I rodent-human chromosome 9 somatic cell hybrid panel showing the cytogenetic location of the deletions in these hybrids that were used to test candidate genes or marker loci to determine if they were located within the critical NBCCS deletion region contained in hybrid GATS7, which is hemizygous for the mutated copy of 9q derived from a NBCCS patient.

54

Abirami Chidarnbararn and Michael Dean

S197

cR

363

cM

102

S196

S280

XPAC YI WI FACC PTC 6937 6378 S287 NCBP TMOD I

369 369 103

104

7447 S180

371 371 373 106

108

Fig. 2 Physical and genetic map of the 9q22.3 region. The genetic markers flanking the NBCCS gene (D9S196 and D9S180) are shown along with other markers, genes, and sequence tagged sites that have been mapped into this interval. FACC, Fanconi anemia complementation group C; PTC, Patched; NCBP, nuclear cap binding protein; TMOD, tropomodulin; XPAC, xeroderma pigmentosum complementation group A. WI numbers refer to Whitehead Institute STS markers (Dib et af., 1995). Below the map are shown physical distance (cR) from a radiation hybrid map, and genetic distance (cM) from linkage mapping. Both cR and cM represent distances from the telomere of 9p.

not restricted to their palms and soles but occurring on any part of the body’s skin surface where an epithelioma occurs and then falls off, leaving a pitlike scar (Goudie et af., 1993). The possibility that either FACC or XPAC could be the NBCCS gene has also been tested, but the results do not support this hypothesis (Bare et af., 1995).

IV. STRATEGIES FOR ISOLATION OF CANDIDATE GENES A. Cosmid Selection a n d Analysis Efforts to identify the NBCCS gene have made used of standard positional cloning approaches. Yeast artificial chromosome (YAC) contigs spanning the region have been characterized (Morris and Reis, 1994; Chidambaram et al., submitted), and used to isolate chromosome %specific cosmids from the region. Sequence analysis of Alu polymerase chain reaction (PCR) products from the ends of the YACs or from cosmids have localized a number of transcribed sequences into the region (Fig. 1).One of these genes (Chidambaram et al., submitted) is a new Kruppel-type zinc finger gene (ZNF269). This family of genes is involved in segmentation and transcriptional regulation, and therefore ZNFZ69 is a potential candidate gene for NBCCS. A second gene in this region is a homolog of the Drosophila Patched (PTC) gene, which is involved in the developmental regulation of segments in DYOsophifa (Hahn et af., 1996).This pathway is conserved in the development of

Nevoid Basal Cell Carcinoma Syndrome

55

mammalian bone and brain tissue, and therefore it is also tempting to consider PTC as an NBCCS candidate gene.

B. Comparative Mapping A considerable amount of data support the fact that mammalian genomes are for the most part composed of chromosomal segments that have been conserved over 100 million years of evolution. While there are approximately 100 such segments, the locations of many genes in the human genome can be predicted by their locations in the mouse genome (Watson and Seldin, 1994). Fifty human chromosome 9 loci have been mapped in the laboratory mouse (Pilz et al., 1995) and this is a valuable resource for focusing attention on evolutionarily conserved syntenic groups in mice and humans that map to the human chromosomal region of interest. A highresolution genetic map of the mouse provides information regarding gene order on the human chromosomal counterpart in addition to suggesting certain markers and loci as candidate genes for disease loci on chromosome 9. Genes in the NBCCS critical region map to both mouse chromosomes 4 and 13 (Fig. 3). The gene for FACC maps to mouse chromosome 13 while the XPAC gene maps to mouse chromosome 4. If D9S287 is taken as the distal boundary for the NBCCS locus (Reis et al., 1992), then the murine homolog is most likely on chromosome 13. Marker information shows that the NBCCS locus on human chromosome 9 is flanked by the FACC (proximal boundary) and XPAC (distal boundary) and therefore must lie somewhere in the breakpoint-fusion region between the two mouse chromosomal segments on 13 and 4 containing these loci (Fig. 3). The maps show the position of the loci with respect to the genetic distances between them in centimorgans and are useful in assessing the strength of linkage between these loci and the likelihood with which that syntenic group will be conserved through evolution from mouse to the human genome. These loci can then be used as markers for construction of physical maps utilizing YAC and cosmid contigs to narrow down the region of the human chromosome 9 containing the NBCCS gene. There are several good candidate loci for NBCCS on both these mouse chromosomes, including ALDOB (aldolase dehydrogenase B), TMOD (tropomodulin), CTSL (cathepsin L), ABCl (ATP-binding cassette transporter l), FPGS (folylpolyglutamate synthetase), and the Drosphilu Notch gene homolog with homeobox gene-like functions. All these loci were tested (Chidambaram et al., submitted) by PCR on a panel of human-rodent somatic cell hybrids enriched for human chromosome 9 with and without deletions in the region containing the NBCCS gene (Fig. 3). Apart from

56

Abirami Chidambaram and Michael Dean

Hu9q

L Mu4

Fig. 3 Syntenic relationships between the region of human chromosome 9 (HSAS) containing the NBCCS locus and mouse chromosomes 4 (MMU4) and 13 (MMU13). TMOD, XPAC, FACC, and the PTC gene map to HSASq22-the regon to which the NBCCS gene has been mapped. Mouse homologs Tmod and Xpa map to MMU4 while Facc and Ptc map to MMU 13, indicating a "split" in the ancestral chromosome corresponding to HSA9q22 (Pilz et al., 1995). In humans, the NCBP gene (which has no known mouse homolog to date) also maps to 9q22, while mouse genes Tpbp and CtlaZa (which have no known human homologs to date) map to the regon of MMU13 which is tightly linked to Facc and Ptc. Such conserved gene orders in syntenic segments provide valuable clues for predicting gene orders in the human chromosomal counterpart, especially when disease or phenotypic markers (e.g., flex tail locus ( f )on MMU13) are closely linked to marker loci of interest. These also provide tentative mouse models for human diseases, which are invaluable tools in understanding the etiology of the disease. This information is further enhanced by the availability of ESTISTS information from data bases (e.g., Whitehead Institute) that help fill in the gaps with human specific DNA sequence information for further analysis.

these genes, there are several intriguing murine phenotypic variants that have been mapped to this region of mouse chromosome 13 (Fig. 2), which include the flex tail (f),which is very closely linked to the Facc locus and involves skeletal and tail anomalies; the Purkinje cell degeneration (pcd) locus, which includes degeneration of parts of the central nervous system;

Nevoid Basal Cell Carcinoma Syndrome

57

and juvenile depilation (jd) and muted (mu), the last two loci involving anomalies of hair/fur growth and appearance.

V. DISCUSSION Hereditary BCCs occur as early as the first decade of life and are associated with a constellation of developmental anomalies and clinical phenotypes that contribute to the NBCCS. Sporadic BCCs, however, are not generally associated with any other pathological condition, occur relatively later in life (around the fifth decade), are significantly fewer in number, and are thought to result from ultraviolet (W)exposure and damage. The gene for both the sporadic and the hereditary forms of BCCs maps to the same region of human chromosome 9q, even though these two forms vary in their age at onset, distribution, number, and perhaps etiology. Once the gene for NBCCS is cloned, molecular analysis will determine the specific mutations that correlate with and contribute to the variability of expression of the clinical phenotypic spectrum observed between and within NBCCS kindreds. Mutation analysis will also determine if sporadic BCCs differ from their hereditary counterparts associated with NBCCS or other inherited disorders, such as the Proteus syndrome or the epidermal nevi syndrome of Jadassohn. (These latter disorders are discussed later in this section.) Mutation analysis will also shed light on the differences between black, Hispanic, and Caucasian NBCCS patients. The clinical features of NBCCS and the wide phenotypic spectrum of these features indicate that the gene for this syndrome must be involved in the normal course of embryogenesis and in addition be involved in cellular regulatory processes such as cell division and differentiation. Malformations involving the skeletal and nervous system and facial dysmorphology indicate that defects in this gene affect early embryonic processes. The gene also affects a variety of tissue types, such as the skin, ovary, and brain-an observation that may be explained by the fact that early on in development, coinciding with the formation of the neural crest, there exists the possibility of a mutation-bearing progenitor cell whose descendants in turn migrate to various parts of the developing embryo, which gives rise to the various tissues that exhibit the effect of this mutation. It has been stated (Bale et a/., 1994) that the finding of developmental defects in a syndrome due to inactivation of a tumor suppressor gene constitutes a paradox because inactivation of one copy of a tumor suppressor gene has little or no effect on cell function and that, in contrast to other disorders caused by tumor suppressors, congenital anomalies are a prominent feature of NBCCS. This aspect of the gene’s function will be clear once the gene is cloned and

58

Abirami Chidambaram and Michael Dean

characterized. The location of the NBCCS gene and the ESS locus flanked by the FACC and XPAC genes suggests a possible mode of action for this gene with regard to developmental pathways or DNA repair defects. NBCCS shares phenotypic features with all three of the above loci: skeletal defects associated with FACC, sensitivity to radiation and pitting of the skin associated with ESS, and UV sensitivity and predisposition to basal and squamous cell carcinomas associated with XPAC. Whether these genes are involved in contributing to the overall NBCCS phenotypic spectrum remains to be determined. The number and distribution of basal skin carcinomas in black NBCCS patients adds another intriguing angle to the study. While the severity and manifestation of most of the congenital anomalies are comparable in Caucasian and black NBCCS patients, it has been documented that, while the former can develop as many as 500-1000 BCCs during the course of their lifetime, the latter exhibit few if any BCCs. However, if black NBCCS patients are treated for medulloblastoma, they can develop hundreds of BCCs in the field of irradiation (Korzcak et af., 1995). This suggests that perhaps multiple mutations or “hits” are required for the development of BCCs, while one inherited mutation is sufficient for causing the systemic-congenital abnormalities. This may also explain the reason why Caucasian NBCCS patients are prone to develop more BCCs; they lack the protection melanin offers and therefore accumulate a larger number of UV radiation-induced mutations (Kraemer, 1995). This in turn predisposes them to skin tumors owing to impaired DNA repair functions, as is the case with xeroderma pigmentosum patients. Correlation of the mutational spectrum of the gene with phenotypic data between and within NBCCS pedigrees will be required to fully understand this aspect of the disorder. There are at least two other well-documented disorders, namely the Proteus syndrome (OMIM #176920) and nevus sebaceous of Jadassohn (OMIM #163200), whose clinical profiles include basal cell nevi or carcinomas as part of the disease phenotype. Large epidermal nevi and linear macular lesions have been observed in patients with the Proteus syndrome, and the affected skin often exhibits basal cell involvement (Viljoen et al., 1988). This syndrome, apart from the presence of the nevi, also shares other features with NBCCS, such as developmental abnormalities involving the skeletal system (e.g., abnormal craniofacial development), redundant skin, and vertebral anomalies (Rizzo et al., 1993; Cohen, 1993). However, whether this represents a somatic cell disorder or an inherited condition is not clear. Nevus sebaceous of Jadassohn (epidermal nevus syndrome) also presents as an autosomal dominant condition with similarities to NBCCS. Multiple developmental abnormalities are observed in patients with this disease, though the primary clinical feature is the presence of hypoplastic sebaceous glands that later turn hyperplastic, from which benign or malignant tumors

Nevoid Basal Cell Carcinoma Syndrome

59

may arise. Familial occurrence of these nevi (usually on the scalp), which turn into BCCs, has been documented (Sahl, 1990). The Proteus syndrome and the epidermal nevus syndrome remain unassigned to a chromosome, and whether there is a common developmental pathway that produces the phenotypes in these three syndromes remains to be seen. However, these observations may provide valuable clues as to the nature and significance of genes controlling development, which, when disturbed, produce such wide-reaching and devastating pathological effects. Note added in proof: Since the time the original manuscript was submitted, mutation analyses of the PTC (human homolog of the Drosophila segment polarity gene patched) in NBCCS patients and related tumors have demonstrated and confirmed the contribution of this gene towards the NBCCS phenotype (Hahn et al., 1996a,b). The loss of one allele apparently leads to developmental anomalies observed in patients with this syndrome while complete loss of the PTC gene function gives rise to tumors associated with NBCCS. As indicated in Fig. 2, the murine homolog (Ptc) of patched maps to mouse chromosome 13 (MMU 13). In addition to the phenotypes closely linked to this locus, a new disorder, mes (mesenchymal dysplasia), has recently been described (Sweet et al., 1996), which also maps to the same region of MMU 13. The spectrum of developmental anomalies associated with the mes mutation make it an interesting candidate for a mouse model for NBCCS.

ACKNOWLEDGMENTS We thank Alisa Goldstein for comments on the manuscript. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

REFERENCES Albrecht, S., von Deimling, A., Pietsch, T., Giangaspero, F., Brander, S., Kleihues, P., and Wiestler, 0. D. (1994).Neuropathol. Appl. Neurobiol. 20, 74-81. Anderson, D. E., and Cook, W. A. (1966)./. Oral Surg. 24, 15-26. Anderson, D. E., McClendon, J. L., and Howell, J. B. (1964).In "Tumors of the Skin," pp. 91127. Year Book Medical Publishers, Chicago. Bale, A. E., Gailani, M. R., and Leffel, D. J. (1994).I. Invest. Dermatol. 103, 126s-130s. Bare, J. W., Xie, J., Quinn, A. G., and Epstein, E. H. Jr. (1995). Am. 1. Hum. Genet. 57, A60. Cancer Facts and Figures. (1993). American Cancer Society, Inc., Atlanta, GA. Chevenix-Trench, G., Wicking, C., Berkman, J., Sharpe, H., Hockey, A., Haan, E., Oley, C.,

60

Abirami Chidambaram and Michael Dean

Ravine, D., Turner, A., Goldgar, D., Searle, D., and Wainwright, B. (1993).Am. /. Hum. Genet. 53,760-767. Chidambaram, A., Gailani, M. R., Gerrard, B., Stewart, C., Goldstein, A., Chumakov, I., Bale, A. E., and Dean, M. (submitted, 1996). Clendenning, W. E., Block, J. B., and Radde, I. C. (1964).Arch. Dermatol. 90,38-53. Cohen, M. M. Jr. (1993).Am. J. Med. Genet. 47,645-652. Compton, J. G., Goldstein, A. M., Turner, M., Bale, A. E., Kearns, K. S., McBride, 0. W., and Bale, S. J. (1994).J. Invest. Dermatol. 103, 178-181. Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J., and Weissenbach, J. (1996). Nature 380, 152-154. Evans, D. G. R., Farndon, P. A., Burnell, L. D., Gattamaneni, H. R., and Birch, J. M. (1991).BY. /. Cancer 64, 959-961. Farndon, P. A., Del Mastro, R. G., Evans, D. G. R., and Kilpatrick, M. W. (1992).Lancet 339, 581-582. Gailani, M. R., Bale, S. J., Leffell, D. J., DiGiovanna, J. J., Peck, G. L., Poliack, S., Drum, M. A., Pastakia, B., McBride, 0. W., Kase, R., Greene, M., Mulvihill, J. J., and Bale, A. E. (1992). Cell 69, 111-117. Goldstein, A. M., Bale, S. J., Peck, G. L., and DiGiovanna, J. J. (1994a).Am. /. Med. Genet. 50, 272-281. Goldstein, A. M., Stewart, C., Bale, A. E., Bale, S. J., and Dean, M. (1994b).Am. /. Hum. Genet. 54, 765-773. Gorlin, R. J. (1987).Medicine 66, 96. Gorlin, R. J. (1982). Personal Communication in OMlM (TM) Online Inheritance in Man. MIM Number 109400. Date last edited: 6/18/96.World Wide Web UR1: http://www3.ncbi.nlm.nih.gov/omim. Gorlin, R. J., and Goltz, R. W. (1960).New Engl. /. Med. 262, 908-912. Goudie, D. R., Yuille, M. A. R., Leversha, M. A., Furlong, R. A., Carter, N. P., Lush, M. J., Affara, N. A., and Ferguson-Smith, M. A. (1993).Nature Genet. 3, 165-169. Hahn, H., Christiansen, J.,Wicking, C., Zaphiropoulos, P., Chidambaram, A., Gerrard, B., Vorechovsky, I., Bale, A. E., Toftgard, R., Dean, M., and Wainwright, B. (1996a).J. Biol. Chem. 271,12125-12128. Hahn, H., Wicking, C., Zaphiropoulos, P., Gailani, M. R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A. B., Gillies, S., Negus, K., Smyth, I., Pressman, C., Leffell, D. J., Gerrard, B., Goldstein, A. M., Wainwright, B., Toftgard, R., Chenevix-Trench, G., Dean, M., and Bale, A. E. (1996b).Cell 85, 1-20. Howell, J. B., and Caro, M. R. (1959).Arch. Dermatol. 79, 67-80. Howell, J. B., and Anderson, D. E. (1982).Commentary: The nevoid basal cell carcinoma syndrome. Arch. Dermatol. 118,824-826. Jarisch, W. (1894). Arch. Dermatol. Syph. (Berlin) 28, 162-222. Kimonis, V. E., Goldstein, A. M., Pastakia, B., Yang, M. L., DiGiovanna, J. J., Bale, A. E., and Bale, S. J. (1995).Am. J. Hum. Genet. 57,A54. Knudson, A. G. (1971).Proc. Natl. Acad. Sci. U.S.A. 68, 820-823. Korzcak, J. F., DiGiovanna, J. J., Brahim, J., Kase, R. G., and Goldstein, A. M. (1995).Am. J. Hum. Genet. 57, A69. Kraemer, K. H. (1995)./. Invest. Dermatol. 7, 887-888. Maddox, W. D., Winkelmann, R. K., Harrison, E. G., Devine, K. D., and Giblisco, J. A. (1964). /. Am. Med. Assoc. 188, 106-111. Morris, D. J., and Reis, A. (1994).Genomics 25, 59-65. Nomland, R. (1932).Arch. Dermatol. Syph. 25, 1002-1008. Pilz, A., Woodward, K., Povey, S., and Abbott, C. (1995).Genomics 25, 139-149.

Nevoid Basal Cell Carcinoma Syndrome

61

Pollard, J. J., and New, P. F. J. (1964).Radiology 82, 840-849. Quinn, A. G., Campbell, C., Healy, E., and Rees, J. L. (1994)./. Invest. Dermatol. 102,300303. Reis, A., Kuster, W., Linss, G., Gebel, E., Hamm, H., Fuhrmann, W., Wolff, G., Groth, W., Custafson, G., Kuklik, M., Burger, J., Wegner, R. D., and Neitzel, H. (1992).Lancet 339, 617. Rizzo, R., Pavone, L., Micah, G., Nigro, F., and Cohen, M. M. Jr. (1993). Am. 1.Med. Genet. 47, 653-655. Sahl, W. J. Jr. (1990)./. Am. Acad. Dermatol. 22, 853-854. Shanley, S., Ratcliffe, A., Hockey, A., Haan, E., Oley, C., Ravine, D., Martin, N., Wicking, C., and Chenevix-Trench, G. (1994). Am. 1.Med. Genet. 50, 282-290. Shanley, S. M., Dawkins, H., Wainwright, B. J., Wicking, C., Heenan, P., Eldon, M., Searle, J., and Chenevix-Trench, G. (1995). Hum. Mol. Genet. 4, 129-133. Springate, J. E. (1986). /. Pediatr. Surg. 21, 908-910. Strong, L. C. (1977).Cancer 40, 1861-1866. Sweet, H. O., Bronson, R. T., Donahue, L. R., and Davisson, M. T. (1996)./. Hered. 87,87-95. Viljoen, D. L., Saxe, N., and Temple-Camp, C. (1988).Pediatr. Dennatol. 5, 14-21. Ward, W. H. (1960). Austr. /. Dermatol. 5 , 204-207. Watson, M. L., and Seldin, M. F. (1994).In “Methods in Molecular Genetics, Vol. 5 , Gene and Chromosome Analysis: Part C” (K. W. Adolph, Ed.), pp. 369-387. Academic Press, San Diego. White, J. C. (1894). 1. Cutan. Genitourin. Dis. 12, 477-484. Wicking, C., Berkman, B., Wainwright, B., and Chenevix-Trench, G. (1994). Genomics 22, 505-5 11.

This Page Intentionally Left Blank

Transforming Growth Factor$ System and Its Regulation by Members of the Steroid-Thyroid Hormone Superfamily Katri Kolil* and lorma Keski-Oia1r2 f

Department

of

Virology, the Haartman Institute, and 2Department of Dermatology and Venereology, University of Helsinki, SF-00014 Helsinki, Finland

1. Introduction 11. Transforming Growth Factor-P

111. IV. V.

VI. VII. VIII. IX.

A. Structure of TGF-P B. Activation of TGF-P C. TGF-p Receptors and Other Binding Proteins Dual Effects of TGF-P on Cell Proliferation Regulation of Cell Differentiation by TGF-p TGF-P in the Regulation of the Immune System The Steroid-Thyroid Hormone Superfamily A. Characteristics of Steroid Receptors B. Nongenomic Actions of Steroid Hormones Steroid Hormone Regulation of TGF-P Isoform Expression A. Vitamin D, and Retinoids in Keratinocyte Differentiation B. Regulation of TGF-Ps in the Mammary Gland Regulation of Plasminogen Activation by Steroids Summary References

I. INTRODUCTION Transforming growth factor-ps (TGF-Ps) are potent regulators of cellular proliferation, differentiation, and morphogenesis as well as extracellular matrix formation, extracellular proteolysis, and inflammation. A major effect of TGF-P is its ability to inhibit cell proliferation. Three different mammalian TGF-P isoforms and many related peptides have been identified. Both TGF-Ps and their receptor molecules are expressed ubiquitously by normal

' Present address: Vanderbilt Cancer Center, MRB 11, Nashville, Tennessee 37232-6838. Advances in CANCER RESEARCH, Vol. 70 Copyright Q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

64

Katri Koli and lorma Keski-Oja

and transformed cells. The diverse activities of the members of the TGF-P family are regulated at the levels of TGF-P expression, secretion, and activity; TGF-P receptor expression; and cellular responsiveness. Members of the steroid hormone superfamily are potent regulators of the expression of TGF-P isoforms. It is proposed that TGF-Ps can act as local mediators of the various actions of steroid hormones. Estrogens and antiestrogens as well as retinoids, androgens, progestins, and vitamin D regulate the production and activity of TGF-p isoforms. In organs such as skin and mammary gland, TGF-P is suggested to have a role in limiting the growth of differentiating cells. Hormonal regulation of TGF-P is mediated mainly by posttranscriptional mechanisms, and a noticeable feature is that a significant fraction of the induced TGF-P is in an active form. Extracellular matrices provide a storage place for growth factors, which can then be activated by targeted proteolysis. Among other factors, plasminmediated proteolysis releases and activates matrix-associated latent forms of TGF-P. Plasminogen activation is under TGF-P regulation in various cell types, thus potentially controlling excessive TGF-p activation. Steroid hormones also participate in the regulation of cellular proteolytic balance, thus providing an additional regulatory step in their action. However, only a fraction of cellular responses to steroid hormones is mediated through TGF-f3 induction. Steroid hormones can elicit opposing effects to those of TGF-P, suggesting that complex regulatory mechanisms are involved in steroid action. At later stages of tumor development, cells often become refractory to the antiproliferative action of TGF-P. A role for TGF-f3 in the progression and escape from hormone dependence has also been suggested. The mechanisms of the regulation of TGF-P expression and activation, in addition to TGF-P signaling pathways, are important in understanding neoplastic transformation and escape from normal growth control. New data are accumulating both on TGF-P activation and its mode of action and on TGF-P regulation by steroid hormones.

11. TRANSFORMING GROWTH FACTOR-p TGF-P1, the prototype growth factor of this superfamily, is produced ubiquitously by various normal and malignant cells. The biologically active TGF-(31 is composed of two identical 112-amino-acid polypeptides each containing seven well-conserved cysteines. Six of these cysteines form a rigid structure known as the cysteine knot. The seventh cysteine forms an interchain disulfide bond with the corresponding residue in the other polypeptide monomer chain. This well-conserved structure is characteristic to all members of the family. Five different members of the TGF-P family have been

Steroid Regulation of TCF-P lsoforms

65

identified. Three of them, TGF-Ps 1-3, are found in mammals; TGF-P4 was isolated from chicken (Jakowlew et al., 1988) and TGF-P.5 from Xenopus (Kondaiah et al., 1990). TGF-Ps are usually found as homodimers, but heterodimeric forms also exist in certain cell types. Porcine platelets, a rich source of both TGF-Pl and TGF-P2, contain the heterodimeric form, TGF-P1,2 (Cheifetz et al., 1987). In addition, TGF-P2,3 was found in bovine bone (Ogawa et al., 1992). The sequence homology between members of the human TGF-P family and family members of different species is very high, ranging between 75% and 95%. The evolutionary conservation of the TGF-P molecules suggests important roles for them in normal physiology. Since the identification of the TGF-P isoforms, many structurally similar polypeptides have been assigned to this superfamily. The active forms of inhibins and activins show about 30% homology to TGF-P1. Inhibins were originally identified as polypeptides that inhibit the production of folliclestimulating hormone in pituitary cells (Ling et al., 1985). Activin has often opposite activity to that of inhibins. Bone morphogenetic proteins (BMPs) induce new bone and cartilage formation when injected under skin or into muscles of rodents (Rosen and Thies, 1992). BMP-2 and BMP-4 show highest degree of identity with the Drosophila decapentaplegic (dpp) gene and may represent mammalian counterparts of this protein. The dpp gene product is required for the dorsoventral axis formation in early embryos (Ferguson and Anderson, 1992). Vgl gene in Xenopus participates in the embryonic axis formation and mesoderm induction (Weeks and Melton, 1987). Mullerian inhibiting substance was identified for its ability to regress the embryonic duct system that develops into oviducts and uterus (Lee and Donahoe, 1993). Many other structurally similar factors that regulate normal growth and development in different organisms have been recognized, including 60A protein from Drosophila, dorsalin from chicken, nodal from mouse, and several growth differentiation factors from human and mouse (Kingsley, 1994).

A. Structure of TGF-f3 The active TGF-P is a 25-kDa protein composed of two identical chains held together by disulfide bonds. Recently, the crystal structure of the monomer fold and dimer association of TGF-P2 was revealed (Schlunegger and Grutter, 1992; Daopin et al., 1992). The monomer consists of two antiparallel pairs of p-strands forming a flat surface and a long a-helix. The monomers are held together by a single disulfide bridge so that the helix of one monomer interacts with the concave P-sheet surface of the other. Loop regions that are exposed might determine receptor specificity. TGF-Ps are usually synthesized and secreted in a latent form. The preproprotein consists of two disulfide-bridged 390- to 412-amino-acid poly-

66

Katri Koli and Jorma Keski-Oia

peptide chains, each containing several glycosylation sites and a mannose 6-phosphate receptor-binding site. The precursor structure is shared by members of the TGF-P superfamily and it seems to be essential for the folding and transport of the complex. The carboxyl-terminal part representing the mature TGF-P is cleaved between two arginine residues. The aminoterminal peptide, also called latency-associated protein (LAP), remains associated with the mature growth factor via noncovalent interactions and is able to render TGF-p latent. TGF-P associated with LAP is frequently called the small latent complex (Olofsson et al., 1992). Additional proteins are frequently associated with small latent TGF-p to yield large latent complexes. Latent TGF-P-binding protein (LTBP), first identified from platelets, binds to LAP covalently by its third 8-cys repeat (Saharinen et al., 1996). LTBP is involved in the secretion of TGF-P and in its association to the extracellular matrix (Taipale et al., 1994b). TGF-p and LTBP are coregulated in several cell systems, and in fibroblasts the assembly and secretion of properly disulfide-bonded TGF-p is dependent on LTBP (Miyazono et al., 1991). LTBP is also likely to play a role in the activation process of TGF-P. The size of LTBP secreted by cultured fibroblasts is 190 kDa, whereas the platelet form appears to be smaller (125-160 kDa), evidently due to proteolytic processing. LTBP is a soluble glycosylated protein containing 16-18 epidermal growth factor (EGF)-like repeats, 3-4 repeats containing eight cysteine residues, and an RGD sequence. The EGF-like repeats found in many proteins are suggested to mediate protein-protein interactions. The eight cysteine motifs are found also in the microfibrillar proteins, fibrillin-1 and fibrillin-2. Abnormalities in these proteins are associated with Marfan syndrome and congenital contractual arachnodactyly, respectively (Tsipouras et al., 1992). Recently, an LTBP homolog (41% at the amino acid level) has been cloned from the human foreskin fibroblast library (Morkn et al., 1994).This protein is called LTBP-2, and it can form a highmolecular-weight complex with small latent TGF-P1. Isolation of LTBP-3, that shares the domain structure of LTBP-1 and -2 was recently reported (Yin et al., 1995). The functions of LTBPs and fibrillins are poorly understood but, in addition to TGF-P binding, the LTBPs appear to have a function in targeting of the latent complexes to extracellular matrix (ECM) (Taipale et al., 1994b; Olofsson et al., 1992). The structural organization of the proteins is very similar, although the amino acid homology is not higher than 25%.

B. Activation of TGF-P Most cells produce TGF-P in a latent form that is not able to interact with the cell surface receptors. The interaction between TGF-P and LAP is electrostatic and can be disrupted in vitro by extremes of pH, chaotropic agents, certain glycosidases, or heat treatment (Brown et al., 1990, Table I). The

67

Steroid Regulation of TGF-P lsoforms

Table I Activation of Latent Forms of TGF-P Reference

Gamma-irradiation

Brown et al. (1990); Lawrence et al. (1985) Wakefield et al. (1988);Taipale et al. (1995) Miyazono and Heldin (1993) Sato and Rifkin (1989); Flaumenhaft et al. (1993) Murphy-Ullrich et al. (1992); Schultz-Cherry et al. (1994) Barcellos-Hoff et al. (1994)

Drug-induced Antiestrogens Glucocorticoids Retinoids Vitamin D

Kna bbe et al. ( 1994) Boulanger et al. (1995) Glick et al. (1989) Koli et al. (1993, 1995)

Extremes of pH Proteases (plasmin, cathepsin G) Glycosidases Cell cocultivation (plasmin mediated) Thrombospondin-mediated

acidic environment in bone tissue and in healing wounds might trigger the release of mature TGF-P, but mechanisms involving proteolysis are more likely to operate in vivo. It was reported that y-irradiation specifically generates active TGF-P in murine mammary gland, but the activation mechanism remains unknown (Barcellos-Hoff et al., 1994). The large latent complex is targeted to the ECM via LTBP (Taipale et al., 1994b). LTBP associates with extracellular fibers morphologically indistinguishable from those of fibronectin-collagen fibers in the pericellular matrix of cultured fibroblasts (Taipale et al., 1995b). It is not clear to which matrix components LTBP binds, but the amino-terminal region of LTBP is involved in this association (Saharinen et al., 1996). Inhibition of LTBP with antibodies or addition of excess free LTBP can inhibit the activation of TGF-p in cocultures of bovine endothelial cells and smooth muscle cells (Flaumenhaft et al., 1993). Evidence for plasmin-mediated activation has been found in many experimental systems (Lyons et al., 1988, 1990). Inhibition of plasmin-mediated proteolysis or transglutaminase prevents the production of active TGF-f3 (Kojima et al., 1993). Cell-cell contacts and targeting of TGF-p and other factors involved in the activation process seem to be crucial for the production of active TGF-p in the coculture system. Also, the retinoidinduced activation of TGF-P in bovine endothelial cells is mediated through an increase in plasmin-mediated proteolysis (Kojima and Rifkin, 1993). Plasmin, a broad-spectrum serine protease, can cleave LAP and thus disrupt the interactions between mature TGF-P and LAP (Lyons et al., 1990). The reaction is self-limiting, since TGF-P induces the production of plasminogen activator inhibitor-1 which decreases the formation of active plasmin (Laiho et al., 1986a,b). Thrombospondin, a platelet a-granule and ECM protein, has recently

68

Katri Koli and lorma Keski-Oja

betaglycan

I1

I

endoglin

SIGNAL

Fig. 1 Schematic illustration of a model of signaling through heteromeric TGF-P receptor complexes. Receptors type 1 and type I1 both contain serine/threonine kinase activity. Type I1 receptor can, in a ligand dependent manner, phosphorylate type 1 receptor. The role of TRlP (TGF-p-receptor interacting protein-1), FKBP12, FT (farnesyl transferase) and ras in signal transduction is still unclear. The main function of betaglycan and endoglin appear to be in ligand presentation for the signaling receptors (Wrana et al., 1994; Miyazono et al., 1993).

been shown to activate latent TGF-P through cell- and protease-independent mechanisms (Schultz-Cherry et al., 1994b). Type one properdin-like repeats of thrombospondin have been found to be the part of the molecule responsible for the binding and activation of TGF-P (Schultz-Cherry et al., 1994a). The activation of TGF-P is central to the regulation of the biological activity of TGF-P, since many cells are able to both produce latent TGF-p and express TGF-p receptors at their surfaces.

C. TGF-P Receptors and Other Binding Proteins Three membrane molecules with sizes of 53,75, and -250 kDa have been termed TGF-P receptors type I, I1 and 111, respectively. The type I and I1 receptors have been cloned and shown to be transmembrane serine-threonine kinases (Franzin et al., 1993; Lin et al., 1992). Type 11 receptor contains a short extracellular domain with several cysteine residues believed to form

Steroid Regulation of TGF-P lsoforms

69

the binding site for TGF-P. The cytoplasmic domain contains the kinase domain and a carboxyl-terminal tail rich in serine and threonine. The kinase domain in type I receptor is distinct from the kinase domain in type I1 receptor. Type I receptor also lacks the carboxyl-terminal tail characteristic for type I1 receptor and contains an additional domain of 29 amino acids called the GS domain. Studies with mutant mink lung epithelial cells devoid of functional receptors suggest that type I1 receptor is needed for the appearance of type I receptor on the cell surface (Laiho et al., 1990b). Type I1 receptor is a constitutively active kinase that is phosphorylated on multiple sites by itself as well as by other cellular kinases (Wrana et al., 1994). It binds TGF-p independent of type I receptor but cannot propagate the growth-inhibitory signal alone. Binding of TGF-p does not significantly alter the phosphorylation status of type I1 receptor. Type I receptor can recognize TGF-P bound to type I1 receptor and form a tight complex (Fig. 1).The binding affinities are at the low picomolar range (5-50 pM). The ability of TGF-Ply432, and -P3 to bind to the receptors diverges in correlation with their ability to inhibit growth in mink lung cells (Laiho et al., 1990b). Type I1 receptor can, in a ligand-dependent manner, phosphorylate type I receptor in the GS-domain, and this seems to be essential for signal propagation (Carcamo et al., 1995). Some proteins that bind to the cytoplasmic parts of the receptors and possibly participate in the propagation of the signal downstream have been characterized. Chen et al. (1995) reported that a WD-domain-containing protein named TGF-P receptor-interacting protein-1 associates with TGF-P type I1 receptor and is phosphorylated by the receptor kinase. Also several proteins, including FKBP-12 and farnesyl transferase, that bind to the cytoplasmic region of type I receptor have been identified (Wang et al., 1994; Kawabata et al., 1995). Their role in TGF-p signaling remains to be clarified. Transmembrane serine-threonine kinase receptors for other members of the TGF-P superfamily have also been characterized, and they can be grouped into type I and type I1 receptor subfamilies. Activins have also been found to signal through complexes of type 1 and type I1 receptors similar to those of TGF-P. Multiple receptors responding to activin have been cloned (Mathews and Vale, 1991; Mathews eta!., 1992). Structurally similar receptors without known ligands have also been assigned to these subfamilies. They could represent receptors for BMPs or other members of the TGF-P superfamily. Type I receptors can form complexes with different type I1 receptors and vice versa, forming complexes with differing ligand specificities and signaling properties. The various effects of TGF-Ps and related molecules on different cell types could result in part from receptor heterogeneity. TGF-P type 111 receptor is a membrane-anchored proteoglycan, also known as betaglycan (Cheifetz et al., 1988a). It is composed of a large extracellular domain containing heparan sulfate and chondroitin sulfate gly-

70

Katri Koli and lorma Keski-Oja

cosaminoglycan chains, a transmembrane domain, and a short cytoplasmic domain with no recognized signaling structures. The affinity of all TGF-P isoforms to betaglycan is relatively high (K,, > 10-9 M). Betaglycan does not directly participate in the signaling, but it is thought to regulate the access of TGF-P to the signaling receptors. Type I1 receptor can bind TGF-Pl tethered to membrane betaglycan better than it binds free TGF-Pl (L6pezCasillas et al., 1993). Binding of TGF-P to betaglycan is mediated through the core protein, and shedding of the glycosaminoglycans does not affect the binding. TGF-P2 binds poorly to type I1 receptor and TGF-P2 signaling is enhanced significantly by betaglycan, whereas TGF-61 and TGF-P3 seem to be less dependent on the presence of betaglycan. Soluble forms of betaglycan can block TGF-P action by sequestering the active form and thus inhibiting the binding to cell surface receptors. Endoglin, another TGF-P-bindingprotein found in endothelial cells, shows amino acid sequence homology to betaglycan and might have a similar function in cells that are devoid of betaglycan (Cheifetz et al., 1992). Endoglin binds TGF-Pl and TGF-P3 efficiently, and can form heteromeric complexes with signaling receptors for TGF-j3 (Yamashita et af., 1994). Mutations in endoglin have recently been linked to a human genetic disorder named hereditary hemorrhagic telangiectasia type l (McAllister et al., 1994). In addition, other matrix components such as fibronectin (Fava and McClure, 1987), thrombospondin (Murphy-Ullrichet al., 1992), type IV collagen (Paralkar et al., 1991), decorin (Yamaguchi et al., 1990), biglycan, and some other proteoglycans can bind TGF-P with varying affinities, but the characteristics of these interactions are unclear. Thrombospondin appears to have an effective role in the activation of latent forms of TGF-p (Schultz-Cherryet al., 1994a,b). Several less well characterized TGF-P receptors or binding proteins have been identified from a variety of sources. The type IV receptor identified only from pituitary cells seems to be a common binding protein for TGF-9, activins, and inhibins (Cheifetz et al., 1988b). Type V receptor is a 400-kDa glycoprotein purified from plasma membranes of bovine liver and appears to be a more frequently expressed binding protein for TGF-P (O’Grady et al., 1991). The binding protein labeled type VI is a 180-kDa glycoprotein, and the binding of TGF-P1 to this receptor appears to be dependent on the presence of TGF-P2 (Segarini et al., 1992). The major binding protein for mature TGF-P in plasma is a,-macroglobulin (O’Connor-McCourt and Wakefield, 1987). It is a circulating large tetrameric protein composed of 180-kDa subunits. It acts as an inhibitor of a wide variety of serum endoproteases, including plasminogen activator, collagenase, and elastase. In addition to TGF-P, it binds platelet-derived growth factor (PDGF) and nerve growth factor, which suggests that it might have a role as a scavenger of inflammatory molecules released by platelets at the site of injury. a,-Macroglobulin has a lower affinity for TGF-P1 than for TGF-P2, which provides a mechanism for differential regulation of their

Steroid Regulation of TGF-P Isoforms

71

biological activities (Danielpour and Sporn, 1990). TGF-P bound to a,macroglobulin cannot bind to cell surface TGF-p receptors, but can possibly modulate cell growth and function through a,-macroglobulin receptor-lowdensity-lipoprotein receptor-related protein. Tissue specific production of a,-macroglobulin has been detected, and it might regulate tissue proteinases and matrix metalloproteinases as well as cytokines in the ECM. Fucoidan, a polyanionic carbohydrate, and heparin can bind TGF-Pl and inhibit its interaction with a,-macroglobulin (McCaffrey et al., 1989). These polyanionic carbohydrates might also protect TGF-Pl from enzymatic and cellular proteolysis, thus leading to the accumulation of TGF-Pl activity (McCaffrey et al., 1994).

Ill. DUAL EFFECTS OF TGF-P ON CELL PROLIFERATION TGF-P, as its name implies was originally identified for its ability to induce morphological transformation and anchorage-independent growth of normal rat kidney fibroblasts (Roberts et al., 1981; Anzano et al., 1983; see also Moses et al., 1981). Since then it has been found that actually quite a few cell types respond to TGF-P by growth stimulation, but in most cells TGF-p acts as a potent growth inhibitor (Moses et al., 1985). The cellular growth response to TGF-P is also dependent on the differentiation and activation state of cells and on the presence of other extracellular and intracellular growth-regulatory molecules. Growth stimulation under certain conditions of mesenchymal cells such as fibroblasts, smooth muscle cells, and osteoblasts, has been reported (Moses et al., 1985; Centrella et al., 1987). The induction of growth appears to be secondary and mediated through induction of growth-stimulatory factors. In fibroblasts, TGF-P can induce the expression of PDGF receptor (Ishikawa et al., 1990) and both genes that code the PDGF chains (Leof et al., 1986; Makela et al., 1987). The kinetics of induction of PDGF and stimulation of DNA synthesis suggest that PDGF mediates the TGF-P-induced growth. In addition, stimulation of basic fibroblast growth factor (bFGF) gene expression has been found in fibroblasts (Pertovaara et al., 1993). TGF-P is a mitogen also for rat glial Schwann cells (Ridley et al., 1989). TGF-P inhibits the proliferation of many cell types, especially epithelial and hematopoietic cells. Recent work indicates that the antiproliferative effect of TGF-P is on late events in the mitogenic pathway. In mink lung epithelial cells, TGF-P arrests cell cycle progression in late GI phase, where it prevents the phosphorylation of the retinoblastoma protein (Rb) (Laiho et al., 1990a). Rb, acting as a tumor suppressor, inhibits the progression of the cell cycle to the S phase when underphosphorylated. Transforming proteins

72

Katri Koli and Jotma Keski-Oja

of some tumor viruses (E1A of adenovirus, T-antigen of simian virus 40 [SV40]) can overcome the block by binding to Rb and preventing its function (Pietenpol et al., 1990). The specific steps required for the TGF-P inhibition of Rb phosphorylation are unclear, but the involvement of cyclincyclin-dependent kinase (cdk) complexes in this process is apparent (for review see Alexandrow and Moses, 1995; Hunter, 1993). TGF-P can downregulate the synthesis of cdk4 and thus prevent the formation of active cyclin D-cdk4 complexes (Ewen et al., 1993). Overproduction of cdk4 in mink lung epithelial cells induces resistance to TGF-P growth inhibition, suggesting an important role in TGF-p signaling. cdk4 also contributes to the activation of cdk2, which forms an active kinase when complexed with cyclin E in the GI phase. TGF-P negatively affects the formation of these complexes (Koff et al., 1993) and prevents the hyperphosphorylation of Rb. In cultured keratinocytes, the expression of cyclin E as well as cdk2 and cdk4 is inhibited by TGF-P. Geng and Weinberg (1993) suggested that TGF-P blocks cell cycle progression primarily by preventing cyclin expression. Interestingly, in cultured epithelial cells hepatocyte growth factor (scatter factor) prevents the suppression of cdk4 and cdk2 but not the induction of p21 by TGF-Pl (Taipale and Keski-Oja, 1996). Inhibition of cyclin complexes by p l 5 protein, recently found to be induced by TGF-P in human keratinocytes, might also play a role in the induction of cell cycle arrest (Hannon and Beach, 1994). In the absence of Rb protein, TGF-P can efficiently inhibit the growth of some breast carcinoma cells, which suggests that additional mechanisms are involved in growth suppression of different cell types (Ong et al., 1991). TGF-P can regulate the expression of immediate early genes like c-fos and members of the jun gene family (Pertovaara et al., 1989). In keratinocytes, TGF-P treatment leads to down-regulation of the proto-oncogene c-myc (Coffey et al., 1988). In these cells, continuous expression of c-myc seems to be required for cell proliferation. Prevention of c-myc action by antisense oligonucleotides leads to a block at late GI comparable to the TGF-f3 growth arrest. Transforming proteins of tumor viruses like SV40, adenovirus 5 , human papillomavirus-16 can abolish the TGF-P-mediated c-myc downregulation (Pietenpol et al., 1990). These proteins must affect primarily cellular functions by binding to Rb and preventing its normal function. This suggests that Rb or related proteins are involved in growth inhibition also in keratinocytes. In contrast to mink lung epithelial cells, the phosphorylation status of Rb is not directly modulated by TGF-p; hypophosphorylation is rather a consequence of growth arrest (Munger et al., 1992). Koike et al. (1994) also reported that Rb is not a primary target for the action of TGF-p in keratinocytes. TGF-P inhibition of macrophage colony-stimulating factor (M-CSF)-induced growth of MAC-1 1 murine myeloid progenitor cells is also liked to c-myc suppression (Chen and Rohrschneider, 1993). M-CSF

Steroid Regulation of TGF-P lsoforms

73

induces c-myc expression through the c-fms receptor, and this can be abrogated by TGF-(3.

IV. REGULATION OF CELL DIFFERENTIATION BY TGF-P TGF-(3 participates in a variety of cell differentiation processes. It can act as an inhibitor or as a stimulator of differentiation with or without concomitant effects on cell growth. TGF-(3 is a potent inhibitor of adipogenic and myogenic differentiation (Ignotz and Massagui, 1985; Heino and MassaguC, 1990). In neither case is the modulation of cell growth by TGF-(3 involved in the differentiation process. Stimulation of chondrogenesis and osteogenesis by TGF-(3 is associated with specific induction of proteoglycans and collagens type I and I1 (Joyce et al., 1990; Centrella et al., 1987). Bone is a very rich source of both TGF-(31 and 432, and the acidic environment in bone tissue might explain the presence of active TGF-(3. TGF-(32 might be more active in vivo than TGF-(31 in stimulating bone formation in association with growth stimulation (Joyce et al., 1990). Administration of active TGF-(3 to the bone in vivo induces extensive bone formation (Noda and Camilliere, 1989). TGF-(3 and related factors can also contribute to bone resorption, suggesting their participation in bone remodeling (Tashjian et al., 1985). Many epithelial cells also respond to TGF-(3 by phenotypic changes. TGF-(3-induced differentiation of bronchial epithelial cells is characterized by an irreversible inhibition of DNA synthesis, an increase in cell surface area, and an increase in extracellular plasminogen activator activity (Masui et al., 1986; Gerwin et al. 1990). In addition, TGF-(3 can participate in the differentiation process of epidermal keratinocytes and intestinal epithelial cells in culture by inducing irreversible growth arrest (Moses et al., 1985; Bascom et al., 1989; Kurokawa et al., 1987). TGF-(3 can also negatively or positively regulate steroid production in specific organs (Feige et al., 1987).

V. TGF-P IN THE REGULATION OF THE IMMUNE SYSTEM TGF-(3s are potent immunomodulatory molecules, which have both immunosuppressive and proinflammatory properties (Wahl, 1992). These dual functions in inflammation seem to be regulated by differential responsiveness of inflammatory cells to TGF-P at different stages of development,

74

Katri Koli and JormaKeski-Oja

maturation, and activation. Large amounts of TGF-(3 are released from platelets at the site of tissue injury, and it functions as a chemoattractant for fibroblasts, monocytes, neutrophils, and T lymphocytes (Postlethwaite et al., 1987; Wahl et al., 1987; Reibman et al., 1991; Adams et al., 1991). TGF-P influences monocyte recruitment by modulating their integrin expression, increasing monocyte-matrix adhesion, and enhancing matrix-specific collagenase expression and chemotaxis (Oppenheim and Neta, 1994). In resting monocytes, TGF-P is known to induce the secretion of inflammatory cytokines such as interleukin (1L)-1, tumor necrosis factor-a (TNF-a), IL-6, bFGF, PDGF, and macrophage inflammatory protein-la (Wahl et al., 1987). After the early phases, during active inflammation, TGF-P acts as a suppressor of immune reactions by several mechanisms. Activated lymphocytes, macrophages, neutrophils, and synovial fibroblasts produce TGF-P, and its mRNA expression is highly elevated during active inflammation. In T lymphocytes undergoing phenotypic modulation, there is an enhancement of TGF-P receptor expression and the cells acquire responsiveness to the growth-inhibitory action of TGF-P. TGF-P is a potent inhibitor of mature CD4+ T lymphocytes, while CD8+ cells are less responsive (Lots et al., 1990). The CD8+ cell population produces IL-4, IL-5, and IL-10, which contribute to the immunosuppressive action. TGF-P inhibits T-cell proliferation primarily by interfering with IL-2-mediated proliferative signals (Ahuja et al., 1993). The proliferation of and immunoglobulin production by B lymphocytes is suppressed by TGF-P (Kehrl et al., 1991). The adhesion of T lymphocytes to the endothelium, macrophage functions, and TNF activities are also modulated by TGF-P (Gamble and Vadas, 1988; Espevik et al., 1987). In addition, TGF-P is a potent down-regulator of natural killer cell activity (Wallick et al., 1990). The potential role of TGF-P as an immunosuppressive agent is reinforced by studies on TGF-P knockout mice. Mice missing TGF-P seem to develop normally, but at the age of about 20 days they succumb to a wasting syndrome accompanied by multifocal, mixed inflammatory cell response and tissue necrosis leading to death (Shull et al., 1992; Kulkarni et al., 1993). Transgenic mice overexpressing TGF-p develop inflammatory or fibrotic lesions in the heart, liver, and kidney (Sanderson et al., 1993). Enhanced TGF-@levels have been identified in the synovial fluids and tissues of arthritis patients, suggesting a role in the pathophysiology of this disease (Lafyatis et al., 1989). In contrast, exogenous TGF-P delays the onset and reduces the severity of experimental autoimmune diseases in animal models of multiple and rheumatoid arthritis (Rache et al., 1991; Kuruvilla et al., 1991). TGF-P could also have a protective role in allogenic rejection (Waltenberger et al., 1991). TGF-f3 inhibits reversibly the proliferation of hematopoietic cells. The hematopoietic progenitor cells responding to IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or colony-stimulating factor-1

Steroid Regulation of TGF-p lsofoms

75

(CSF-1) are growth inhibited by TGF-P (Ohta et al., 1987; Keller et al., 1988). TGF-P knockout mice appear to have no defects in hematopoiesis, but have abnormally high levels of circulating monocytes and neutrophils, suggesting that TGF-p might limit early hematopoiesis (Schull et al., 1992). Granulopoiesis, in contrast, is stimulated by TGF-P in the presence of GMCSF (Keller et al., 1991). Myeloid leukemia cell proliferation is also inhibited by TGF-P. Phorbol-12-myristate-13-acetate or retinoic acid-induced differentiation of these cells is associated with coordinated up-regulation of genes involved in TGF-P signal transduction (TGF-P and its receptors), making them more susceptible to TGF-P-mediated growth inhibition (Taipale et al., 1994a).

VI. THE STEROID-THYROID HORMONE SUPERFAMILY Steroid and thyroid hormones as well as vitamins A and D participate in the regulation of cell growth and differentiation, as well as diverse events involved in embryonic development. They mediate various effects through changing the patterns of cellular gene expression. Steroid and thyroid hormones as well as vitamins A and D are small lipophilic molecules that diffuse into cells through plasma membrane and bind to their cognate receptors. Nuclear receptors for steroids function as ligand-specific transcription factors, which recognize specific elements in DNA and enhance or repress gene transcription (Karin et al., 1993). Also, nongenomic effects are part of the cellular response, and at least for vitamin D the existence of membrane “receptors” has been suggested. Steroid hormones can be divided into three major classes: the adrenal steroids, sex steroids, and vitamin D,. Together with vitamin A metabolites and thyroid hormone, although not structurally or biosynthetically related, they constitute a family that mediates their effects through receptors with a common structure (Evans, 1988). These agents have wide influence on development and normal body physiology. The adrenal steroids participate in the control of glycogen and mineral metabolism as well as mediate the stress response (Tempel and Leibowitz, 1994; Munc et al., 1984). Also, androgens have multiple effects in the nervous and immune systems. The sex steroids are crucial for the reproductive system and have profound effects on cells of the breast, ovary, prostate, and testis (Josso, 1992). Vitamin A and its derivatives, commonly called retinoids, are important for the function of the visual circle and participate in the regulation of growth and differentiation of various cell types, including most epithelial cells (McCaffery and Drager, 1993). Vitamin D, has a role in controlling body calcium homeostasis as well as regulating growth and differentiation in many cell types (Bikle, 1992). In

76

Katri Koli and JomaKeski-Oja

intestine, bone, and skin, vitamin D3 is important for the proper development and differentiation processes (Suda et al., 1990).

A. Characteristics of Steroid Receptors The nuclear receptor superfamily includes receptors for adrenal and sex steroids as well as thyroid hormone, retinoids, and vitamin D3 (Evans, 1988). The cellular localization of receptors of this superfamily varies. The unoccupied glucocorticoid receptor is located in the cytosol, whereas unoccupied receptors for retinoids, thyroid hormone, and vitamin D, are already nuclear. Nuclear receptors can be associated with DNA in the absence of ligand. The DNA-binding domain of these receptors contains nine highly conserved cysteine residues, eight of which form two zinc finger structures. In the first zinc finger, there is a critical region that determines sequencespecific DNA binding of each receptor (Danielsen et al., 1989). The carboxyl-terminal region of the receptor contains a ligand-binding domain, which confers high-affinity binding for the ligand. The transactivation and nuclear localization properties also map to the carboxyl-terminal region. The zinc finger region of vitamin D, receptor (VDR) binds to DNA as a dimer, and sequences in both the carboxyl termini and in the DNA-binding domain participate in the formation of homo- and heterodimers (Rosen et al., 1993). Receptor phosphorylation might also participate in the transcriptional regulation of target genes. VDR has been shown to be phosphorylated in a ligand-dependent manner, and the phosphorylation correlates with transcriptional activation (Jurutka et al., 1993). Most steroid-inducible genes contain at least one hormone response element (HRE) in the regulatory region of the gene. Two classes of vitamin D response elements have been characterized. The motif GGGTGA, arranged as a direct repeat with a spacing of six nucleotides or as a palindrome without spacing, or as an inverted palindrome with a 12-nucleotide spacing, confers vitamin D inducibility mediated by VDR alone. The other class of response elements, composed of directly repeated pairs of motifs (GGTCCA, AGGTCA, or GGGTGA) spaced by three nucleotides (DR-3), is synergistically activated by VDR and retinoid X receptor (RXR) (Carlberg et al., 1993). RXR is a coregulator that can heterodimerize with retinoic acid receptors (RAR) as well as with thyroid hormone and vitamin D3 receptors (Yu et al., 1991; Kliewer et al., 1992). VDR localizes to the nucleus and is probably associated with DNA in the absence of ligand. Residues in the DNA-binding and ligand-binding domains of the receptor participate in the formation of the dimerization interfaces, and protein-protein interactions are critical for the target recognition (Towers et al., 1993). Retinoid receptors and thyroid receptor (TR) also recognize very similar response elements of six nucleotides arranged as direct repeats with prefer-

Steroid Regulation of TGF-P lsoforms

77

ential spacing of five or four nucleotides, respectively (Umensono et a/., 1991). The spacing between half-sites appears to be more important for DNA-binding selectivity than sequence differences. It has been demonstrated that, in addition to associating with RXR, VDR can interact with RAR and TR (Schrader et al., 1994a).The polarity of the binding of heterodimeric nuclear receptor complexes to response elements seems to be an additional regulatory property (Schrader et al., 1994b). This interactive network of nuclear receptors provides a large number of transcription factors with distinct functional properties. The binding of ligands to homo- and heterodimeric receptors determines the transactivation capacity of the complex (Fig. 2). In contrast to VDR, TR, and retinoid receptors, steroid receptors (glucocorticoid, progesterone, androgen, mineralocorticoid, and estrogen receptors) bind to their response elements as homodimers. The orientation of the consensus sequence and spacing is also different. The consensus estrogen response element contains a half-site sequence AGGTCA, whereas the functional glucocorticoid response element (GRE) contains AGGACA or

@ Ligands

transcription

+ Cell

@ Response elements

Nucleus

@

Receptors

* dimeric composition * dimeric polarity * ligand occupancy

* direct repeat * palindrome * inverted palindrome * half site spacing

Fig. 2 Signal transduction pathways used by steroid and thyroid hormones and vitamins A and D (Karin et al., 1993; Evans, 1988). Lipophilic ligands can enter cells passively and interact with receptor proteins either in the cytoplasm (glucocorticoid receptor) or nucleus (retinoid and vitamin D receptors). Receptor dimers of homo- or heteromeric composition can bind to response elements on target genes.

78

Katri Koli and lorma Keski-Oja

AGAACA (Zilliacus et al., 1995). The GRE can also mediate gene activation by progesterone and androgen (Beato et al., 1989). Most response elements characterized are positive regulatory elements in the promoter regions of steroid-inducible genes. Steroids do also downregulate the expression of many genes, and the response elements differ somewhat from the HREs found in positively regulated genes. Different molecular mechanisms might be involved in mediating repression than gene induction. Competition by receptor binding to its cognate DNA sequences for binding of other essential transcription factors or positive modulatory factors is likely to be involved (Beato et al., 1989). Interference of the function of other transcription factors by direct protein-protein interactions is also an important mode of action, as in the case of glucocorticoid repression of collagenase gene. The glucocorticoid-receptor complex binds the activating protein-1 (AP-I) protein complex and prevents it from inducing activation of the target gene (Krane, 1993). AP-1 binding sequences in some genes are also target sequences to regulation by steroids. The use of common regulatory sequences by two distinct classes of transcription factors can explain differential gene expression in proliferating and differentiating cells. Transcription factors fos and jun, which are expressed in proliferating cells, form stable heteromeric complexes that interact with AP-1 sites in a sequence-specificmanner. It has been suggested that in proliferative cells the response elements of genes involved in differentiation are occupied by the AP-1 complex and their transcription is suppressed (Lian et al., 1991). The specific interactions between DNA and different protein complexes are unclear, but experimental data of fos-junmediated suppression of the osteocalcin and alkaline phosphatase genes in bone support the idea of phenotypic suppression (Schule et al., 1990).

B. Nongenomlc Actions of Steroid Hormones Steroid hormones can elicit rapid changes in signal transduction pathways, including regulation of intracellular calcium concentration, protein kinase C activity, phospholipid metabolism, and cyclic nucleotide formation (de Boland and Nemere, 1992). The existence of a membrane response element for vitamin D distinct from nuclear receptor has been suggested, but no membrane receptors have been identified yet (Norman et al., 1992). Differential effects of vitamin D, analogs on genomic and nongenomic responses are in favor of the consideration that associated receptors are distinctly different from one another (Norman et al., 1993). Cellular responses to steroids are a combined action of both receptor-mediated changes in transcription and rapid nongenomic effects. These two pathways are not completely separate; nongenomic actions can reinforce genomic responses and vice versa.

Steroid Regulation of TGF-f3 lsoforms

79

VII. STEROID HORMONE REGULATION OF TGF-P ISOFORM EXPRESSION Members of the steroid hormone superfamily are potent regulators of the expression of TGF-P isoforms (Table 11) (Wakefield et al., 1990; Roberts and Sporn, 1992). TGF-Ps might have a role as local mediators of the various actions of steroid hormones. In most cells the regulation is mediated by posttranscriptional mechanisms, although in some cell types mRNA induction is associated with increased protein levels. A notable feature is that steroids can induce TGF-P in an active form, which can function in an autocrine or paracrine manner. In the skin and mammary gland, the differentiation-associated regulation of TGF-P isoforms is striking and can be linked to phenotypic changes that cells undergo in response to steroid hormones. Androgens and synthetic progestins as well as glucocorticoids are also potent regulators of the expression of TGF-p isoforms (Table 11), and the regulation is mediated mainly by posttranscriptional mechanisms (Benz et al., 1991; Colletta et al., 1991). Progesterone can induce TGF-P2 mRNA in human endometrium and it can mediate the suppression of the matrix metalloproteinase matrilysin (Bruner et al., 1995).TGF-P2 expression in the prostate seems to be down-regulated by androgens, since withdrawal of the hormone in cell culture or by castration in animals leads to a significant stimulation of TGF-P2 mRNA (Knabbe et al., 1993; Bacher et al., 1993). Glucocorticoid regulation of TGF-6s is highly cell type dependent. In fetal lung fibroblasts, cortisol can increase TGF-P3 expression, and it is possibly important in the stimulation of lung maturation (Wang et al., 1995).

A. Vitamin D,

and Retinoids

in Keratinocyte Differentiation The differentiation of epidermal keratinocytes involves many phases, which are regulated by growth factors and hormones in an autocrine or paracrine manner (Bikle and Pillai, 1993). The basal layer of epidermal keratinocytes rests on the basal lamina and consists of continually proliferating cells. Cells migrate upward from this basal layer, cease to proliferate, and gradually acquire the characteristics of fully differentiated corneocytes. Cells of the spinous layer (above basal cells) are programmed to produce insoluble keratins K1 and K10. In addition, involucrin and transglutaminase are needed in the formation of the cornified envelope. The granular layer contains electron-dense granules containing profilaggrin or loricrin as well as lipid-filled lamellar bodies that fuse with the plasma membrane. In the cornified layer, cellular organelles are destroyed and the highly resistant

80

Katri Koli and lorma Keski-Oia

Table II Steroid Hormone Regulation of TGF-f3 Expression and/or Activity Hormone GIutocorticoids

Isoform

Cell type

Reference

TGF-P1 f TGF-P2 t TGF-P1 f

T lymphocytes Osteoblasts Brain cells Lung carcinoma Rat kidney fibroblasts Lung carcinoma

Ayanlar Batuman et al. (1991) Oursler et al. (1993) Nichols and Finch (1994) Danielpour et a/. (1991)

TGF-$2

1

Estrogens

TGF-$2 J Mammary carcinoma Jeng et a/. (1993) Arrick et a/. (1990) TGF-$3 1 Komm e t a / . (1988) TGF-P1 f Osteosarcoma

Antiestrogens

TGF-P1

Norethindrone (progestin)

TGF-$2 J. TGF-P3 J.

T

Gestodene TGF-PI t (synthetic progestin) TGF-$2 t TGF-$3 f Androgens

TGF-61 TGF-P2 t TGF-Pl J TGF-P2 J

Retinoic acid

TGF-P2

t

TGF-P2 J. Vitamin D,

Mammary carcinoma Fetal fibroblasts

Knabbe et a/. (1987) Colletta et a/. (1990)

Mammary carcinoma Jeng and Jordan (1991) Mammary carcinoma

Colletta e t a / . (1991)

Thymocytes Osteosarcoma Osteoblastic Rat prostate cells Prostate cells

Olsen et a/. (1993) Benz e t a / . (1991) Kasperk et a/. (1990) Kyprianou and lsaacs (1989) Knabbe et al. (1993)

Epidermal keratinocytes Chondrocytes Rat kidney fibroblasts Lung carcinoma Myocytes

Glick et al. (1989)

Arrick et al. (1994)

TGF-pl TGF-P2

t t

Epidermal keratinocytes

TGF-pl

t

Mammary carcinoma

Jakowlew et al. (1992) Danielpour et a/. (1991) Jakowlew et al. (1992) Kim et al. (1992) Koli and Keski-Oja (1991, 1993a) Koli and Keski-Oja ( 1995)a

Vitamin D, also enhances the production of the binding protein LTBP-1.

corneocytes are eventually shed off. Calcium seems to be an important modulator of these pathways. Calcium gradients exist in the epidermis so that in the basal and spinous layers calcium is found primarily intracellularly, but in the upper layers calcium accumulates in large amounts in the intercellular matrix (Menon et al., 1985). Keratinocytes cultured in low calcium proliferate readily, but when switched to high calcium start to differentiate (Hennings and Holbrook, 1983). High extracellular calcium increases intracellular free calcium concentrations, which correlate with the ability of keratinocytes to form cornified envelopes (Pillai and Bikle, 1991).

,

Steroid Regulation of TGF-P lsoforms

81

TGF-P1 and TGF-62 are expressed in the differentiating layers of epidermis, and likely have a role in keratinocyte withdrawal from the cell cycle and maintenance of the quiescent state. Studies of Glick et al. (1993) suggest that TGF-Pl is likely to be a negative regulator of keratinocyte proliferation in the basal layer, while TGF-P2 may inhibit DNA synthesis in the suprabasal, terminally differentiated cells. TGF-p alone cannot induce keratinocyte differentiation but is important for growth arrest, which is a prerequisite for irreversible commitment of the cells to terminal differentiation. Overexpression of TGF-P in the epidermis of transgenic mice results in neonatal lethality, further supporting an important role for TGF-P in the epidermis (Sellheyer et al., 1993). TGF-Pl knockout mice have a three- to fivefold increase in the epidermal labeling index compared to normal mice (Kulkarni et al., 1993). Loss of expression of TGF-P in skin tumors is associated with hyperproliferation and high risk for malignant conversion (Glick et al., 1993). Tumor- and stromally produced TGF-p can have different effects on tumor cell growth. In the skin, genetic deletion of autocrine TGF-p in the tumor cells results in accelerated, multifocal progression to squamous carcinoma, whereas autocrine TGF-Pl expression suppresses malignancy. Expression of TGF-Pl in dermal fibroblasts, in contrast, enhances epithelial tumor cell proliferation (Glick et al., 1994). Vitamin D, is produced by keratinocytes in association with early events of epidermal differentiation. When terminal differentiation markers are detected in keratinocyte cultures, the amount of la,25-dihydroxy vitamin D, declines and increased levels of the inactive metabolite 24,25-dihydroxy vitamin D, are observed (Pillai et al., 1988). A concomitant decrease in receptor number is observed in more differentiated cells without notable changes in receptor affinity. Vitamin D, is a strong inhibitor for keratinocyte growth (Matsumoto et al., 1990). Production of involucrin, expression of transglutaminase activity, and cornified envelope formation are induced by endogenously produced vitamin D, in vitro (Matsumoto et al., 1991). Calcium and vitamin D3 interact in a complex manner to bring about the inhibition of keratinocyte proliferation and stimulation of the expression of various differentiation markers (Su et al., 1994). Keratinocytes are more sensitive to the antiproliferative effects of vitamin D, in a high-calcium environment and vice versa. Vitamin D, acts mainly on the intermediate differentiation layers of the epidermis, where TGF-Ps are produced. Vitamin D, and also its analogs, which are strong inhibitors of keratinocyte proliferation, can efficiently regulate the production and activation of TGF-Pl and TGF-P2 in cultured murine keratinocytes (Koli and Keski-Oja, 1993a). Significant amounts of active TGF-p are secreted in response to vitamin D, treatment. Neutralizing TGF-P antibodies partially inhibit vitamin D,induced inhibition of growth, suggesting autocrine or paracrine functions. The mechanism of activation of TGF-P during vitamin D3 treatment is unclear at present. In epithelial and fibroblastic cells, vitamin D, strongly

82

Katri Koli and lorma Keski-Oia

down-regulates plasminogen activator activity (Koli and Keski-Oja, 1993b, 1996), indicating that other than plasmin-mediated mechanisms are likely to be involved. These effectsof vitamin D, and its synthetic analog calcipotriol most likely play a role in the therapeutic efficacy of these compounds in the treatment of psoriasis (Lea and Goa, 1996). Retinoic acid (RA) seems to have a dual function in keratinocyte proliferation. Choi and Fuchs (1990) reported that RA increases the proliferation and migration of human keratinocytes. In mouse keratinocytes, RA appears to inhibit cell growth in a dose-dependent manner (Glick et al., 1989). RA and high calcium specifically induce the expression of TGF-P2 in murine epidermal keratinocytes through posttranscriptional mechanisms (Glick et al., 1989, 1990). However, in epidermal differentiation events RA acts as a negative regulator. RA receptors unique to skin have been characterized, which suggests that retinoids participate in the control of cell differentiation in vivo (Krust et al., 1989). In other cell types, such as human lung carcinoma cells and normal rat kidney fibroblasts, RA also induces TGF-P2 production (Danielpour et al., 1991). It has been shown using neutralizing antibodies that TGF-P2 mediates the growth-inhibitory response of RA (Glick et al., 1989).

B. Regulation of TGFeQs in the Mammary Gland The growth of ductal structures of the mammary gland is tightly controlled in different stages of mammary development. During puberty and sexual maturation, significant elongation and branching of ductal epithelium takes place, and a patterning with typical interductal spaces is formed. During pregnancy, further ductal growth and formation of secretory alveoli generate the lactating phenotype. After weaning, the secretory tissue degenerates and the mammary gland reverts to a state similar to that of a virgin. This highly regulated balance between proliferation, differentiation, and degeneration requires fine control by hormones and growth factors as well as cross-talk between epithelial cells and stromal fibroblasts of the mammary gland. TGF-@isoforms are expressed in a developmentally regulated manner in the mammary gland, thus maintaining the open pattern of ductal branching by restricting the formation and growth of lateral buds (Robinson et al., 1991; Pierce et al., 1993). A complex epithelium-matrix interaction on TGF-@activation might be involved in the regulation of ductal outgrowth (Howlett and Bissell, 1993). There is an overlapping expression pattern of the three TGF-P isoforms within the epithelium of actively growing mammary end buds during branching morphogenesis as well as within the epithelium of growth-quiescent ducts (Robinson et al., 1991). TGF-@3is the only isoform detected in mouse mammary myoepithelial cells. There is a dramatic down-regulation of all TGF-@sduring lactation. In a transgenic mouse

Steroid Regulation of TGF-p lsoforms

83

model where TGF-p is overexpressed in the mammary gland under M M r V promoter, a severe hypoplasia of the mammary epithelium is detected. However, TGF-@overexpression does not inhibit mammary development during pregnancy, and alveolar differentiation and lactation can occur (Pierce et a1.,1993). If TGF-@expression is driven under WAP promoter, which targets the transgene expression to the pregnant gland, alveolar development and lactation are inhibited (Jhappan et al., 1993).TGF-P might function to limit the accumulation of milk proteins in pregnancy, since the expression of TGF-@2and 433 is increased during pregnancy but drops dramatically at the onset of lactation (Robinson et al., 1993; Jhappan et al., 1993). Mammary epithelial cells in culture, as well as several breast carcinoma cell lines, express TGF-P and are responsive to its growth-inhibitory effects. TGF-P functions as an autocrine negative regulator of breast cancer cell proliferation (Arteaga et al., 1990). Estrogens and antiestrogens regulate TGF-@ production in mammary carcinoma cells and in fetal fibroblasts (Knabbe et al., 1987; Jeng et al., 1993; Colletta et al., 1990). Estrogeninduced growth is associated with down-regulation of TGF-P2 and TGF-P3 in several breast carcinoma cell lines (Arrick et al., 1994; Jeng et al., 1994), whereas the antiestrogens tamoxifen and toremifen can enhance the expression of TGF-Pl (Knabbe et al., 1987; Warri et al., 1993). Interestingly, in a clinical study in which breast tumor biopsies from tamoxifen-treated patients were studied for TGF-@by immunohistochemistry, the induction of TGF-Pl was detected in stromal fibroblasts but not in tumor epithelium (Butta et al., 1992). However, since mRNA expression was not analyzed, the actual synthesis site of TGF-P is unclear. Studies trying to establish the role of antiestrogen-induced TGF-@ in the growth-inhibitory response have failed. It seems that autocrine TGF-p is not necessary for the induction of growth arrest by antiestrogens (Arteaga et al., 1988; Dugger et al., 1994). However, paracrine effects of the induced TGF-@ on host cells have been poorly characterized. Estrogens are critically involved in the progression of breast cancer. In later stages of tumor development, cells acquire hormone independence. Following progression toward estrogen independence, there is a large increase in T G E p production and change in cellular responses toward this growth factor. Acquisition of steroid autonomy was paralleled with acquired sensitivity to stimulation by TGF-p, and inhibition by TGF-p-neutralizing antibodies, in an in vitro model of hormone independence (Daly et al., 1990). TGF-p actions depend on cell type but also on the state of differentiation and other factors present. TGF-f.3mRNA is expressed in breast cancers as well as nonneoplastic breast tissues, but the levels are higher in breast cancer (Barrett-Lee et al., 1990). No correlation with estrogen receptor expression in tumors was found in this study. Several studies suggest that TGF-P can indirectly promote breast cancer cell progression (Arteaga et al., 1993a; Gorsch et al., 1992; Welch et al., 1990; for review, see Koli and Arteaga, 1996).

84

Katri Koli and lorma Keski-Oja

Mechanisms likely to favor tumor cell growth involve down-regulation of immune functions (Arteaga et al., 1993b) and promotion of angiogenesis as well as stromal effects. A. M. Thompson et al., (1991) reported that breast tumors unresponsive to the antiestrogen tamoxifen express significantly higher levels of TGF-Pl mRNA than do clinically responsive tumors, which further suggests a role for TGF-P in breast cancer progression. Some vitamin D, analogs are under clinical evaluation for the treatment of breast cancer patients. The strong immunosuppressive and growth-inhibitory effects of these compounds are likely to have therapeutic value. Several breast cancer cell lines express a high-affinity VDR and respond to vitamin D, treatment by inhibition of growth. Vitamin D, and its analogs induce TGF-Pl mRNA and protein expression in cultured cells in a concentrationand time-dependent manner (Koli and Keski-Oja, 1995).The induction kinetics are rather slow, which suggests post-transcriptional mechanisms for the vitamin D3 response. Importantly, a significant part of the secreted TGF-p is active, suggesting autocrine or paracrine functions. A concomitant vitamin D3-dependent increase in the secretion of LTBP protein supports the role of vitamin D, as an important regulator of the TGF-j3 system. Whether TGF-P mediates the antiproliferative effect is still unclear. Of note, however, in an unresponsive breast cancer cell line there was no induction of TGF-P by vitamin D, (Koli and Keski-Oja, 1995).

VIII. R E G U M I O N OF PLASMINOGEN ACTIWTION BY STEROIDS Plasmin is a wide-spectrum serine protease that can dissolve fibrin clots and cleave various extracellular and basement membrane proteins. In addition, plasmin activates metalloproteinases and latent elastase, which further contribute to the degradation of matrices (Tryggvasonet al., 1986; Vassalli et al., 1991).Plasmin is generated from an abundant proenzyme, plasminogen, through proteolytic cleavage by plasminogen activators (PAS).Urokinasetype (u-PA) and tissue-type (t-PA) PAS are products of different genes, but the homology at the amino acid sequence level is about 40% (Dan0 et al., 1985). Urokinase is produced in a proenzyme form, which can be activated by plasmin. Urokinase activation leads to further activation of plasminogen to plasmin and an autocatalytic loop is formed (Wun et al., 1982). The production of active plasmin is negatively regulated by efficient inhibition of PAS by plasminogen activator inhibitors (PAIs), or of plasmin by o12-antiplasmin or a,-macroglobulin. PAIs are serine protease inhibitors and pseudo-substrates of PAS that are proteolytically cleaved and subsequently form covalent complexes with the enzymes (Andreasen et al., 1990). u-PA produc-

Steroid Regulation of TGF-P lsoforms

85

tion and activity have been associated with reproduction, inflammation, and cell migration, while t-PA is regarded as an important mediator of fibrinolysis and thrombolysis (Rijken et al., 1982). The most potent in vivo function of the PA system is fibrinolysis, the removal of fibrin clots. PAS and plasmin also play roles in various processes associated with proteolytic modulation of ECMs and basement membranes. During ovulation, embryonic implantation, and development, there is a distinct pattern of expression of PAS and PAIs, suggesting involvement in the regulation of reproduction (Saksela and Rifkin, 1988). The production of PAS by inflammatory cells, such as monocytes and macrophages, is also associated with cell migration and degradation of affected tissues. Increased expression of u-PA and u-PA receptor parallels induction of migration (Pollinen et al., 1991). Migrating keratinocytes at the edges of a wound also express enhanced u-PA activity (Morioka et al., 1987). Plasminogen activation is also involved in prohormone processing (Virji et al., 1980). In addition, there is evidence that the activation of plasmin is involved in cell invasion and angiogenesis. Certain matrix-bound growth factors can be released and activated through plasmin-mediated proteolysis (Taipale et al., 1992, 1994b). Several hormones, growth factors, and cytokines are involved in the regulation of pericellular proteolytic activity (Laiho and Keski-Oja, 1989). The activity can be regulated at the levels of expression, activation, and localization of the components of the proteolytic system. The activation of plasminogen can be a local event with cell surface-bound components. Enhanced PA activity is often associated with rapidly growing cells. Malignant cells express high levels of proteolytic activity, and u-PA is implicated in cancer invasion and metastasis. u-PA has been demonstrated to be a bad prognostic marker, at least in breast cancers (Duffy et al., 1990). Interestingly, primary breast tumors also have higher levels of PAI-1, and it has been shown to be a strong prognostic factor for early relapse in breast cancer patients (Foekens eta!., 1994). Estrogen is known to positively regulate t-PA activity in dimethylbenzanthracene-induced mammary carcinomas (Yamashita et al., 1992). The regulation by steroid hormones of PA and PA1 production seems to be highly dependent on cell type (Table 111). The TGF-fl signaling system seems to be affected in many cases by the plasminogen activation system, which itself is under hormonal and cytokine control. Plasmin-mediated release and activation of latent TGF-p forms has been observed in various cell systems (see Section 1I.B). In cocultures of bovine endothelial cells and smooth muscle cells, TGF-P activation can be prevented by inhibition of plasminogen activation (Kojima et al., 1993). In addition, retinoid-induced activation of TGF-p in bovine endothelial cells depends on plasminogen activation (Kojima and Rifkin, 1993). The release and activation of TGF-P from extracellular storage sites constitutes an important regulatory site for TGF-p-mediated signaling. The presence of latent

86

Katri Koli and JomaKeski-Oja

n b l e 111 Steroid Regulation of PA and PA1 Activities Hormone

Glucocorticoids

Estrogens Progesterone Androgens Retinoic acid

Vitamin D3

Cell type

U-PA

t-PA PAL1

Monocytes Granulosa cells Hepatoma cells Prostate cells Mammary carcinoma cells Mammary carcinoma cells Endothelial cells Endometrial stromal cells Granulosa cells Embryonal carcinoma cells Endothelial cells Epidermal keratinocytes Epidermal keratinocytes

t

t t

t

t

J

.

t

t t

l

Reference Hamilton et al. (1993) Jia et al. (1990) Bruzdzinski et al. (1993) Heaton et al. (1992) Freeman et al. (1990) Henderson and Kefford (1993) Yamashita et al. (1992)

t t

Blei et al. (1993) Casslen et al. (1992)

t

Jia et al. (1990) Tienari et al. (1991) Kratzschmar et af. (1993) Thompson et al. (1991) Medh et al. (1992) Koli and Keski-Oja (1996)

TGF-P complexes in the matrix may provide the cells with an easily activated resource to obtain TGF-P.

IX. SUMMARY TGF-Ps and their receptors are expressed ubiquitously, and they act as key regulators of many aspects of cell growth, differentiation, and function. Steroid action on target tissues is often associated with increase in TGF-P isoforms. Regulation of TGF-P expression and activation is crucial for normal development and growth control. The loss of responsiveness of different tumor cells to the antiproliferative effects of TGF-p is a common feature in carcinogenesis. Multiple changes are required for the cells to gain complete resistance to TGF-P growth inhibition (Fynan and Reiss, 1993; Kimchi et al., 1988; Samuel et al., 1992). Although many tumor cells are not growth inhibited by TGF-P, they respond to TGF-P treatment by changes in the expression of matrix components and enhanced proteolytic activity (KeskiOja et al., 1988). Agents that induce TGF-p production in target tissues can have a chemopreventive or chemotherapeutic value for the management of epithelial malignancies.

Steroid Regulation of TGF-p lsoforms

87

Conversely, data supporting a positive role for TGF-P in established tumor progression are beginning to emerge (Arteaga et al., 1993a,b; Barrett-Lee et al., 1990; Arrick et al., 1992; E. A. Thompson et al., 1991).In later stages of tumor development, cell proliferation is often not inhibited by TGF-P, and tumor cells secrete large amounts of this growth factor (Fynan and Reiss, 1993). In vivo TGF-P secreted by tumor or stromal cells can influence host responses such as natural killer cell function and thus indirectly support tumor cell viability (Arteaga et al., 1993b). TGF-P may also affect tumor growth indirectly by stromal effects and promotion of angiogenesis. TGF-P may also be involved in the progression of breast tumors from the steroidsensitive to steroid-insensitive state (King et a1.,1989). Understanding of the net effect of TGF-P in different stages of tumor development is critical for the evaluation of its therapeutic value in cancer treatment.

ACKNOWLEDGMENTS Our original work was supported by the Academy of Finland, the University of Helsinki and Biocentrum Helsinki, the Finnish Cancer Organizations, the Sigrid Juselius Foundation, and the Alfred Kordelin Foundation. We thank Dr. Carlos Arteaga for expert review of the manuscript.

REFERENCES Adarns, D. H., Hathaway, M., Shaw, J., Burnett, D., Elias, E., and Strain, A. J. (1991). J. Immunol. 147, 609-612. Ahuja, S. S., Paliogianni, F., Yamada, H., Balow, J. E., and Boumpas, D. T. (1993).1.Immunol. 150,3109-3118. Alexandrow, M. G., and Moses, H. L. (1995). Cancer Res. 55, 1452-1457. Andreasen, P. A., Georg, B., Lund, L. R., Riccio, A., and Stacey, S. N. (1990). Mol. Cell. Endocrinol. 68, 1-19. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B., and DeLarco, J. E. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 6264-6268. Arrick, B. A., Korc, M., and Derynck, R. (1990). Cancer Res. 50, 299-303. Arrick, B. A., Lopez, A. R., Elfman, F., Ebner, R., Damsky, C. H., and Derynck, R. (1992).J. Cell Biol. 118, 715-726. Anick, B. A. Grendell, R. L., and Griffin, L. A. (1994). Mol. Cell. Biol. 14, 619-628. Arteaga, C. L., Tandon, A. K., Von Hoff, D. D., and Osborne, C. K. (1988). Cuncer Res. 48, 3898-3904. Arteaga, C. L., Coffey, R. J. Jr., Dugger, T.C., McCutchen, C. M., Moses, H. L., and Lyons, R. M. (1990). Cell Growth Differ. 1, 367-374. Arteaga, C. L., Carty-Dugger, T., Moses, H. L., Hurd, S. D., and Pietenpol, J. A. (1993a). Cell Growth Differ. 4, 193-201. Arteaga, C. L., Hurd, S. D., Winnier, A. R., Johnson, M. D., Frendly, B. M., and Forbes, J. T. (1993b). J. Clin. Invest. 92, 2569-2576. Ayanlar Baturnan, O., Ferrero, A. P., Diaz, A., and Jimenez, S. A. (1991). 1. Clin. Invest. 88, 1574-1 580.

88

Katri Koli and Jorma Keski-Oia

Bather, M., Rausch, U., Goebel, H. W., Polzar, B., Mannherz, H. G., and Aumuller, G . (1993). Exp. Clin. Endocrinol. 101, 78-86. Barcellos-Hoff, M. H., Derynck, R., Tsang, M. LA., and Weatherbee, J. A. (1994). /. Clin. Invest. 9, 892-899. Barrett-Lee, P., Travers, M., Luqmani, Y., and Coombes, R. C. (1990). Br. J, Cancer 61,612617. Bascorn, C. C., Wolfshohl, J. R., Coffey, R. J. Jr., Madisen, L., Webb, N. R., Purchio, A. R., Derynck, R., and Moses, H. L. (1989). Mol. Cell Biol. 9,5508-5515. Beato, M., Chalepakis, G., Schauer, M., and Slater, E. P. (1989).J. Steroid. Biochem. 32,737747. Benz, D. J., Haussler, M. R., Thomas, M. A., Speelman, B., and Komm, B. S. (1991). Endocrinology 128, 2723-2730. Bikle, D. D. (1992). Endocr. Rev. 13, 765-784. Bikle, D. D., and Pillai, S. (1993). Endocr. Rev. 14, 3-19. Blei, F., Wilson, E. L., Mignatti, P., and Riflcin, D. B. (1993).J. Cell. Physiol. 155, 568-578. Brown, P. D., Wakefield, L. M., Levinson, A. D., and Sporn, M. B. (1990). Growth Factors 3, 35-43. Bruner, K. L., Rodgers, W. H., Gold, L. I., Korc, M., Hargrove, J. T., Matrisian, L. M., and Osteen, K. G. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 7362-7366. Bruzdzinski, C. J., Johnson, M. R., Goble, C. A., Winograd, S. S., and Gelehrter, T. D. (1993). Mol. Endocrinol. 7, 1169-1177. Butta, A., MacLennan, K., Flanders, K. C., Sacks, N. P., Smith, I., McKinna, A., Dowsett, M., Wakrfield, L. M., Sporn, M. B., Baum, M., et al. (1992). Cancer Res. 52,4261-4264. Circamo, J., Zentella, A., and Massagut, J. (1995). Mol. Cell. Biol. 15, 1573-1581. Carlberg, C., Bendik, I., Wyss, A., Meier, E., Sturzenbecker,L. J., Grippo, J. F., and Hunziker, W. (1993).Nature (London), 361, 657-660. Casslen, B., Urano, S., and Ny, T. (1992). Thromb. Res. 66, 75-87. Centrella, M., McCarthy, T. L., and Canalis, E. (1987).1.Biol. Chem. 262, 2869-2874. Cheifetz, S., Weatherbee, J. A., Tsang, M. L.-S., Anderson, J. K., Mole, J. E., Lucas, R., and Massagui, J. (1987). Cell 48, 409-415. Cheifetz, S., Andres, J. L., and MassaguC, J. (1988a).J. Biol. Chem. 263, 16984-16991. Cheifetz, S., Ling, N., Guillemin, R., and MassaguC, J. (1988b).J. Biol. Chem. 263, 1722517228. Cheifen, S., Bellon, T., Calks, C., Vera, S., Bernabeu, C., Massagui, J., and Letarte, M. (1992). J. Biol. Chem. 267, 19027-19030. Chen, A. R., and Rohrschneider, L. R. (1993). Blood 81,2539-2546. Chen, R.-H.,Miettinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995). Nature (London),377,548-552. Choi, Y., and Fuchs, E. (1990).Cell Regul. 1,791-809. Coffey, R. J. Jr., Sipes, N. J., Bascom, C. C., Graves-Deal, R., Pennington, C. Y., Weissman, B. E., and Moses, H. L. (1988). Cancer Res. 48, 1596-1602. Colletta, A. A., Wakefield, L. M., Howell, F. V., van Roozendal, K. E. P., Danielpour, D., Ebbs, S. R., Sporn, M. B., and Baum, M. (1990). J. Cancer 62,405-409. Colletta, A. A., Wakefield, L. M., Howell, F. V., Danielpour, D., Baum, M., and Sporn, M. B. (1991).J. Clin. Invest. 87, 277-283. Daly, R. J., King, R. J. B., and Darbre, P. D. (1990).J. Cell. Biochem. 43, 199-211. Danielpour, D., and Sporn, M. B. (1990).J. Biol. Chem. 265, 6973-6977. Danielpour, D., Kim, K. Y., Winokur, T. S., and Sporn, M. B. (1991).J.Cell. Physiol. 148,235244. Danielsen, M., Hinck, L., and Ringold, G. M. (1989). Cell 57, 1131-1138. Danra, K., Andreasen, P., Grrandahl-Hansen,J., Kristensen, K., Nielsen, L., Skriver, L. (1985). Adu. Cancer Res. 44, 139-266.

Steroid Regulation of TGF-(3 lsoforms

89

Daopin, S., Piez, K. A., Ogzwa, Y., and Davies, D. R. (1992).Science 257, 369-372. de Boland, A. R., and Nemere, I. (1992).J. Cell. Biocbem. 49,32-36. Dugger, T. C., Meggouh, F., and Arteaga, C. L. (1994). Proc. Am. Assoc. Cancer Res. 35, 265. Duffy, M. J., Reilly, D., O’Sullivan, C., O’Higgins, N., Fenelly, J. J., and Andreasen, P. (1990). Cancer Res. 50, 6827-6829. Espevik, T., Figari, I. S., Shalaby, M. R., Lackides, G. A., Lewis, G. D., Shepard, M., and Palladino, M. A. Jr. (1987).J. Exp. Med. 166, 571-576. Evans, R. M. (1988).Science 240, 889-895. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J.-Y., and Livingstone, D. M. (1993). Cell 73,487-497. Fava, R. A., and McClure, D. B. (1987).J. Cell. Pbysiol. 131, 184-189. Feige, j.-J.,Cochet, C., Rainey, W. E., Madani, C., and Chambaz, E. M. (1987).J. Biol. Chem. 262, 13491-13495. Ferguson, E. L., and Anderson, K. V. (1992). Cell 71, 451-461. Flaumenhaft, R., Abe, M., Sato, Y., Miyazono, K., Harpel, J., Heldin, C.-H., and Rifkin, D. B. (1993).J. Cell Biol. 120, 995-1002. Foekens, J. A., Schmitt, M., van Putten, W. L. J., Peters, H. A., Kramer, M. D., Janicke, F., and Klijn, J. G. M. (1994).J. Clin. Oncol. 12, 1648-1658. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H., and Miyazono, K. (1993).Cell 75, 681-692. Freeman, S. N., Rennie, P. S., Chao, J., Lund, L. R., and Andreasen, P. A. (1990). Biocbem. J. 269, 189-193. Fynan, T.M., and Reiss, M. (1993). Crit. Ra! Oncog. 4, 493-540. Gamble, J. R., and Vadas, M. A. (1988).Science 242, 97-99. Geng, Y., and Weinberg, R. A. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 10315-10319. Gerwin, B. I., Keski-Oja, J., Seddon, M., Lechner, J. F., and Harris, C. C. (1990).Am.J. Physiol. 259, L262-L269. Glick, A. B., Flanders, K. C., Danielpour, D., Yuspa, S. H., and Sporn, M. B. (1989).CeflRegul. 1, 87-97. Glick, A. B., Danielpour, D., Morgan, D., Sporn, M. B., and Yuspa, S. H. (1990). Mol. Endocrinol. 4,46-52. Glick, A. B., Kulkarni, A. B., Tennenbaum, T., Hennings, H., Flanders, K. C., O’Reilly, M., Sporn, M. B., Karlsson, S., and Yuspa, S. H. (1993).Proc. Natl. Acad. Sci. U.S.A.90,60766080. Glick, A. B., Lee, M. M., Darwiche, N., Kulkarni, A. B., Karlson, S., and Yuspa, S. H. (1994). Genes Dev. 8,2429-2440. Gorsch, S. M., Memoli, V. A., Stukel, T. A., Gold, L. I., and Arrick, B. A. (1992). Cancer Res. 52,6949-6952. Hamilton, J. A., Whitty, G. A., Whojta, J., Gallichio, M., McGrath, K., and Ianches, G. (1993). Cell. Immunol. 152, 7-17. Hannon, G. J., and Beach, D. (1994). Nature (London),371,257-261. Heaton, J. H., Kathju, S., and Gelehrter, T. D. (1992).Mol. Endocrinol. 6, 53-60. Heino, J., and MassaguC, J. (1990).J. Biol. Cbem. 265, 10181-10184. Henderson, B. R., and Kefford, R. F. (1993). BY.J. Cancer 67, 99-101. Hennings, H., and Holbrook, K. A. (1983). Exp. Cell Res. 143, 127-142. Howlett, A. R., and Bissell, M. J. (1993). Epithelial Cell Biol. 2, 79-89. Hunter, T. (1993).Cell 75, 839-841. Ignotz, R. A., and Massaguk, J. (1985). PYOC.Natl. Acad. Sci. U.S.A. 82, 8530-8534. Ishikawa, O., LeRoy, E. C., and Trojanowska, M. (1990).J. Cell Physiol. 145, 181-186. Jakowlew, S. B., Dillard, P. j., Sporn, M. B., and Roberts, A. B. (1988). Mol. Endocrinol. 2, 1186-1 195.

90

Katri Koli and lorma Keski-Oja

Jakowlew, S. B., Cubert, J., Danielpour, D., Sporn, M. B., and Roberts, A. B. (1992).1. Cell. Physiol. 150, 377-385. Jeng, M.-H. and Jordan, V. C. (1991). Mol. Endocrinol. 5, 1120-1128. Jeng, M.-H., ten Dijke, P., Iwata, K. K., and Jordan, V. C.'(1993).Mol. Cell Endocrinol. 97, 115-123. Jeng, M.-H., Jiang, S. Y., and Jordan, V. C. (1994). Cancer Lett. 82, 123-128. Jhappan, C., Geiser, A. G., Kordon, E. C., Bagheri, D., Hennighausen, L., Roberts, A. B., Smith, G. H., and Merlino, G. (1993).EMBO J. 12, 1835-1845. Jia, X. C., Ng. T., Hsueh, A. J. (1990).Mof. Cell Endocrinol. 68, 143-151. Josso, N. (1992). Semin. Perinatol. 16,279-288. Joyce, M. E., Roberts, A. B., Sporn, M.B., and Bolander, M. E. (1990).]. Cell. Biol. 110,21952207. Jurutka, P. W., Terpening, C. M., and Haussler, M. R. (1993). Biochemistry 32, 8184-8192. Karin, M., Yang-Yen, H.-F., Chambard, J.-C., Deng, T., and Saatcioglu, F. (1993). Eur. J. Clin. Pharmucol. 45(Suppl I), S9-S15. Kasperk, C., Fitzsimmons, R., Strong, D., Mohan, S., Jennings, J., Wergedal, J., and Baylink, D. (1990).1. Clin. Endocrinol. Metab. 71, 1322-1329. Kawabata, M., Chytil, A., Engel, M., Miyazono, K., and Moses, H. L. (1995).Abstracts of the 9th International Conference on Second Messengers and Phosphoproteins, #590. Kehrl, J. H., Thevenin, C., Rieckmann, P., and Fauci, A. S. (1991).J. Immunol. 146, 40164023. Keller, J. R., Mantel, C., Sing, G. K., Ellingsworth, L. R., Ruscem, S. K., and Ruscetti, F. W. (1988).J. Exp. Med. 168, 735-750. Keller, J. R., Jacobsen, S. E. W., Still, K. T., Ellingsworth, L. R., and Ruscetti, F. W. (1991).Proc. Natl. Acad. Sci. U.S.A. 88, 7190-7194. Keski-Oja, J., Blasi, F., Leof, E. B., and Moses, H. L. (1988).J. Cell Biol. 106,451-459. Kim, S.-J., Park, K., Koeller, D., Kim, K. Y., Wakefield, L. M., Sporn, M. B., and Roberts, A. B. (1992).J. Biol. Chem. 267,13702-13707. Kimchi, A., Wang, X.-F., Weinberg, R. A., Cheifetz, S., and MassaguC, J. (1988).Science, 240, 196-198. King, R. B. J., Wang, D. Y., Daly, R. J., and Darbre, P. D. (1989).J. Steroid. Biochem. 34,133138. Kingsley, D. M. (1994). Genes Dev. 8, 133-146. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992). Nature (London) 355,446-449. Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kasid, A., Derynck, R., and Dickson, R. B. (1987). Cell 48,417-428. Knabbe, C., Klein, H., Zugmaier, G., and Voigt, K. D. (1993).J. Steroid Biochem. Mol. Biol. 47,137-142. Koff, A., Ohtsuki, M., Polyak, K., Roberts, J. M., and MassaguC, J. (1993).Science, 260,536539. Koike, M., Ishino, K., Huh, H.-H., and Kuroki, T. (1994). Biochem. Biophys. Res. Commun. 201, 673-681. Kojima, S., Nara, K., and Rifkin, D. B. (1993).J. Cell Biol. 121, 439-448. Kojima, S., and Rifkin, D. B. (1993).J, Cell Physiol. 155, 323-332. Koli, K., and Keski-Oja, J. (1991). J. Cell Bio. 115(2), 224a. Koli, K., and Keski-Oja, J. (1993a). Growth Factors 8, 153-163. Koli, K., and Keski-Oja, J. (1993b). J. Invest. Dermatol. 101, 706-712. Koli, K., and Keski-Oja, J. (1995). Cancer Res. 55, 1540-1546. Koli, K., and Keski-Oja, J. (1996). J. Invest. Dermatol. Symp. Proc. 1, 33-38. Koli, K., and Arteaga, C . L. (1996)./. Mammary Gland Biol. Neoplasia, 1, 371-378, 1996. Komm, B. S., Terpening, C. M., Benz, D. J., Graeme, K. A., Gallegos, A., Korc, M., Greene, G. L., O'Malley, B. W., and Hauuler, M. R. (1988). Science 241, 81-84.

Steroid Regulation of TGF-p Isoforms

91

Kondaiah, P., Sands, M. J., Smith, J. M., Fields, A., Roberts, A. B., Sporn, M. B., and Melton, D. A. (1990).J.Biol. Chem. 265, 1089-1093. Krane, S. M. (1993). Br. /. Rheumatol. 32 (Suppl.), 3-5. Kratzschmar, J., Haendler, B., Kojima, S., Rifkin, D. B., and Schleuning, W. D. (1993). Gene 125,177-183. Krust, A., Kastner, P., Petkovich, M., Zelent, A., and Chambom, P. (1989).Proc. Natl. Acad. Sci. U.S.A. 86, 5310-5314. Kulkarni, A. B., Huh, C.-G., Becker, D., Geiser, A., Lyght, M., Flanders, K. C., Roberts, A. B., Sporn, M. B., Ward, J. M., and Karlsson, S. (1993).Proc. Natl. Acad. Sci. U.S.A. 90,770774. Kurokawa, M., Lynch, K., and Podolsky, D. K. (1987).Biochem. Biophys. Res. Commun. 142, 775-782. Kuruvilla, A. P., Shah, R., Hohwald, G. M., Liggitt, H. D., Palladino, M. A., and Thorbecke, G. J. (1991).Proc. Natl. Acad. Sci. U.S.A. 88, 2918-2921. Kyprianou, N., and Isaacs, J. T. (1989).Mol. Endocrinol. 3, 1515-1522. Lafyatis, R., Thompson, N. L., Remmers, E. F., Flanders, K. C., Roche, N. S., Kim, S.-J., Case, J. P., Sporn, M. B., Roberts, A. B., and Wilder, R. L. (1989).J. Immunol. 143, 1142-1148. Laiho, M., and Keski-Oja, J. (1989). Cancer Res. 49, 2533-2553. Laiho, M., Saksela, O., Andreasen, P. A., and Keski-Oja,J. (1986a).J. Cell Biol. 103, 24032410. Laiho, M., Saksela, O., and Keski-Oja, J. (1986b).Exp. Cell Res. 164, 399-407. Laiho, M., DeCaprio, J. A., Ludlow, J. W., Livingston, D. M., and Massagut, J. (1990a). Cell 62, 175-185. Laiho, M., Weis, F. M. B., and Massagut, J. (1990b)./. Biol. Chem. 265, 18518-18524. Lea, A. P., and Goa, K. L. (1996).Clin. Immunotherapeutics 5, 230-248. Lee, M. M., and Donahoe, P. K. (1993).Endocr. Rev. 14, 152-164. Leof, E. B., Proper, J. A., Goustin, A. S., Shipley, G. D., DiCorleto, P. E., and Moses, H. L. (1986). Proc. Natl. Acad. Sci. U.S.A. 83,2453-2457. Lian, J. B., Stein, G. S., Bortell, R., and Owen, T. A. (1991).J. Cell Biochem. 45, 9-14. Lin, H. Y., Wang, X.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992).Cell 68,775785. Ling, N., Ying, S. Y., Ueno, N., Esch, F., Denoroy, L., and Guillemin, R. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 7217-7221. Lopez-Cassillas,F., Wrana, J. L., and Massagut, J. (1993). Cell 73, 1435-1444. Lots, M., Kekow, J., and Carson, D. A. (1990).J. Immunol. 144,4189-4192. Lyons, R. M., Keski-Oja, J., and Moses, H. L. (1988)./. Cell Biol. 106, 1597-1605. Lyons, R. M., Gentry, L., Purchio, A. F., and Moses, H. L. (1990). /. Cell Biol. 110, 13611367. Makela, T., Alitalo, R., Paulsson, Y., Westermarck, B., Heldin, C.-H., and Alitalo, K. (1987). Mol. Cell. Biol. 7, 3656-3662. Masui, T., Wakefield, L. M., Lechner, J. F., LaVeck, M. A., Sporn, M. B., and Harris, C. C. (1986). Proc. Natl. Acad. Sci. U.S.A. 83,2438-2442. Mathews, L. S., and Vale, W. W. (1991).Cell 65, 973-982. Mathews, L. S., Vale, W. W., and Kinter, C. R. (1992). Science 255, 1702-1705. Matsumoto, K., Hashimoto, K., Nishida, Y., Hashiro, M., and Yoshikawa, K. (1990).Biochem. Biophys. Res. Commun. 166, 916-923. Matsumoto, K., Azuma, Y., Kiyoki, M., Okumura, H., Hashimoto, K., and Yoshikawa, K. (1991).Biochim. Biophys. Acta 1092, 311-318. McAllister, K. A.., Grogg, K. M., Johnson, D. W., Gallione, C. J., Baldwin, M. A., Jackson, C. E., Helmbold, E. A., Markel, D. S., McKinnon, W. C., Murrell, J., McCormick, M. K., Pericak-Vance, M. A., Heutink, P., Oostra, B. A., Haitjema, T., Westerman, C. J. J., Portenous, M. E., Guttmacher, A. E., Letarte, M., and Marchuk, D. A. (1994).Nature Genet. 8, 354-351.

92

Katri Koli and Jorma Keski-Oia

McCaffery, P., and Drager, U. C. (1993).Adv. Exp. Med. Biol. 328, 181-190. McCaffrey, T.A., Falcone, D. J., Brayton, C. F., Agarwal, L. A., Welt, F. G. P., and Weksler, B. B. (1989).J. Cell Biol. 109, 441-448. McCaffrey, T. A., Falcone, D. J., Vicent, D., Du, B., Consigli, S., and Borth, W. (1994).J. Cell. Physiol. 159, 51-59. Medh, R. D., Santell, L., and Levin, E. G. (1992). Blood 80, 981-987. Menon, G. K., Grayson, S., and Elias, P. M. (1985).J. Invest. Dematol. 84, 508-512. Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991).EMBOJ. 10,1091-1101. Morin, A., Olofsson, A., Stenman, G., Sahlin, P., Kanzaki, T., Claesson-Welsh, L., ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1994).J. Biol. Chem. 269, 32469-32478. Morioka, S., Lazarus, G. S., Baird, J. L., and Jensen, P. (1987).J. Invest. Dermatol. 88,418423. Moses, H. L., Branum, E. B., Proper, J. A., and Robinson, R. A. (1981). Cancer Res. 41,28422848. Moses, H. L., Tucker, R. F., Leof, E. B., Coffey, R. J. Jr., Halper, J., and Shipley, G. D. (1985). In Cancer Cells 3 (I. Feramisco, B. Ozanne, and C. Stiles Eds.), pp. 65-71. Cold Spring Harbor Press, New York. Munc, A., Guyre, P. M., and Holbrook, N. J. (1984). Endocr. Rev. $25-44. Miinger, K., Pietenpol, J. A., Pittelkow, M. R., Holt, J. T., and Moses, H. L. (1992). Cell Growth Differ.3, 291-298. Murphy-Ullrich, J. E., Schultz-Cherry,S., and Hook, M. (1992). Mol. Biol. Cell 2 , 181-188. Nichols, N. R., and Finch, C. E. (1994). Ann. N.Y. Acad. Sci. 746, 145-156. Noda, M., and Camilliere, J. J. (1989). Endocrinology 124, 2991-2994. Norman, A. W., Nemere, I., Zhou, L.-X., Bishop, J. E., Lowe, K. E., Maiyar, A. C., Collins, E. D., Taoka, T., Segreev, I., and Farach-Carson, M. C. (1992).J. Steroid Biochem. Mol. Biol. 41,231-240. Norman, A. W., Bouillon, R., Farach-Carson, M. C., Bishop, J. E., Zhou, L.-X., Muralidharan, K. R., and Okamura, W. H. (1993).J. Biol. Chem. 268,20022-20030. O’Connor-McCourt, M. D., and Wakefield, L. M. (1987).J. Biol. Chem. 262,14090-14099. Ogawa, Y., Schmidt, D. K., Dasch, J. R., Chang, R.-J., and Glaser, C. B. (1992).J. Biol. Chem. 267,2325-2328. O’Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991).J. Biol. Chem. 266,8583-8589. Ohta, M., Greenberger, J. S., Anklesaria, P., Bassols, A., and Massagui, J. (1987). Nature (London) 329,539-541. Olofsson, A., Miyazono, K., Kanzaki, T., Colosetti, P., Engstrom, U., and Heldin, C.-H. (1992). I. Biol. Chem. 267,19482-19488. Olsen, N. J., Zhou, P., Ong, H., and Kovacs, W. J. (1993).J. Steroid Biochem. Mol. Biol. 45, 327-332. Ong, G., Sikora, K., and Gullick, W. (1991). Oncogene 6, 761-763. Oppenheim, J. J., and Neta, R. (1994). FASEB J. 8, 158-162. Oursler, M. J., Riggs, B. L., and Spelsberg, T. C. (1993). Endocrinology 133, 2187-2196. Paralkar, V. M., Vukicevic, S., and Reddi, A. H. (1991). Dev. Biol. 143, 303-308. Pertovaara, L., Sistonen, L., Bos, T. J., Vogt, P. K., Keski-Oja, J., and Alitalo, K. (1989). Mol. Cell. Biol. 9, 1255-1262. Pertovaara, L., Saksela, O., and Alitalo, K. (1993). Growth Factors 9, 81-86. Pierce, D. F. Jr., Johnson, M. D., Matsui, Y., Robinson, S. D., Gold, L. I., Purchio, A. F., Daniel, C. W., Hogan, B. L., and Moses, H. L. (1993). Genes Dev. 7, 2308-2317. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R. M., Pittelkow, M. R., Miinger, K., Howley, P. M., and Moses, H. L. (1990). Cell 61, 777-785. Pillai, S., and Bikle, D. D. (1991).J, Cell Physiol. 146, 94-100. Pillai, S., Bikle, D. D., and Elias, P. M. (1988).J. Biol. Chem. 263, 5390-5395. Pollanen, J., Stephens, R. V., and Vaheri, A. (1991). Adv. Cancer Res. 57,273-328.

Steroid Regulation of TGF-P lsoforms

93

Postlethwaite, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987).J. Exp. Med. 165, 25 1-256. Rache, M. K., Dhib-Jalbut, S., Cannella, B., Albert, P. S., Raine, C. S., and McFarlin, D. E. (1991).J. Immunof. 146, 3012-3017. Reibman, J., Meixler, S., Lee, T. C., Gold, L. I., Cronstein, B. N., Haines, K. A., Kolasinski, S. L., and Weissmann, G. (1991).Proc. Natl. Acud. Sci. U.S.A. 88, 6805-6809. Ridley, A. J., Davis, J. B., Stroobant, P., and Land, H. (1989).J. Cell Biol. 109, 3419-3424. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982).J. Biol. Chem. 257, 2920-2925. Roberts, A. B., and Sporn, M. B. (1992). Cancer Surv. 14, 205-220. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., and Sporn, M. B. (1981).Proc. Natl. Acad. Sci. U.S.A. 78,5339-5343. Robinson, S. D., Silberstein, G. B., Roberts, A. B., Flanders, K. C., and Daniel, C. W. (1991). Development 113,867-878. Robinson, S. D., Roberts, A. B., and Daniel, C. W. (1993).J. Cell Biol. 120, 245-251. Rosen, E. D., Beninghof, E. G., and Koenig, R. J. (1993).J. Biol. Chem. 268, 11534-11541. Rosen, V., and Thies, R. S. (1992).Trends Genet. 8, 97-102. Saez, S., Faletta, N., Guillot, C., Meggouh, F., Lefebvre, M.-F., and Crepin, M. (1993). Cancer Res. Treat. 27, 69-81. Saharinen, J., Taipale, J., and Keski-Oja, J. (1996). EMBO J. 15, 245-253. Saksela, O., and Rifkin D. B. (1988).Annu. Rev. Cell Biol. 4, 93-126. Samuel, S. K., Hurta, R. A. R., Kodaiah, P., Khalil, N., Turley, E. A., Wright, J. A., and Greenberg, A. H. (1992).EMBO J. 11, 1599-1605. Sanderson, N., Nagy, P., Murakami, H., Kondaiah, P., Roberts, A. B., Sporn, M. B., and Thorgeirsson, S. S., (1993).In: Regulation of Liver Gene Expression in Health and Disease. Cold Spring Harbor Laboratory Press, New York. Schlunegger, M. P., and Griitter, M. G. (1992). Nature (London) 358, 430-434. Schrader, M., Miiller, K. M., and Carlberg, C. (1994a).J. Biol. Chem. 269,5501-5504. Schrader, M., Miiller, K. M., Nayeri, S., Kahlen, J.-P., and Carlberg, C. (1994b). Nature (London) 370,382-386. Schule, R., Umensono, K., Mangelsdorf, D. J., Bolano, J., Pike, J. W., and Evans, R. M. (1990). Cell 61,497-504. Schull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D.,. Annunziata, N., and Doetschman, T. (1992).Nature (London) 358,693-699. Schultz-Cherry, S., Lawler, J., and Murphy-Ullrich, J. E. (1994a).J. Biol. Chem. 269,2678326788. Schultz-Cherry, S., Ribeiro, S., Gentry, L., and Murphy-Ullrich, J. E. (1994b).J. Biol. Chem. 269,26775-26782. Segarini, P. R., Ziman, J. M., Kane, J. M., and Dasch, J. R. (1992).J. Biol. Chem. 267, 10481053. Sellheyer, K., Bickenbach, J. B., Rothnagel, J. A., Bundman, D., Longley, M. A., Krieg, T., Roche, N. S., Roberts, A. B., and Roop, D. R. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 5237-524 1. Studzinski, G . P., McLane, J. A., and Uskokovic, M. R. (1993). Crit. Rev. Eukaryotic Gene Expression 3, 279-312. Su, M. J., Bikle, D. D., Mancianti, M. L., and Pillai, S. (1994).J. Biol. Chem. 269, 1472314729. Suda, T., Shinki, T., and Takahashi, N. (1990). Annu. Rev. Nutr. 10, 195-211. Taipale, J., and Keski-Oja, J. (1996).J. Biol. Chem. 271, 4342-4348. Taipale, J., Koli, K., and Keski-Oja, J. (1992).J. Biol. Chem. 267, 25378-25388. Taipale, J., Matikainen, S., Hurme, M., and Keski-Oja, (1994a). Cell Growth Difer. 5, 13091319. Taipale, J., Miyazono, K., Heldin, C.-H., and Keski-Oja, J. (1994b).J. CellBiol. 124,171-181.

94

Katri Koli and Jorma Keski-Oja

Taipale, J., Lohi, J., Saarinen, J., Kovanen, P. T., and Keski-Oja,J. (1995a).J. Biol. Chem. 270, 4689-4696. Taipale, J., Saharinen, J., Hedman, K., and Keski-Oja, J. (199Sb) Mol. Biol. Cell S(supp1.) 3 11A. Tashjian, A. H., Voelkel, E. F., Lazzaro, M., Singer, F. R., Roberts, A. B., Derynck, R., Winkler, M. E., and Levine, L. (1985). Proc. Nutl. Acud. Sci. U.S.A. 82,4535-4538. Tempel, D. L., and Leibowitz, S. F. (1994).1.Neuroendocrinol. 6, 479-501. Thompson, A. M., Kerr, D. J., and Steel, C. M. (1991). Br.]. Cancer 63,609-614. Thompson, E. A., Nelles, L., and Collen, D. (1991). Eur. J. Biochem. 201, 627-632. Tienari, J., Alanko, T., Lehtonen, E., and Saksela, 0. (1991). Cell Regul. 2, 285-297. Towers, T. L., Luisi, B. F., Asianov, A., and Freedman, L. P. (1993).Proc. Nutl. Acud. Sci. U.S.A. 90,6310-6314. Tryggvason, K., Hoytya, M., and Salo, T. (1986). Biochim. Biophys. Actu 907, 191-217. Tsipouras, P., Del Mastro R., Sarfarazi, M., Lee, B., Vitale, E., Child, A., Godfrey, M., Devereux, R., Hewett, D., Steinman, B., Viljoen, D., Sykes, B. C., Kilpatrick, M., and Ramirez, F. (1992). New Engl. J. Med. 326, 905-909. Umensono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991). Cell 65,12551266. Vassalli, J.-D., Sappino, A.-P., and Belin, D. (1991).J. Clin. Invest. 88, 1067-1072. Virji, M. A. G., Vassalli, J. D., Estensen, R. D., and Reich, E. (1980). Proc. Nutl. Acud. Sci. U.S.A. 77, 875-879. Wahl, S. M. (1992).J. Clin. Immunol. 12, 61-74. Wahl, S. M., Hunt, D. A., Wakefield, L. M., McCartney-Francis, N., Wahl, L. M., Roberts, A. B., and Sporn, M. B. (1987). Proc. Nutl. Acud. Sci. U.S.A. 84,5788-5792. Wakefield, L., Kim, S.-J., Glick, A., Winokur, T., Colletta, A., and Sporn, M. B. (1990).J. Cell Sci. Suppl. 13, 139-148. Wallick, S . C., Figari, I. S., Morris, R. E., Levinson, A. D., and Palladino, M. A. (1990).J. Exp. Med. 172,1777-1784. Waltenberger,J., Wanders, A., Fellstrom, B., Miyazono, K., Heldin, C.-H., and Funa, K. (1993). J. Immunol. 151, 1147-1157. Wang, J., Kuliszewski, M., Yee, W., Sedlackova,L., Xu, J., Tseu, I., and Post, M. (1995).J. Biol. Chem. 270,2722-2728. Wang, T., Donahoe, P. K., and Zervos, A. S. (1994). Science 265,674-676. Warri, A. M., Huovinen, R. L., Laine, A. M., Martikainen, P. M., and Harkonen, P. L. (1993). J. Nutl. Cancer Inst. 85, 1412-1418. Weeks, D. L., and Melton, D. A. (1987). Cell 51, 861-867. Welch, D. R., Fabra, A., and Nakajima, M. (1990). Proc. Nutl. Acud. Sci. U.S.A. 87,76787682. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994). Nature (London) 370,341-347. Wun, T.-C., Ossowski, L., and Reich, E. (1982).J. Biol. Chem. 257, 7262-7268. Yamaguchi, Y., Mann, D. M., and Ruoslahti, E. (1990). Nature (London) 346, 281-284. Yamashita, J., Inada, K., Yamashita, S., Matsuo, S., Nakashima, Y., and Ogawa, M. (1992). Horm. Metab. Res. 24, 565-569. Yamashita, H., Ichijo, H., Grimsby, S., Morkn, A., ten Dijke, P., and Miyazono, K. (1994).J. Biol. Chem. 269,1995-2000. Yin, W., Smiley, E., Germiller, J., Mecham, R. P., Florer, J. B., Wenstrup, R. J., and Bonadio, J. (1995)I. Biol. Chem. 270,10147-10160. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, 0. V., Naar, A. M., Kim, S. Y., Boutin, J.-M., Glass, C. K., and Rosenfeld, M. G. (1991).Cell 67, 1251-1266. Zilliacus, J., Wright, A. P. H., Carlstedt-Duke, J., and Gustafsson, J.-A. (1995). Mol. Endocrinol. 9, 389-400.

c+Myc in the Control of Cell Proliferation and Embryonic Development lean-Marc Lemaitre,' Robin S.Buckle,* and Marcel MCchali' 'lnstitut]. Monod CNRS, 75251 Paris cedex 05, France, and 2The Randall Institute, King's College London, London WC2 SBRL, England

1. Introduction 11. The c-myc Gene A. Discovery of the c-myc Gene B. Family Members C. Promoters and Transcripts D. Protein Products Ill. Structural and Functional Features of the c-Myc Protein A. Transforming Domain B. Autoregulation Domain C. Nuclear Localization D. Transactivation and Transcriptional Repression Domains E. DNA-Binding Domains F. Interaction with Other Proteins IV. c-Myc as a Transcription Factor A. c-Myc as a Transcriptional Activator B. c-Myc as a Transcriptional Repressor V. c-Myc and Cell Proliferation A. c-Myc Is Required Throughout the Cell Cycle B. Signal Transduction and Induction of c-myc Transcription C. Cell Cycle Targets of c-Myc D. c-Myc and DNA Replication VI. c-Myc in Embryonic Development VII. c-Myc and Differentiation VIII. c-Myc and Apoptosis A. Induction of Apoptosis or Proliferation Appears Mechanistically Related B. c-Myc and pS3 in Apoptosis C. c-Myc and bcl-2 in Apoptosis D. Apoptosis or Proliferation? References

Advances in CANCER RESEARCH, Vol. 70 Copyright 8 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

96

Jean-MarcLemaitre et al.

I. INTRODUCTION Much of the recent progress in understanding the mechanisms of cell growth has highlighted the critical importance of checkpoints in guaranteeing that each step of the cell cycle is carried out in a precise order. Embryonic development requires cellular multiplication coupled to diversification of the genetic program, and therefore additional levels of control are required to provide a tight coordination between proliferation and differentiation. In addition, during either embryogenesis or adult life, a precise balance between cell division and cell death governs the establishment and integrity of tissues. It appears that several proto-oncogenes are involved in the control of these processes. C-myc was the first nuclear proto-oncogene discovered and has been the subject of intense investigation during the past 16 years. The initial property attributed to c-myc was its ability to transform normal cultured cells, but a great deal of evidence since then has indicated that this proto-oncogene is involved in a variety of different cellular processes, such as proliferation, differentiation, and apoptosis. The current focus of investigation into c-myc is therefore primarily concerned with deciphering how one gene can both participate in and discriminate between these seemingly divergent activities. We review here recent knowledge on the cell cycle regulation of c-myc expression and c-Myc activity, as well as structural features of the c-Myc protein that give new insights into the understanding of c-Myc function during the cell cycle and embryonic development.

11.

THE c-myc GENE

A. Discovery of the c-myc Gene The c-myc proto-oncogene was characterized as the cellular homolog of the gene borne by the retrovirus MC29 responsible for a chicken leukemia (Roussel et af., 1979; Sheiness and Bishop, 1979; Sheiness et al., 1980). For a long time, it was described as a gene specific to the vertebrate phylum, where it has been identified in several species ranging from fish and amphibians to humans (Kato and Dang, 1992; see Fig. 1). However, it has more recently been identified in a nonvertebrate organism, the sea star (Walker et af., 1992), and indirect evidence suggests that a related protein might exist in insects (Papoulas et af., 1992; Lemaitre et af., 1994). Surprisingly, despite several attempts, no related myc sequences were cloned in Drosophifa. A cDNA clone exhibiting some similarities to c-myc has also been isolated

97

c-Myc in Cell Cycle and Embryonic Development

HOMOLOGY

SPECIES 439

1

Human

100%

Mouse

92%

Chicken

71 %

Xenopus 1

68%

Trout

64%

Zebra Fish

61 %

Sea Star

46%

II

MYC FAMILY

N-Myc (human)

43% (with human c-Myc)

L-Myc (human)

41 % (with human c-Myc)

B-Myc (rat)

67% (with rat C-MYC) 30% (with rat C-Myc)

S-Myc (rat) 50-90%

of homology

> 90% of homology

Fig. 1 Sequence homologies of the c-Myc protein in different species, as compared to human c-Myc (Bernard et al., 1983; Stanton et al., 1984; Van Beneden et al., 1986; Vriz et al., 1989; Walker et al., 1992; Schreiber-Agus et al., 1993). A comparison to the other Myc family members is also shown (Kohl et al., 1986; DePinho et al., 1987; Ingvarsson et al., 1988; Sugiyama et al., 1989). The bars represent Myc amino acid sequence: black bars indicate regions of 90-100% amino acid sequence identity, grey bars regions of 50-90% sequence identity, and white bars regions containing less than 50% sequence identity. Sequences are from Genbank. Modified from Kato, G. J., and Dang, C. V. (1992). FASEB J.

98

lean-Marc Lemaitre et al.

from the eastern oyster and could be derived from the same ancestral gene that gave rise to vertebrate c-myc (Marsh and Chen, 1995). The structure of the c-myc gene is conserved between species, consisting of three exons (Fig. 2). An open reading frame (ORF) in exons 2 and 3 encodes the c-Myc protein, which normally exists in two forms (p64 and p67) in humans. This gene is well conserved from sea star to human, exhibiting 46% sequence identity at the amino acid level, while certain regions exhibit 90-100% identity among all species and permit the definition of boxes of strong homologies, indicating they play an essential role in c-Myc function. The three boxes (A, B, and C) present in the N-terminal part of the protein have already been described (Van Beneden et al., 1986; Kato et al., 1990). The others are in the middle part of the protein and in the B-HLH domain (Fig. 1).

B. Family Members C-Myc is one of three highly related proteins expressed in eukaryotic cells. The other Myc family members were initially identified as genes possessing an amplified copy number in two specific transformed cell types, N-myc from human neuroblastomas (Schwab et a1.,1983) and L-myc from lung carcinomas (Nau et al., 1985). As well as possessing sequence homology, these genes were found to be structurally similar to c-myc, with the corresponding proteins being encoded by exons 2 and 3. Highly homologous regions have been conserved in all three proteins (see Fig. 1). Two further family members have since been isolated form a rat genomic library, termed B-myc and S-myc (Ingvarsson et al., 1988; Sugiyama et al., 1989). B-myc exhibits sequence homology with exon 2 of the c-myc gene and encodes for a polypeptide of 168 amino acids, while S-myc has sequence similarity with exons 2 and 3 of c-myc, although in this case the ORF is present as one contiguous exon. These genes have not been well characterized, but ectopic expression of both B-myc and S-myc in cell lines has indicated that they are able to antagonize c-Myc activity (Sugiyamaet al., 1989; Resar et al., 1993), although the existence of the corresponding endogenous proteins has not yet been demonstrated in vivo.

C. Promoters and Transcripts Expression of the c-myc gene is regulated at several different levels, encompassing transcriptional initiation and elongation, mRNA stability, and translation (reviewed in Spencer and Groudine, 1991). RNA polymerase I1 initiates c-myc transcription from distinct promoters

99

c-Myc in Cell Cycle and Embryonic Development

I c-myc gene organisation

A

I

c-myc gene Exon 1

B

c-myc gene expression

Promoters

Exon 2

I Proteins

Messengers Size

Proportion

Exon 3

Amino acid number

size

3 , l kb

<5%

439 aa 454 aa 114aa 188 aa

64 KDa 67 KDa 15 KDa 29 KDa

2,s kb

<1%

439 aa 454 aa 114aa

64 KDa 67 KDa 15 KDa

10-25 %

439 aa 454 aa

64 KDa 67 KDa

- 90%

439 aa 454 aa

64 KDa 67 KDa

PO

P1

2,4kb

P2

2,2 kb

75

P3

2,3 kb <5% 439 aa 64 KDa C-myc gene organization (A) and expression (B). The c-myc gene consists of three exons, and the different promoters (PO, P1, P2, P3), translation initiation codons (CUG, AUG), and polyadenylation sites (AAAl, AAA2) are indicated. The size and proportion of the c-myc messenger RNAs produced, as well as the corresponding promoter usage and translation products, are also shown. The major c-myc transcripts and protein products are shown in bold type.

Rg. 2

that are relatively well conserved in evolution (Fig. 2). In the human gene, the major promoters P1 and P2 are separated by 161 bp and give rise to 95% of total c-myc messenger RNA. Another promoter, PO, is located 550 bp upstream of P1 and yields less than 5 % of total c-myc mRNA, while transcription from the P3 promoter found in the human, mouse, and rat

100

Jean-Marc Lemaitre et af.

c-myc intron 1is also detected, again yielding less than 5% of c-myc mRNA. Antisense transcripts from several different sites of the human and mice c-myc genes have also been described in vitro, although their relevance and function in vivo is unknown (Piechaczyk et al., 1988). Another source of mRNA heterogeneity is provided by the presence of two conserved polyadenylation sites, separated by 150 nucleotides, that are found in the human, rat, and chicken c-myc genes. The downstream site is the most commonly used.

D. Protein Products Despite the heterogeneous nature of the transcripts produced from the c-myc gene, one ORF is observed in all mRNA species, spanning 439 amino acids in humans. It starts at an AUG initiation codon located at the 5' end of exon 2 and codes for the major c-Myc protein observed in vivo, of 64 kDa (Hann and Eisenman, 1984; Ramsay et al., 1984). A minor species of 67 kDa is also observed, and is translated from a CUG codon located at the 3' end of exon 1 in frame with the previous ORF. The p67 protein therefore corresponds to an additional 14 amino acids at the N-terminus of the p64 sequence (Hann et al., 1988). Both forms are relatively short-lived nuclear phosphoproteins, and the conserved existence of these two forms in several species would suggest different functions for both proteins. It is noteworthy that in Burkitt lymphomas p64 c-Myc is the only one synthesized, since initiation at the CUG codon is suppressed by a rearrangement. This has led to the notion that the p67 form might have a growth-inhibitory function, a view supported by the observation that there is a 5- to 10-fold increase in p67 synthesis as cells approach quiesence (Hann et al., 1992). However, there appears to be little, if any, functional difference between the p64 and p67 forms (Blackwood et al., 1994). A study as indicated that in certain circumstances they can differ in their abilities to induce transcription (Hann et al., 1994; discussed in Section 1II.A). Two additional ORFs were identified in some of the minor mRNA species. In vitro translation of these transcripts produces corresponding proteins of 114 and 188 amino acids (Bentley and Groudine, 1986). However, the major transcripts from the P1 and P2 promoters do not contain these ORFs, and minor transcripts containing them were identified only in some cell types. These transcripts were localized in polysomes, suggesting their translation in vivo (Bentley and Groudine, 1986a), and a protein product from the 188amino-acid ORF has been identified in HeLa cells, existing as a protein dimer of 58 kDa (Gazin et al., 1986). However, no biological significance has been defined for either ORF.

c-Myc in Cell Cycle and Embryonic Development

101

111. STRUCTURAL AND FUNCTIONAL FEATURES OF THE C-MYCPROTEIN The biochemical analysis of c-Myc activity has been hampered by the extreme difficulty in obtaining a soluble form of the purified full-length protein. This problem has been largely alleviated by the production of soluble fragments of the protein using recombinant DNA technology, and, in conjunction with mutagenesis experiments, this approach has helped to define functional domains within the c-Myc protein (Fig. 3).

A. Transforming Domain Regions necessary for neoplastic transformation were initially identified in rat embryonic fibroblasts (Stone et al., 1987; Sarid et al., 1987). The first 143 N-terminal and the last 89 C-terminal amino acids of the human protein are necessary for cellular transformation. Mapping at higher resolution indicated that amino acids 106-143 are essential for this activity, while some deletions are permissible in the first 91 residues. Mutations in the central region of the protein (amino acids 250-260) decrease but do not abolish the transforming potential, in accordance with the cell type analyzed (Heaney et al., 1986). Naturally occurring mutations at threonine 58, observed in Burkitt lymphoma and in avian v-Myc, can increase the transforming properties of the protein (Rabbitts et al., 1983; Fryberg et al., 1987). Furthermore, site-directed mutagenesis of either threonine 58 or serine 62, which are sites of phosphorylation in the c-Myc N-terminal domain, has also been shown to influence transformation by c-Myc (Henriksson et al., 1993; Pulverer et al., 1994). The protein sequences required for transformation largely overlap those necessary for the inhibition of differentiation (Freytag et al., 1990) and c-Myc-induced apoptosis (Evan et al., 1992), emphasizing regulatory links between these functions.

B. Autoregulation Domain Under physiological conditions, c-Myc can negatively regulate its own expression (Grignani et al., 1990). This autosuppression occurs at the level of transcriptional initiation (Penn et al., 1990a), and protein sequences involved in such a mechanism are identical to those required for cellular transformation and the inhibition of differentiation (Penn et al., 1990b).

CK II .

*

**

..

Functions -Transformation -Autossupression -Differentiation inhibition

3 4 1-1

250 m 2 6 0

355 -439

-Apoptosis

Domain of activity -Nuclear localisation -Transactivation

3 4 1-1

-Transcription repression 92

-Non specific DNA binding

do6 122 265

-Specific DNA binding

-

320

355 1 3 6 8

-Dimerisation with Max 369 -5

-Interaction with YY1 250

435

-Interaction with AP2 I345 - :

439

-Interaction with pl07Rb 41 -176

-Interactionwith TBP -Interaction with Q tubulin

1 -204

48 -134

Fig. 3 Prominent functional and structural features of the human c-Myc protein. The major motifs and phosphorylation sites are indicated at the top. Black bars represent the regions involved in known c-Myc functions, as well as the domains required for interaction with other proteins in vivo. The precise amino acid residues involved are numbered.

c-Myc in Cell Cycle and Embryonic Development

103

C. Nuclear Localization c-Myc is a nuclear protein that has been found to associate with the nuclear matrix in certain instances (Eisenman et al., 1985; Evan and Hancock, 1985; Von Straaten and Rabbitts, 1987). Its exact localization is unknown, although in mitotic cells c-Myc is not bound to chromatin (Winquist et al., 1984). c-Myc contains two different nuclear translocation signals (reviewed in Garcia-Burtos et al., 1991), termed M1 and M2 (Stone et a/., 1987; Dang and Lee, 1988). The M1 region (amino acids 320-328 in humans) is the dominant signal and displays sequence homology with the previously characterized nuclear localization signal (NLS) in the simian virus 40 (SV40)and polyoma T antigens (Kalderon et al., 1984; Richardson et al., 1986). This region is able to direct nuclear accumulation of the normally cytoplasmic pyruvate kinase when fused to it (Dang and Lee, 1988). A similar sequence in N-Myc exhibits the same properties (Dang and Lee, 1989). L-Myc does not contain a related sequence, but does contain the M2 NLS. However, this sequence displays very little activity (Dang and Lee, 1988), suggesting that L-Myc might be localized to nuclei by an alternative mechanism. Deletion of the M2 region in c-Myc (amino acids 364-374 in humans) has no effect on its localization, due to dominance of the M1 NLS.

D. Transactivation and Transcriptional Repression Domains A transactivation domain was first identified by Kato and coworkers (1990), who analyzed the ability of different parts of the c-Myc protein to stimulate transcription when fused to the DNA-binding domain of Gal4 protein. The N-terminal 143 amino acids of c-Myc was the only region able to transactivate a reporter gene containing Gal4 DNA-binding sites in its promoter. This transactivation domain can be divided into three parts. The first (amino acids 1-41 in humans) is acidic and glutamine rich, and displays sequence homologies with the transactivation domains of the human Sp1 and herpesvirus VP16 proteins (Mitchell and Tijan, 1989). The second part (amino acids 42-103 in humans) resembles the CTF1-NF1 transactivation domain (Mitchell and Tijan, 1989) but has low activity. The third part (amino acids 104-143 in humans) was shown to exhibit a strong transactivation activity without exhibiting any sequence homology to other known transactivation domains (Kato et al., 1990). Mutagenesis experiments in the homologous region of N-Myc and L-Myc led to similar phenotypes, indicating that they also contain this transactivation domain. Regions within this N-terminal domain have also been implicated in transcriptional repression. c-Myc mediates this repression through the initiator

104

lean-Marc Lemaitre et al.

region (Inr) DNA sequence element, as found for both the adenovirus major late (Roy et al., 1993; Li et al., 1994) and cyclin D1 (Philipp et al., 1994) promoters (see Section 1II.C). Surprisingly the N-terminal sequences required for this repressive activity have been mapped to different regions, corresponding to residues 122-143 of human c-Myc for transcriptional inhibition at the adenovirus major late promoter (Roy et al., 1993), and residues 92-106 in the case of the cyclin D1 promoter (Philipp et al., 1994). The reasons for this difference are unknown, although different inhibitory mechanisms may be involved in the two cases.

E. DNA-Binding Domains 1. NONSPECIFIC INTERACTION

c-Myc can bind to single- as well as double-stranded DNA (Persson and Leder, 1984; Watt et al., 1984; Beimling et al., 1985). The protein sequence required for this binding is located between amino acids 265 and 318 in human c-Myc and has been shown to bind DNA nonspecifically in vitro (Dang et al., 1989). This region contains four Ser-Pro-X-X motifs (X often corresponds to a lysine), which were initially identified as the residues responsible for nonspecific DNA binding in histone H1 (Churchill and Suzuki, 1989; Suzuki, 1989). It has been proposed that this motif might facilitate the sliding of proteins along DNA until a high-affinity binding site is located (Suzuki, 1989). No function has been attributed to this domain in vivo, although its deletion reduces the ability of c-Myc to transform cells (Stoneet al., 1987), and sequences overlapping this region are required for c-Myc to interact with the transcription factor YY-1 (Shrivastava et al., 1993). 2. DlMERlZATlON AND SEQUENCE-SPECIFIC INTERACTION

The identification of a specific interaction between c-Myc and DNA in 1990-1991 was a significant breakthrough in determining the precise molecular mechanisms underlying its activity. Protein sequence analysis between the Myc family of proteins and related proteins initially led to the identification of three functionally conserved structural motifs: a leucine zipper (LZ) motif (amino acids 411-439 in humans); a helix-loop-helix (HLH) motif (amino acids 368-410 in humans); and a basic (B) motif (amino acids 355-367 in humans). Associated (B-LZ) and (B-HLH) motifs have been found in many sequence-specific DNA-binding proteins, and define two large families of proteins involved in transcriptional control (Fig. 4). The c-Myc protein exhibited particularly strong structural homology to two B-HLH-LZ transcription factors, USF

c-Myc in Cell Cycle and Embryonic Development

I05

B/HLH/U

c-Myc H-Hyc L-Myc Max Mad Mix1

AP-4 USF TFE3 TFEB

Cons.

$

-

L , I , V , M, F

Fig. 4 Amino acid sequences of the DNA-binding and dimerization domains of B-LZ, BHLH, and B-HLH-LZ proteins. Sequence homologies are shaded. The dashes represent spaces inserted into the sequence for purposes of alignment. A consensus sequence is indicated for B-LZ and B-HLH-LZ proteins (cons.).

(Gregor et al., 1990) and TFE3 (Beckmann et al., 1990). These proteins specifically bound the so-called E-box DNA sequence CANNTG, and it was therefore reasoned that the c-Myc basic domain might also recognize an E-box sequence. This was confirmed by two distinct in vitro studies, which identified the c-Myc-binding site as the palindromic sequence 5 '-CACGTG3' (Blackwell et al., 1990; Prendergast and Ziff, 1991). In addition to USF and TFE3, this binding site is also recognized by other Myc family members as well as a growing number of nuclear factors containing the By HLH, and

106

lean-Marc Lemaitre et a/.

LZ motifs in one contiguous region: N-Myc, L-Myc (Alex et al., 1992; Ma et al., 1993), Max (Blackwood and Eisenman, 1991), Mad (Ayer et al., 1993), Mxil (Zervos et al., 1993),TFEB (Carr and Sharp, 1990), and AP-4 (Y.F. Hu et a1.,1990). The presence of both the HLH and LZ motifs within a single protein appears to stabilize protein dimerization and set up interaction specificity (Beckmann and Kadesch, 1991). The activity of c-Myc is dependent upon its specific interaction with DNA. Sequence-specific DNA binding by Myc family proteins requires dimerization with Max, a ubiquitous nuclear protein containing a B-HLH-LZ domain homologous to that found in c-Myc (Blackwood and Eisenman, 1991; Prendergast et al., 1991).Max normally exists in two forms as a result of alternative splicing: p21 Max (151 amino acids) and p22 Max (160 amino acids), which differ by a 9-amino-acid insertion upstream of the basic region. Both forms heterodimerize with c-Myc with similar efficiency, and are expressed at equivalent levels in cultured cells (Blackwood and Eisenman, 1991).Alternatively spliced transcripts encoding other Max isoforms have also been identified, although the corresponding endogenous proteins have not been detected (Makela et al., 1992; Vastrik et al., 1995). These variants are C-terminally truncated but possess the B-HLH-LZ motif and are still able to interact with c-Myc and bind DNA in vitro (Fig. 5).Another splicing variant has been described that lacks the DNA-binding basic region. This protein, termed dMax, can still associate with c-Myc but is unable to bind DNA. dMAX is detected in cell lysates and may function as a dominant negative repressor of c-Myc activity (Arsura et al., 1995). Myc/Max heterodimers and Max/Max homodimers form both in vitro and in vivo and bind the same E-box DNA sequence, CACG(A/T)G (Blackwood et al., 1992; Kato et al., 1992), although bases flanking this core sequence may differentially influence their binding (Fisheret al., 1993; Solomon et al., 1993). Noncanonical sequences can also be bound with high affinity (Blackwellet al., 1993; Hann et al., 1994).Earlier reports indicating that c-Myc could specifically bind to a 7-bp sequence in the human c-myc promoter, in a region containing transcriptional enhancer and replication origin activity (Ariga et al., 1989), have now been attributed to a singlestranded DNA-binding protein, MSSP-1 (Negishi et al., 1994), which may tether c-Myc to this sequence (H. Ariga, personal communication). Nevertheless this sequence has not been identified as a c-Myc-binding site by several in vitro DNA-binding site selection studies, using either recombinant or endogenous c-Myc- and Max-containing complexes (Papoulas et al., 1992; Blackwell et al., 1993; Solomon et al., 1993; Buckle and MCchali, 1995). The c-Myc homodimer is not observed in vivo. This is presumably due to steric constraints within the full-length protein, since truncated c-Myc proteins containing just the C-terminal B-HLH-LZ domain can dimerize in vitro (Blackwell et al., 1990). The formation of Myc/Max heterodimers is

107

c-Myc in Cell Cycle and Embryonic Development

Amino acid number Transactivation and Repression domain

B/HLH/LZ

I

NLS

C-MYC

439

p22 Max

*

* *

*

*

160

p21 Max

151

A Max

110

dMax

124

Mad

221

Mxil-SR

220

Mxil-WR

192

Phosphorylation sites

Fig. 5 Schematic representation of the B- ILH-LZ proteins involved in the c-MydMax network. The protein size and relevant functional domains are indicated. Phosphorylationsites are represented by an asterisk.

kinetically favored over Max homodimerization, and consequently the majority of c-Myc is associated with Max in vivo (Blackwood et ul., 1992; Kato et al., 1992; Littlewood et al., 1992). Max also interacts with N-Myc and L-Myc in vivo (Wenzel et al., 1991; Mukherjee et al., 1992). In contrast to c-Myc, which exhibits a short half-life of 20-30 minutes and whose expression varies with the cell cycle, Max is a stable protein that is constitutively expressed at constant levels irrespective of the cell cycle (Blackwood et al., 1992; Ayer et al., 1993; Larsson et al., 1994). Thus it is likely that Max homodimers are the predominant form at times of the cell cycle when c-Myc is limiting, but that, upon stimulation of c-Myc synthesis during mitogenesis, the increasing levels of c-Myc sequester Max into a heterodimeric complex.

108

Jean-MarcLemaitre et af.

F. Interaction with Other Proteins Max is the major protein found to associate in a stable fashion with c-Myc, and in many cases has been considered to be its obligatory partner. However, c-Myc has also been found associated with high-molecular-weight protein complexes. These complexes were detected either by chemical crosslinking (Gillespsie and Eisenman, 1989) or by sucrose gradient sedimentation (Studzinski et al., 1991; Lemaitre et al., 1994, 1995).The nature of the proteins directly interacting with c-Myc in these complexes is unknown, and the proportion of the endogenous c-Myc engaged in such complexes appears different in different cell systems. A variety of proteins that interact with c-Myc in vivo have now been identified (Fig. 3). The c-Myc N-terminal region may stably associate with TBP (Hateboer et al., 1993; Maheswaran et al., 1994), the retinoblastoma (Rb)-related protein p107 (Beijersbergen et al., 1994; Gu et al., 1994; Hoang et al., 1995), and a-tubulin (Alexandrova et al., 1995), while the C-terminal region has been shown to bind the transcription factors YY-1 (Shrivastava et al., 1993) and AP-2 (Gaubatz et al., 1995). Interactions with c-Myc in vitro have also been demonstrated for plO5Rb Rustgi et al., 1991), TFII-I (Roy et al., 1993), the extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinases (Gupta and Davis, 1994), and the ERK-related kinase Mxi2 (Zervos et al., 1995).

IV. C-MYCAS A TRANSCRIPTION FACTOR A. c-Myc as a Transcriptional Activator In 1992, Amati and coworkers showed that, when ectopically expressed in yeast, c-Myc was able to activate transcription of a reporter gene containing the CACGTG Myc-binding site in its promoter. Similar results were reported in mammalian cells (Kretzner et al., 1992). Sequence-specific binding by c-Myc was strictly dependent on dimer formation with Max, while transactivation required the c-Myc N-terminal region. Overexpression of Max was unable to activate transcription of this reporter gene, although Max dimers could bind to the CACGTG motif, suggesting that Max homodimers might have a functional role in blocking transactivation by c-Myc/Max. By deleting the N-terminal region of c-Myc, without affecting its ability to dimerize with Max, it was further possible to form transcriptionally inactive heterodimers able to act as dominant negative mutants for c-Myc transformation (Mukherjee et al.,1992; Amati et al., 1993a), highlighting the importance of the c-Myc N-terminal domain in this process. The essential role for Max in c-Myc transactivation has been demonstrated in PC12 cells, which lack a

c-Myc in Cell Cycle and Embryonic Development

I09

functional Max protein due to aberrant splicing of Max mRNA (Hopewell and Ziff, 1995). In such cells, transactivation by c-Myc is only observed upon ectopic expression of Max. Two forms of c-Myc are normally present in cells, differing by 14 amino acids at the N-terminus. Both bind Max with equal affinities and activate transcription from reporter genes containing the CACGTG motif in their promoters (Blackwood et af., 1994; Hann et al., 1994). However, Myc/Max dimers can also bind noncanonical sequences, and, at one such site, the EFII enhancer element from the Rous sarcoma virus long terminal repeat, the p67 and p64 c-Myc proteins have differential effects. In this case the more abundant c-Myc form, p64, is unable to activate transcription when this sequence is linked to a reporter gene, in contrast to p67, which efficiently stimulates transcription (Hann et al., 1994). The observation of different transactivating abilities for the two c-Myc forms, according to the binding site employed, suggests that, while sharing many functions (Blackwood et al., 1994), the two proteins may have distinct cellular roles.

I . REGULATION OF C-MYC TRANSACTlVATlON ACTIVITY AT THE LEVEL OF DNA BINDING Several studies have shown that Max can inhibit the transactivation activity of c-Myc (Reddy et al., 1992; Kretzner et al., 1992; Gu et al., 1993; Amin et al., 1993). This inhibition is proportional to the level of Max expression (Mukherjee et af., 1992; Prendergast, et af., 1992; Amati et af., 1993a) and results from competition between the c-Myc/Max heterodimer and the transcriptionally inactive Max homodimer for binding at the CACGTG sequence motif. Max does not contain a functional transactivation domain (Kato et af., 1992).Max may therefore be considered as both an activator and a repressor of c-Myc activity, depending upon the relative abundance of the two proteins (Fig. 6). Although Max is a key effector of Myc function, it may also interact with other B-HLH-LZ proteins. The Madl and Mxil proteins were isolated by screening for such factors, and these two proteins appear to be important members of the c-Myc regulatory network (Ayer et af., 1993; Zervos et af., 1993). M a d l and Mxil display strong sequence identity with each other, and two other Mad family members, Mad3 and 4, have now been identified (Hurlin et al., 1995b). The Mad and Mxil proteins possess interaction specificities that are very similar to c-Myc; they homodimerize poorly and do not interact with Myc family members, but interact strongly with Max. Consequently, Mad proteins compete with c-Myc for Max heterodimer formation (Figs. 5 and 6). Both Mad/Max and Myc/Max complexes also bind the c-Myc/Max E-box sequence with an equal affinity. Functional analysis

c-Myc Max

Max Max

Mad(Mxi1-SR)

/- /L,transcription

c-Myc dMax

Fig. 6 Different levels of regulation for transactivation by c-Myc. c-Myc/Max heterodimers bind at the CACGTG recognition sequence and activate transcription by interacting directly with the basal transcription machinery (+ +). In contrast, all the other complexes indicated act as antagonists of c-Myc transcriptional activity (- - or -). Double arrows indicate equilibria, which are highly regulated during the cell cycle.

111

c-Myc in Cell Cycle and Embryonic Development

has indicated that M a d l does not contain a transactivation domain, and Madl is able to suppress transcriptional activation from a promoter containing the CACGTG consensus sequence and Eisenman, 1993).Thus, as for Max homodimers, Mad/Max and Mxil/Max are able to inhibit c-Myc/Max transcriptional activity. The network of interactions with Max provided by the Myc and Mad families may be fundamental to the choice between cellular proliferation and differentiation. Max is a constitutively expressed protein that is highly stable, possessing a half-life in excess of 24 hours (Blackwood et al., 1992). In contrast, its preferred dimerization partners, Myc and Mad, display very short half-lives, while their expression is highly regulated throughout the cell cycle (Ayer and Eisenman, 1993). When quiescent cells are stimulated by mitogenic agents, c-myc expression is induced, leading at the protein level to an equilibrium shift from Max/Max to Myc/Max heterodimer formation (Figs. 6 and 7). Conversely, withdrawal of mitogenic agents or addition of differentiation agents represses c-myc expression, concomitant with the loss of Myc/Max heterodimers. Moreover, Madl and Mxil expression is induced upon differentiation (Ayer and Eisenman, 1993; Zervos et al., 1993; Larsson et al., 1994), and in human myeloid U937 cells Mad can be detected in complexes with Max in less than 2 hours following the induction of differentiation. Mad/Max heterodimers therefore accumulate to become the only Max-containing complex present in these differentiated cells. Thus the short half-life of c-Myc, coupled to the stability of Max and the Mad or Mxil ratio, provides a very sensitive mechanism for the regulation of c-Myc activity. In this way cell cycle exit or entry may be controlled by the formation or disruption of transcriptionally active c-Myc/Max heterod'imers. Two new proteins that interact with Madl have recently been identified using the yeast two-hybrid system (Ayer et al., 1995; Schreiber-Agus et al., 1995). These two proteins, termed mSin3A and mSin3B, are closely related to the yeast transcriptional repressor Sin3, and contain four conserved paired amphipatic helix (PAH) domains (Wang and Stillman, 1993). Both interact with an N-terminal region of M a d l and also the related protein Mxil (Fig. 5). Mutations in this region eliminate interaction of M a d l with mSin3 proteins and also block Madl transcriptional repression (Ayer et al., 1995). Two Mxil isoforms, Mxi-SR and Mxi-WR, can also be expressed as a result of mRNA alternative splicing. Mxi-WR lacks the Sin3 interaction motif, and the absence of this sequence drastically reduces its suppressive activity (Schreiber-Agus et al., 1995). The degree of antagonism of Mad on c-Myc transactivation might therefore be mediated by formation of a Max/Mad/mSin3 ternary complex, using mSin3 as a corepressor. Moreover, the level of repression by Mxil could be modulated by generating different forms of the protein (e.g., Mxi-SR or Mxi-WR), able or not to bind the (Ayer

112

lean-Marc Lernaitre et al. C-Myc

Expression C-Myc

Mad Mxil

Max

-0 GO

G1

S

G2/M Cell cycle

4

Mitogenic agents

Fig. 7 cycle.

D Differentiation

Expression of c-Myc and associated proteins (Mad, Max, Mxil) throughout the cell

corepressor mSin3 (Fig. 6). In addition, it is proposed that reported cell line differences in the strength of Mad repression might be explained by their relative levels of mSin3 expression (Cerni et af., 1995). An alternative mechanism by which c-Myc transactivation can be inhibited is provided by the transcription factor AP-2, which specifically interacts with the B-HLH-LZ domain of c-Myc in vivo (Gaubatz et af., 1995).AP-2 is able to form a ternary complex with Myc/Max heterodimers, and inhibits c-Myc transcriptional activity by destabilizing the interaction of Myc/Max with DNA. The binding of Myc/Max dimers to the E-box motif can also be prevented by overlapping AP-2-binding sites, which are found adjacent to the E-box in the promoters of two Myc target genes, a-prothymosin and

c-Myc in Cell Cycle and Embryonic Development

113

ornithine decarboxylase (ODC) (Gaubatz et al., 1995). This provides an additional, albeit indirect, mechanism by which AP-2 can inhibit transcriptional induction by c-Myc. When considering the pivotal role of Max in mediating c-Myc activity, it should also be considered that Max might regulate the expression of some genes that are not activated by c-MycIMax. Indeed, the sequence specificity of binding for Max homodimers is not as strict as that for Myc/Max heterodimers, which are affected by the nucleotide composition adjacent to the E-box consensus sequence CACGTG (Fisher et al., 1993; Solomon et al., 1993; Prochownick and Van Antwerp, 1993). Moreover, Max can dimerize with other proteins of the Myc family, L-Myc and N-Myc (Blackwood and Eisenman, 1991; Wenzel et al., 1991; Mukherjee et al., 1992), which may have alternative targets to c-Myc, as suggested by the observation of differential outcomes when c-Myc and L-Myc were constitutively expressed within the same cell type (Morgenbesser et al., 1995). Another possibility for regulating c-Myc activity is provided by the observation that Max DNA binding can be modulated by phosphorylation at its N-terminus, at least in vitro. Serines 2 and 11, which are located next to the DNA-binding basic domain, are targets for casein kinase I1 (CKII) in vitro (Berberich and Cole, 1992), and they are also phosphorylated in vivo (Bousset et al., 1993; Koskinen et al., 1994). Phosphorylation at these serines increases both the on and off rates for DNA binding of both Max homodimers and Myc/Max heterodimers, suggesting that Max phosphorylation increases dimer exchange rather than modifying the DNA-binding affinity (Bousset et al., 1993). The role of these modifications in vivo remains unclear, however, as Max phosphorylation does not appear to be modulated during cell growth of differentiation (Koskinen et al., 1994). The c-Myc protein is also a substrate for CKII in vitro, at two conserved sites adjacent to both the specific and nonspecific DNA-binding regions. However, the interaction of c-Myc with DNA is unaffected by their phosphorylation in vitro. These sites are also phosphorylated in vivo, although no functional significance has been attributed to these modifications (Liischer et a/., 1989).

2. REGULATION OF C-MYCTRANSACTIVATION ACTIVITY THROUGH ITS TRANSACTIVATION DOMAIN The ability of c-Myc to transactivate can vary with the cell cycle. Transfection studies using a c-Myc-inducible reporter gene indicated two peaks of c-Myc transactivation, the major one arising in the G2 phase and the minor one in G1 (Seth et af., 1993). This result suggests that the role of c-Myc is not only restricted to the G1 phase but may be important throughout the cell cycle. Two phosphorylation sites within the N-terminal domain, threonine 5 8 and serine 62, have been implicated in the regulation of c-Myc transac-

114

Jean-Marc Lernaitre et al.

tivation (Gupta et al., 1993; Albert et al., 1994), and their phosphorylation is correlated with the two peaks of c-Myc activity observed in G1 and G2 (Tamemoto et al., 1992; Seth et al., 1993). However, other studies found that mutation of these sites had little effect on transactivation (Lutterbach and Hann, 1994; Henriksson et al., 1994). The importance of the threonine 58 and serine 62 residues for c-Myc function is indicated by their evolutionary conservation from sea star to human. Both sites, as well as serine 71, are phosphorylated in vivo (Gupta et al., 1993; Lutterbach et al., 1994). In vitro they can be targets for several different kinases: glycogen synthase kinase 111 (Lutterbach and Hann, 1994); ERK MAP kinases (Alvarez et al., 1991; Gupta and Davis, 1994); cyclindependent kinase 1 (cdkl; p34cdc2) (Seth et al., 1991); and a pl07-cyclin A-cdk2 complex (Hoang et al., 1995). Furthermore, ERK MAP kinase can stably bind to the c-Myc N-terminus in vitro, the interaction being sensitive to phosphorylation (Gupta and Davis, 1994). The regulation of N-terminal phosphorylation in vivo appears hierarchical, with threonine 5 8 phosphorylation being dependent upon prior serine 62 phosphorylation (Lutterbach et al., 1994). The same workers also showed that mutation of threonine 58 increased serine 62 phosphorylation, although a different study found that threonine 58 mutition prevented any N-terminal phosphorylation (Gupta et al., 1993). Mutations at or close to threonine 58 are frequently found in vim, as in Burkitt and AIDS-related lymphomas (Bhatia et al., 1993; Yano et al., 1993; Albert et al., 1994) or in the v-myc allele of the MC29 retrovirus (Papas and Lautenberger, 1985). Transformation by c-Myc is positively regulated in vivo by serine 62 phosphorylation and negatively by phosphorylation at threonine 58 (Pulverer et al., 1994; Henriksson et al., 1994). This contrasts with c-Myc transactivational activity, which is negatively regulated when either site is mutated, suggesting that transformation by c-Myc results only partially from its transactivation activity. Threonine 58 is also subject to glycosylation (Chou et al., 1995), which might provide another level of control, although as yet this modification is poorly characterized. The c-Myc transactivation domain may also be regulated by its interaction with other proteins. Transcriptional induction by c-Myc can be inhibited by the pl05Rb-related tumor suppressor protein pl07, which specifically interacts with the N-terminal domain of c-Myc in vivo (Beijersbergen et al., 1994; Gu et al., 1994). The c-Myc transactivation domain also binds plOSRb in vitro (Rustgi et al., 1991), although this has not been confirmed in vivo. Interaction between c-Myc and p107 is mediated by the Rb hydrophobic pocket region, which is also required for Rb family proteins to bind the adenovirus E1A protein and SV40 T antigen (Q. Hu et al., 1990; Qin et al., 1992; Zhu et al., 1993). The binding of p107 to these viral transforming proteins, in addition to c-Myc, may thus represent a common mechanism to suppress cell growth (Fig. 6).

c-Myc in Cell Cycle a n d Embryonic Development

115

The c-Myc N-terminal region is also able to interact directly with the TATA box-binding protein (TBP), both in vitro and in vivo (Hateboer et al., 1993; Maheswaren et al., 1994). Thus c-Myc has the ability to interact physically and functionally with basal components of the transcription machinery. Immunoprecipitation of the c-Myc-TBP complex from cell lysates is prevented by the addition of a peptide homologous to the Rb pocket region (Maheswaran et al., 1994), and protein sequences required for the c-Myc-TBP interaction are identical to those involved in the interaction of c-Myc with p l O W in vitro (Rustgi et al., 1991; Hateboer et al., 1993). Thus the ability of p107 to inhibit transactivation by c-Myc may be mediated by direct competition for c-Myc-TBP binding (Fig. 6). Interestingly, ectopic expression of the Myc family member B-Myc inhibits both c-Myc transcriptional activity and c-Myc-Ras cotransformation (Resar et al., 1993). B-Myc is homologous to the c-Myc transactivation domain but lacks the B-HLH-LZ motif, and may therefore suppress transcription by associating with the basal transcription machinery in a nonfunctional manner (Fig. 6 ) .

B. c-Myc as a Transcriptional Repressor In addition to its transactivational ability, the c-Myc protein can also directly repress transcription, via a mechanism dependent on the Inr promoter sequence of susceptible genes. For a promoter that does not contain a TATA box, transcriptional initiation by RNA polymerase I1 is dependent upon a conserved DNA motif, the Inr, which overlaps the transcriptional start site. Transcriptional repression by c-Myc was first detected at the adenovirus major late promoter, which contains both an Inr and TATA box, as well as an upstream c-Myc (USF)-binding site, CACGTG. In this peculiar promoter, the initiation of transcription may occur by either TBP-TFII-I or TBP-TFII-A interactions. Using such a system in vitro, it was determined that c-Myc interacts specifically with TFII-I at the Inr region to inhibit transcriptional initiation, whereas TFII-A-dependent transcription was not sensitive to c-Myc (Roy et al., 1993). The interaction of c-Myc with TFII-I requires the C-terminal HLH-LZ domain of c-Myc, while transcriptional repression from the Inr sequence is dependent upon a small region within the c-Myc N-terminal domain (amino acids 122-143 in humans). Interestingly, a mutant c-Myc protein lacking these residues fails to cooperate with ras in cellular transformation (Li et al., 1994). This region resides within the same c-Myc domain that interacts with TBP in vitro, suggesting that repression depends upon a direct interaction of a c-Myc-TFII-I complex with TBP. Repression of the cyclin D1 promoter by c-Myc is also mediated by an Inr element, but in this case the c-Myc N-terminal region

I16

lean-Marc Lernaitre et al.

responsible mapped to residues 92-106 (Philipp et al., 1994). The reason for this difference is unclear, and might indicate that transcriptional inhibition is mediated by different mechanisms in the two cases. Another c-Myc-interacting factor is YY-1, which was identified in a yeast two-hybrid screen using the c-Myc C-terminal domain. This interaction was confirmed in vivo (Shrivastava et a1.,1993). YY-1 is a ubiquitous B-HLH transcription factor that is able to bind the Inr promoter sequence and either activate, initiate, or repress transcription according to the promoter context (Seto et al., 1991; Gualberto et al., 1992; Riggs et al., 1993). c-Myc interferes with both the activator and repressor functions of YY-1. This function of c-Myc occurs by a direct interaction with YY-1 and is independent of Max, whose dimerization with Myc is prevented by YY-1 binding (Shrivastava et al., 1993). The interaction of YY-1 requires the c-Myc residues 250-439 in the human protein, encompassing the B-HLH-LZ domain as well as the nonspecific DNA-binding region, NLS domain, and CKII phosphorylation sites. It is noteworthy that YY-1 is an activator of c-myc transcription (Riggs et al., 1993), and it could be possible that its interaction with c-Myc participates in the process of c-myc autosuppression. The overexpression of c-Myc also represses promoters dependent on the CTF-NF1 transcriptional activator. In this case inhibition is mediated in an indirect manner via a modification of this factor (Yang et al., 1993).

V. C-MYCAND CELL PROLIFERATION

A. c-Myc Is Required Throughout the Cell Cycle One of the best documented functions of c-Myc is its role in promoting cellular proliferation. C-myc belongs to the family of immediate early response genes, which are rapidly induced upon mitogenic stimulation of resting (GO) cells. Its expression is activated by a variety of mitogens during the GO-G1 phase of the cell cycle, without any requirement for ongoing protein translation (Cochran et af., 1983; Kelly et al., 1983; Greenberg et af., 1985; Lau and Nathans, 1987). Expression of c-myc alone is sufficient to carry out the GO-G1 transition, without coincident expression of the other immediate early response genes (Eilers et af., 1991). However, its inhibition in proliferative cells by injection of antisense oligonucleotides does not prevent G1 events but rather blocks progression beyond G1 phase (Coffey et al., 1988; Heikkila et al., 1987; Holt et al., 1988; Loke et a1.,1988). Ectopic c-myc expression allows the alleviation of such an inhibition (Coppola and Cole, 1986; Eilers et al., 1991). Similarly, mouse fibroblasts unable to express c-myc as a consequence of possessing a mutated colony-stimulating factor (CSF-1) receptor exhibit G1 arrest, which can be relieved by ecto-

c-Myc in Cell Cycle a n d Embryonic Development

1 I7

pically expressed c-myc (Roussel et al., 1991). Thus, although c-Myc is able to induce the GO-G1 transition, its essential role is to drive cells throughout the G1 phase. Mitogenic induction of c-myc is both slower and more prolonged in comparison to the archetypal immediate early genes c-fos and c-jun. While its expression is highly induced during the first 2 hours following growth factor stimulation of quiescent cells, it is not restricted to this phase, and in exponential cells a basal level is maintained continuously throughout the cell cycle (Hann et al., 1985; Rabbitts et al., 1985; Thompson et al., 1985; see Fig. 7). This basal level of expression is dependent upon continuous growth factor stimulation (Dean et al., 1986b; Waters et al., 1991), and at least in some cases is maintained by posttranscriptional regulation (Blanchard et al., 1985; Dean et al., 1986a). Indeed, serum starvation induces a rapid disappearance of c-myc mRNA and protein, leading to a block in G1 phase, regardless of the phase in which starvation starts (reviewedin Pardee, 1989). Cell types expressing a high level of exogenous c-Myc display a shortened G1 phase (Karn et al., 1989) and an increased growth rate (Palmieri et al., 1983; Langdon et al., 1988), in addition to very reduced needs for growth factors (Armelin et al., 1984; Sorrentino et al., 1986; Stern et al., 1986; Langdon et al., 1988). The continued expression of c-myc throughout the cell cycle suggests that the role of c-Myc is not simply restricted to the G1 phase. Indeed, a requirement for this protein in the G1 phase has been demonstrated by Shibuya and coworkers (1992), who observed that BAF-B03 pre-B cells unable to express c-myc in response to stimulation with epidermal growth factor (EGF) could not exit G2. Ectopic expression of c-myc relieved this block and allowed progression into mitosis. Evidence for a role for c-Myc in G2 is also supplied by studies of c-Myc transactivation, where biphasic activation of a reporter gene containing a c-Myc-binding site was observed in both G1 and G2 (Seth et al., 1993). This transcriptional activation by c-Myc also correlated to the cell cycle-regulated phosphorylation of the c-Myc transactivating domain. Furthermore, coexpression of c-myc and an activated form of H-ras leads to transcriptional induction from the cdc2 gene promoter during the period of the S-to-G2-M phase transition (Born et al., 1994), in a process requiring both an intact c-Myc dimerization domain and the phosphoacceptor serine 62 in its transactivating domain. Thus c-Myc appears to play a role in at least two distinct phases of the cell cycle.

B. Signal Transduction and Induction of c-myc Transcription The induction of c-myc by mitogenic agents has been characterized in several cell types, but most extensively in quiescent fibroblasts, where c-myc

I18

lean-Marc Lemaitre et a/.

is readily induced after stimulation with platelet-derived growth factor (PDGF),fibroblast growth factor (FGF),epidermal growth factor (EGF),and the phorbol ester 12-0-tetradecanoy1phorbo1-13-acetate (Kelly et al., 1983; Dean et al., 1986b; Cutry et al., 1989). Although various signal transduction pathways have been implicated in the control of c-myc transcription, including those using protein kinase C (PKC) (Coughlin et al., 1985) and CAMP-dependent protein kinase (PKA) (Ran et al., 1986), as well as routes independent of PKA and PKC activation (Frick and Scher, 1990), the precise mechanisms that govern this process are only now beginning to be defined.

I . PDGF-SK PATHWAY One approach indicating the requirement for c-Myc in G1 progression was the observation that ectopic c-myc expression restored the ability of a mutant CSF-1 to induce cell proliferation (Roussel et al., 1991). This mutant receptor failed to induce c-myc expression, although induction of c-fos and c-jtm was normal. The mutant CSF-1 receptor was also found to poorly activate the nonreceptor tyrosine kinase src, normally involved in CSF-1 signaling (Courtneidge et al., 1993). The link between STC activation and c-myc induction has now been confirmed by analyzing the PDGF-induced proliferation of quiescent fibroblasts (Barone and Courtneidge, 1995). Upon activation of the PDGF receptor, SIC family kinases mediate S-phase entry, a process that can be blocked by a dominant negative form of c-Myc. Overexpression of c-Myc can also overcome the S-phase block effected by a dominant negative src mutant, and furthermore, the induction of c-myc transcription by PDGF can be inhibited by src-inhibitory antibodies. 2. V-abl

c-Myc has also been implicated as a major effector of transformation by the v-abl oncogene, a homolog of the ubiquitous nonreceptor tyrosine kinase c-abl. For example, transdominant negative forms of c-Myc can block the transformation of fibroblasts by v-abl (Sawyers et al., 1992). One major consequence of the activation of c-a61 and the viral homolog v-a6l is the induction of c-myc transcription from both the P1 and P2 promoters (Cleveland et al., 1989; Renshaw et al., 1992; Wong et al., 1995a). Recent evidence suggests that the induction of c-myc transcription, which can occur in the absence of protein synthesis, is mediated through the E2F site in the c-myc promoter (Wong et al., 1995b). This study also showed that both the v-abl tyrosine kinase and src homology region 2 (SH2) domains were required for this process, consistent with the observation that overexpression of c-Myc complements mutations in the SH2 domain of BCR/Abl in transformation assays (Afar et al., 1994).

c-Myc in Cell Cycle a n d Embryonic Development

119

3. Vav AND Ras Ras oncoproteins are ubiquitous guanine exchange factors (GEFs) that act as a fulcrum for a variety of signal transduction pathways mediating cell proliferation. In conjunction with c-myc, activated Ras leads to cellular transformation (Land et al., 1983,1986; Lee et al., 1985), a process that can be blocked by dominant negative forms of c-Myc. Evidence now suggests that c-Myc function is also required for cellular transformation mediated by Vav, a haematopoiesis-specific GEF. Furthermore, Ras and Vav appear to use distinct but overlapping pathways that converge upon c-Myc to induce transformation in fibroblasts (Katzav et al., 1995).

4. E2F AND C-mgC INDUCTION A further link between the cell cycle and c-myc induction is provided by the E2F transcription factor, which is pivotal in activating genes essential for cell cycle progression (La Thangue, 1994). A highly conserved E2F binding site is located between the P1 and P2 c-myc promoters. E2F binding at this site is essential for c-myc induction by the adenovirus E1A protein (Thalmeier et al., 1989) and serum stimulation (Mudryj et al., 1990). Furthermore, the E2F site mediates the induction of c-myc by cyclins A and D1, which can be abrogated by p105Rb (Oswald et al., 1994). Indeed, it had previously been shown that c-myc transcription can be repressed by p l O W (Hamel et al., 1992),which may sequester E2F in a transcriptionally inactive form (Weintraub et al., 1992; Nevins, 1992).

C. Cell Cycle 'Ihrgets of c-Myc 1. REGULATION OF CYCLIN TRANSCRlPTION

Cell cycle progression is controlled by various cyclin-cdk complexes. During G1, cyclin D1 and cyclin E-cdk complexes cooperate to phosphorylate and thereby inactivate the pl05Rb protein, allowing progression through G1. The transcriptional activation of the various cyclin or cdk genes, which are regulated in a cell cycle-dependent manner, is one mechanism by which c-Myc might exert its mitogenic effect. Indeed, the activation of a conditional c-myc gene in quiescent cells strongly induces transcription of cyclin A, one of the S-phase cyclins (Jansen-Durr et al., 1993). Of the G1 cyclins, cyclin E mRNA is modestly induced while the RNA levels of cyclins D1, D2, and G1 cdks 2 and 4 are unaffected under such conditions (Jansen-Durr et al., 1993). The role of increased cyclin E transcription in mitogenic activation by Myc is unclear, since its induction appears to follow rather than precede plOSRb phos-

I20

Jean-Marc Lernaitre et al.

phorylation, while high levels of cyclin E protein preexist in quiescent cells (Steiner et al., 1995). Support for the regulation of cyclins A and E by c-myc is provided by studies in fibroblasts lacking one copy of the endogenous c-myc gene, wherein cyclin A and E RNA induction is delayed following mitogenic stimulation of quiescent cells (Hanson et al., 1994). Conflicting evidence exists concerning the effect of c-Myc expression on the induction of cyclin D1 mRNA. Constitutive expression of c-Myc or a Myc-estrogen receptor (MycER) chimera represses cyclin D1 induction (Jansen-Durr et al., 1993; Philipp et al., 1994). The cyclin D1 gene contains an Inr sequence as the transcription initiation site but no TATA box (Philipp et al., 1994). Transcription of this gene is normally activated by the BHLH-LZ transcription factor USF, which is thought to bind at the Inr sequence via an interaction with the basal transcription factor TFII-I, as observed for the adenovirus major late promoter (Roy et al., 1991,1993; Du eta!., 1993). c-Myc was observed to repress USF transactivational activity in a Max-independent manner, most probably by competing for TFII-I binding (Philipp et al., 1994). However, another study found MycER to strongly induce cyclin D1 in serum-starved cells in the presence of estrogen (Daksis et al., 1994). It now appears that this latter finding might be artefactual as a consequence of transactivation by the estrogen receptor part of the MycER chimera, suggesting that, although c-Myc may interact with cyclin D1 regulatory elements, it cannot activate transcription (Solomon et al., 1993).

2. DEREPRESSION OF THE cdk INHIBITORS Overexpression of c-Myc can override p53-induced cell cycle arrest in G1 without elevating the expression of cyclins-cdks involved in the G1-S transition (Hermeking et al., 1995)..p53 inhibits progression into S phase by transactivating the p21 cdk inhibitor, and it appears c-Myc abrogates the effect of p53 by interfering with the inhibitory action of p21 and thereby derepressing cyclin-cdk complexes. Activation of the conditional Myc allele MycER in quiescent cells leads to the rapid activation of cyclin E- and cyclin D1-dependent kinases in the absence of significant changes in the amounts of the cyclin-cdk complexes (Steiner et al., 1995). This kinase activation is blocked by transcriptional inhibitors and requires the c-Myc DNA binding and dimerization domains, although the precise mechanism remains to be determined. However, activation of the preassembled cyclin E-cdk2 complex correlates with its release from a larger inactive complex that contains the cdk inhibitor p27, leading to speculation that Myc might effect the dissociation of p27 from this complex, possibly by promoting its proteolysis. This suggests an indirect way by which c-Myc might facilitate cell cycle progression.

c-Myc in Cell Cycle and Embryonic Development

121

3. ORNlTHlNE DECARBOXYLASE This gene encodes a rate-limiting enzyme involved in polyamine biosynthesis that is essential for cell proliferation. Both specific inhibitors of ODC activity and polyamine depletion block the cell cycle in its G1 phase (Pohjanpelto et al., 1981; Tabor and Tabor, 1984; Pegg, 1986; Heby and Persson, 1990; Balasundaram et al., 1991). ODC transcription is induced by the MycER chimera upon addition of estrogen to quiescent cells, even in the absence of protein synthesis, indicating that O D C is a direct c-Myc target gene (Wagner et al., 1993). ODC transcription is strongly induced by overexpression of c-Myc, a process mediated by two conserved c-Myc-binding sites present in the first intron (Bello-Fernandez et al., 1993; Tobias et al., 1995). While these E-box motifs readily bind Myc/Max dimers in vitro, these two studies provide contradictory evidence concerning the requirement for Max heterodimerization in the transactivation process in viva ODC expression is stimulated both in G1, just before DNA synthesis, and in G2-M, and thus strictly correlates with c-Myc transactivational activity (Seth et al., 1993). Moreover, ODC cooperates with ras to induce cellular transformation (Hibshoosh et al., 1991; Auvinen et al., 1995; Moshier et al., 1993). 4. p53

p53 is a tumor suppressor protein intimately involved in cell cycle control. It has been identified as both a transcription factor and the major regulator of the G1-S checkpoint, whereby cells may arrest in G1 as a result of DNA damage (reviewed in Cox and Lane, 199.5).The p.53 promoter contains an essential c-Myc binding site within its promoter, which mediates c-MycMax transactivation in cotransfection assays (Reisman et al., 1993). The notion that p.53 may be a target gene for c-Myc is supported by studies in which the presence of antisense c-Myc RNA leads to reduced expression from the endogenous p53 promoter (Roy et al., 1994). Furthermore, the relative levels of c-Myc protein and p.53 mRNA expression appear to correlate in a variety of tumor cell lines (Roy et al., 1994).

5. OTHER POTENTIAL C-MYCTARGET GENES Several other potential c-Myc target genes have now been identified that contain functionally important Myc/Max consensus sequences (CACGTG) in their promoters. However, screening for such genes may prove to be a complicated process, since Myc/Max complexes can also bind to noncanonical sequences with high affinity (Blackwell et al., 1993), and in one instance it has been shown that such a site can mediate c-Myc transactiva-

I22

lean-Marc Lemaitre et al.

tion (Hann et al., 1994). c-Myc can also interact with several other DNAbinding proteins, which may further expand its range of potential targets. One approach has been to undertake differential screening to identify mRNAs specifically induced by c-Myc. The ECA39 gene was identified in such a way, and has been characterized as a gene that is amplified in teratocarcinoma cells and expressed during embryonic development. Its function is so far unknown. As for the ODC gene, it contains a Myc/Max E-box motif in the 5 ’ untranslated region of the gene, which is functional in transfection assays (Benvenisty et al., 1992). The a-prothymosin gene is another c-Myc-inducible gene identified by a genetic screen (Eilers et al., 1991), and its expression correlates with c-myc levels during differentiation (Mori et al., 1993). It encodes the precursor of the small nuclear protein thymosin (Watts et al., 1989), which is structurally related to several acidic nuclear proteins such as nucleolin and proliferating cell nuclear antigen (Gomez-Marquez and Sega, 1988). a-Prothymosin expression is restricted to growing cells (Eschenfeldt and Berger, 1986) and it is essential for proliferation (Sburlati et al., 1991). It too contains an E-box motif in its first intron, although recent data indicate that deletion of this sequence in transfection assays does not affect transcriptional induction of the gene. Furthermore, the a-prothymosin gene is unresponsive to differing levels of c-Myc in the same system (Mol et al., 1995), casting doubt as to whether this gene is truly a direct c-Myc target. The cad gene codes for an enzyme involved in pyrimidine synthesis, and contains a Myc/Max-binding site in an important regulatory element specifying cell cycle control of its transcription. cad mRNA is induced at the G1S transition, and Myc/Max complexes appear to play a role in this process (Miltenberger et al., 1995). Another key metabolic enzyme that is essential for DNA synthesis and S-phase progression is dihydofolate reductase (DHFR), which provides cells with dTTP. This is encoded by the DHFR gene, which is also a potential target gene for c-Myc (Mai and Jalava, 1994). A MycIMax E-box consensus sequence is located within the DHFR promoter, and in vitro studies suggest c-Myc-containing complexes can bind to this site. However, no functional data concerning transactivation yet exist for this gene. Sequence analysis in data banks has identified other potential c-Myc target genes containing the E-box consensus sequence in their promoter. The c-sis gene, which codes for the B polypeptide of PDGF and is itself involved in the induction of c-myc expression, contains the E-box motif in its transcriptional enhancer region (Van den Ouweland et al., 1987; Rao et al., 1988; Ratner, 1989). The same situation is observed for the nucleolin gene, whose protein is involved in synthesis and assembling of ribosomes (Bourbon et al., 1988; Srivastava et al., 1990), and also for the U3B RNA gene, which codes for a small nuclear RNA mostly localized in the nucleolus that

c-Myc in Cell Cycle and Embryonic Development

123

is involved in pre-rRNA processing (Mazan and Bachellerie, 1988; Busch et al., 1982). Could c-Myc be involved in the regulation of ribosome synthesis, a process essential for cellular growth? Interestingly, c-Myc-transformed cells exhibit hypertrophy of the nucleolus when compared to cells transformed by other oncogenes (Palmieri et al., 1983). Protein synthesis is controlled by both the levels of both ribosomes and translation initiation factors (Hershey, 1991). The expression of the eIF-4E initiation factor and the a-subunit of the eIF-2 complex correlates with the stimulation of c-myc expression by growth factors (Rosenwald et al., 1993), although it is not clear whether c-Myc acts directly to transactivate these genes in G1 or whether late response genes are responsible. Nevertheless, stimulation of translational initiation factors by c-Myc might provide a link between immediate early response genes and late response genes, which are essential for cell cycle progression beyond G1.

6. INTERACTION WITH THE ~ 1 0 AND 5 ~p107 ~ TUMOR SUPPRESSOR PROTEINS The initial observation suggesting that c-Myc could be a target for the tumor suppressor protein p l 0 . F was made in mouse keratinocytes, where down-regulation of c-myc by transforming growth factor$ (TGF-P) appeared to be mediated by pl05Rb. This effect was blocked by the viral proteins adenovirus ElA, SV40 T antigen, and human papillomavirus-16 (HPV-16) E7 in a manner dependent upon their pl05Rb-binding domains (Pietenpol et al., 1990). In vitro binding studies using recombinant proteins indicated that the c-Myc N-terminal transactivation domain can specifically interact with plO5Rb (Rustgi et al., 1991), and a possible cellular interaction between these two proteins is supported by the results of a two-hybrid assay, wherein p l O W interacts with c-Myc to stimulate reporter gene transcription. This result was dependent upon the integrity of the c-Myc transactivation domain (Adnane and Robbins, 1995). Furthermore, microinjection of c-Myc into cells can overcome the G1 growth arrest induced by p105Rb (Goodrich and Lee, 1992). However, a direct interaction between plOSRb and c-Myc cannot be detected in vivo, although the highly related protein p107 does bind c-Myc in vivo (Beijersbergen et al., 1994; Gu et al., 1994). The association of p107 with c-Myc requires the c-Myc N-terminal domain, and binding of p107 was found to suppress c-Myc transactivation. Beijersbergen and coworkers further demonstrated that ectopic expression of c-Myc could alleviate pl07-induced growth arrest, but not that induced by plOSRb. This latter finding contrasts with that of Goodrich and Lee (1992), but may be explained by the different levels of c-Myc used in the two studies. It is also noteworthy that, while p107 inhibits c-Myc transactivation, the

124

Jean-Marc Lemaitre et al.

study of Adnane and Robbins (1995) found that in certain cell lines interaction between ~ 1 0 . and 5 ~c-Myc ~ stimulated transcription. The reasons for this difference are not clear. The transactivational potential of c-Myc can be modulated by phosphorylation within its N-terminal domain, and a study has shown that serine 62 can be phosphorylated in vitro by a pIO7-cycIin A-cdk complex (Hoang et al., 1995). This observation may provide an explanation for how mutant c-Myc proteins isolated from Burkitt lymphomas can escape transactivational repression by p107, even though they still bind the protein with affinities similar to those of wild-type c-Myc (Gu et al., 1994). It is proposed that mutation within the N-terminal transactivation domains might prevent a functional interaction with the associated cyclin A-cdk complex (Hoang et al., 1995). The differential cellular interaction between c-Myc and p l O P and p107 may be explained since the targets of these two growth suppressor proteins appear different. This is indicated because only p l OW-induced cell cycle arrest in G1, and not that induced by p107, can be alleviated by overexpression of either E2F-1 or cyclins A or E (Zhu et al., 1993). A further argument against the sequestration of plO5Rb by c-Myc is the observation that, although cyclin D1-associated kinases are required for G1 progression in Myc-transformed cells, p105Rb-/p10SRb- cells do not require cyclin D1associated kinase activity to enter S phase (Lukas et al., 1994). It is perhaps more likely that activation of cdks is required for the inactivation of plOSRb by Myc, since dominant negative alleles of cdk2 (Van den Heuvel and Harlow, 1993) or overexpression of the cdk inhibitors p16, p21, and p27 can inhibit the G1 progression effected by c-Myc (Xiong et al., 1993; El-Deiry et al., 1994; Polyak et al., 1994; Toyoshima and Hunter, 1994).

D. c-Myc and DNA Replication The strong correlation observed between c-Myc expression and proliferation has driven several groups to look for a possible role of c-Myc in DNA replication. This was a rather attractive possibility as the control of initiation of DNA replication remains rather elusive in multicellular organisms, and only a limited number of proteins are known to be involved in this regulation. One possible link between c-Myc and DNA replication was provided by the identification of a region within the human c-myc promoter displaying both transcriptional enhancer and replication origin activity and containing a putative c-Myc binding sequence (Ariga et al., 1989). However, although other studies confirmed that the upstream region of the c-myc gene appears to contain an initiation zone for DNA replication (McWhinney and Leffak, 1990; Berberich et al., 1995), this was not localized to the region identified by Ariga and coworkers. Furthermore, the ability of c-Myc to bind the site

c-Myc in Cell Cycle a n d Emblyonic Development

125

identified within the human c-myc promoter has not been confirmed by binding site selection studies (Buckle and Mtchali, 1995; see Section 1I.E). Initial investigations into the effects of different anti-c-Myc antibodies on DNA synthesis at the replication fork and on the initiation of replication in SV40 did not provide conclusive results (Studzinskiet al., 1986; Gutierrez et al., 1987, 1988). However, further studies of SV40 DNA replication in human lymphoid cells showed that overexpression of c-Myc in these cells increased the replication of SV40 DNA (Classon et al., 1990). Analysis of the c-Myc domains involved in this stimulation identified the basic region and the LZ motif. Interestingly, the HLH domain is not necessary (Classon et al., 1993), suggesting that the stimulation of SV40 DNA replication by c-Myc does not require its association with Max. This observation suggests that, if c-Myc is directly involved in DNA replication, it would be through a mechanism distinct from c-Myc/Max transcriptional activation. Observations on the localization of c-Myc during Xenopus early development lend support to this possibility. During the early cleavage stage, a large store of cytoplasmic c-Myc migrates into cell nuclei (Gusse et af., 1989). This developmental period is characterized by intensive cell division coupled to a complete absence of transcription within the embryo, and therefore the nuclear uptake of c-Myc in these circumstances might be more related to cycles of DNA replication. Furthermore c-Myc DNA binding is not detected in association with Max during this developmental stage, and the formation of c-Myc/Max complexes is inhibited in embryonic nuclei (Lemaitre et al., 1995), again suggesting a separate function for c-Myc independent of its association with Max, However, there is so far no conclusive evidence for a direct role of c-Myc in DNA replication. Indeed, the ability of c-Myc to stimulate cyclin-cdk activity (Steiner et al., 1995) also provides an indirect mechanism by which c-Myc might induce DNA replication. Nevertheless, the characterization of c-Myc as a transcription factor does not preclude it from also functioning as a replication factor. A great deal of evidence has accumulated for a role of some transcription factors in DNA replication, as exemplified by the viral protein SV40 T antigen, which represents one of the oldest known examples in eukaryotes (see DePamphilis, 1988; Heintz, 1992, for reviews). A dual function for some nuclear proto-oncogenes in both transcription and DNA replication might also explain the dramatic consequences of their deregulation.

VI. C-MYCIN EMBRYONIC DEVELOPMENT The majority of studies concerning c-myc expression in adult tissues or embryos are related to the production of mRNA, although gene expression

126

lean-Marc Lemaitre et al.

does not always strictly correlate with the temporal pattern and level of protein expression during development. In addition, regulation of c-Myc may differ according to the developmental strategy used in different organisms. It might not, therefore, be surprising that no real convergence has emerged from these studies. In adult tissues, c-myc expression is generally consistent with the proliferative rate of the tissue. For example, in mice, tissues that contain several proliferative cell types, such as the kidneys, intestine, thymus, and spleen, exhibit a high level of c-Myc expression. In contrast, low expression is detected in the heart, liver, brain, and lungs (Zimmerman et af., 1986), possibly as a consequence of posttranscriptional control (Morello et af., 1989). Regenerative processes are also accompanied by a rise in c-myc expression, both in rat liver (Makino et af., 1984) and in Xenopus forelimb (GCraudie et af., 1990; Lemaitre et al., 1992). In this case the pattern of expression is more consistent with commitment to proliferation rather than active proliferation. During embryonic development, some correlation of c-myc expression with cell proliferation is detected, but this is clearly not the rule, and large variations are observed in different tissues as well as different organisms. The expression of c-myc during embryonic development has been characterized in several organisms: mice (Zimmerman et al., 1986; Downs et al., 1989; Schmid et al., 1989); chicken (Vandenbunder et al., 1989; Jaffredo et af., 1989); Xenopus (King et af., 1986; Taylor et af., 1986; Nishikura, 1987; Hourdry et al., 1988; Vriz et af., 1989; Gusse et al., 1989; Lemaitre et af., 1995); and zebra fish (Screiber-Aguset af., 1993). In these animals c-Myc expression is maximal in proliferative tissues of mesodermal origin. However, endodermal and ectodermal tissues exhibit very low or no c-Myc expression, even though these may be highly proliferative. The lack of correlation between c-myc levels and cell division is further highlighted during the formation of placods and optical vesicles in Xenopus (Hourdry et al., 1988). Expression of c-myc in both proliferating and differentiating embryonic chicken lens cells was also observed (Harris et al., 1992), in accordance with previous observations obtained in vivo (Nath et af., 1987). During mouse lens cell differentiation, a decrease of c-myc expression is observed; however, differentiation is unaffected by the forced expression of c-myc (Morgenbesser et al., 1995). In humans, a high level of c-myc expression is detected in the highly proliferative cytotrophoblast, but no correlation with cell proliferation is generally detected in embryonic tissues (Pfeifer-Ohlsson et al., 1985). The early developmental period of an organism is characterized by a phase of rapid cell division. In mouse, where zygotic transcription begins at the two-cell stage, the maternal store of c-myc mRNA in the oocyte or egg is rather low (1to 10 copies per egg), and expression of c-myc is detected from

c-Myc in Cell Cycle and Embryonic Development

127

the four-cell stage at both the mRNA and protein levels (Pal et al., 1993; Paria et al., 1992). A different developmental strategy is used in Xenopus, where during the initial 12 cell divisions that occur without accompanying zygotic transcription, the embryo utilizes its maternal store of proteins and mRNAs (see Davidson, 1986, for a review). In this case c-myc is already highly expressed in the oocyte both at the mRNA and protein levels (Taylor et al., 1986; King et al., 1986; Gusse et al., 1989). However, the protein exhibits an unusual cytoplasmic localization during this stage, and upon fertilization it migrates into and saturates the embryonic nuclei, resulting in an exceptionally high c-Myc:DNA ratio (Lemaitre et al., 1995). In this case c-Myc function may be linked to the rapid cycles of DNA replication that characterize this developmental period. Early development in zebra fish follows a pattern similar to that of Xenopus, with early cell divisions occurring in the absence of zygotic transcription. In this case maternally derived c-myc mRNA is barely detectable, and these early cleavage stages are characterized by extremely high levels of L-myc mRNA (Schreiber-Agus et al., 1993). This suggests that in zebra fish L-Myc might function to promote the rapid cell divisions of early development. The possibility that in certain instances L-Myc might functionally substitute for c-Myc during embryogenesis is supported by observations in mice, where embryos bearing homozygous null mutations for either c-Myc or N-Myc survive until early gestation (Davis et al., 1993; Charron et al., 1992; Stanton et al., 1992). Studies with transgenic mice, where c-myc can be placed under the control of various inducible promoters, show that an ectopic expression contributes to an increased incidence of tumors during development (Leder et al., 1986; Langdon et al., 1986; Schoenenberger et al., 1988). However, a number of tissues overexpressing c-myc do not develop tumors, and the synergistic action of other factors (e.g., growth factors, cooperating oncogenes) is necessary for malignancy (Sinn et al., 1987; Murakami et al., 1993). Hyperplastic growth is detected by constitutive c-myc expression in cardiac myocytes (Jackson et al., 1990). While these analyses are important in our understanding of the oncogenic properties of c-Myc in vivo, they do not show a specific function for c-Myc during embryogenesis. Rather, they tend to suggest that c-Myc is not directly involved in the differentiation of a restricted set of tissues. A more direct approach in the elucidation of c-Myc function during development has been to use homologous recombination to generate a null c-myc mutation in mice (Davis et al., 1993). The mutation is lethal after 10 days of gestation in the homozygote embryos, which exhibit several abnormalities. This result suggests that c-myc is not essential for cell proliferation during early development. However, the early embryos are smaller and developmentally retarded, and this does not exclude a role for c-Myc in regulating cell division during early embryogenesis. Some compensatory mechanism

I28

Jean-Marc Lemaitre et al.

might occur in these embryos to replace c-Myc function, possibly carried out by another member of the Myc family.

VII. C-MYCAND DIFFERENTIATION While proliferating cells exhibit stable c-myc RNA levels, abrupt changes in c-myc expression are often observed during exit from the cell cycle as cells undergo terminal differentiation (Fig. 7). However, no simple conclusion can be drawn from the numerous studies characterizing these changes, as different results are obtained according to the cell type, differentiation state, and differentiation agent used (Kelly and Siebenlist, 1986; Marcu, 1987; Zimmerman and Alt, 1990). In the majority of cases a decrease in c-myc mRNA and protein levels is observed with cell growth arrest and the initiation of differentiation (Westin et al., 1982; Reitsma et al., 1983; Lachman and Skoultchi, 1984; Dony et al., 1985; Einat et al., 1985; Bentley and Groudine, 1986b; Bianchi-Scarra et al., 1986; Siebenlist et al., 1988), while for some cell types this decrease occurs only during terminal differentiation (Campisi et al., 1984; Gonda and Metcalf, 1984; Resnitzky et al., 1986). In mouse erythroleukemia (MEL) cells induced to differentiate by chemical agents, c-Myc expression displays a biphasic mode. The mRNA level initially decreases 10-fold as cells start to differentiate, before returning to a proliferative cell-like value and then finally declining to below basal levels at the onset of terminal differentiation (Mechti et al., 1986; Nepveu et al., 1987; Watson, 1988). A similar biphasic phenomenon was observed in the rat muscle cell line (L6E9-B)by Endo and Nadal-Ginard (1986), and also in murine P19 embryonic carcinoma cells (St. Arnaud et al., 1988). Such descriptive studies that correlate changes in c-myc expression to the differentiation process do not address the key issue as to whether the observed variations are causal or simply consequential to the start and maintenance of the differentiated state. More direct evidence for c-Myc playing a role in controlling the onset of differentiation has been provided by several studies using overexpressingof the c-myc gene or antisense oligonucleotides. Indeed, deregulated c-myc expression can block the induction of differentiation in a number of cultured cell lines (Freytag, 1988; Griep and Westphal, 1988; Larsson et al., 1988; Hoffman-Libermann and Libermann, 1991; Miner and Wold, 1991; Chisholm et al., 1992). Overexpression of c-myc inhibits cell differentiation of preadipocyte cells (3T3-L1), a reaction that is reversed by c-myc antisense RNAs. Interestingly, the overexpression of c-myc does not induce uncontrolled proliferation; cells still arrest in a GOG1 stage, but this stage is distinct from the GO-G1 stage required for differentiation, and the cells are not irreversibly withdrawn from the cell

c-Myc in Cell Cycle and Embryonic Development

129

cycle (Freytag, 1988). This approach has also been extended to transgenic mice, where the constitutive ectopic expression of c-Myc driven by a B-cellspecific promoter leads to an abnormally increased population of proliferative pre-B cells, unable to enter the differentiated state (Langdon et al., 1988; Schmidt et al., 1988). Differentiation was initiated in human HL60 cells by the presence of c-myc antisense RNA or DNA, which induced a decrease in the level of c-myc mRNA and therefore c-Myc protein (Yokoyama and Imamoto, 1987; Holt et al., 1988). Furthermore, c-myc antisense oligonucleotides made MEL cells more sensitive to differentiating agents (Lachman et al., 1986; Prochownick et al., 1988). These experiments were corroborated by constitutively expressing c-Myc from a plasmid in MEL cells, which made the cells insensitive to these agents (Dmitrovsky et al., 1986; Prochownick et al., 1988; Coppola et al., 1989). Overall these results suggest that a decrease in c-Myc expression is directly involved in the initiation and maintenance of the differentiated state. However, several contrary observations have been documented where c-myc is still expressed at relatively high levels during cellular differentiation in culture (Dotto et al., 1986; St. Arnaud et al., 1988; Bernard et al., 1992; Younus and Gilchrest, 1992; Craig et al., 1993). C-myc mRNA undergoes a transient elevation in differentiating cell lens (Nath et al., 1987), and studies of lens maturation in transgenic mice overexpression c-myc indicate that differentiation can occur in the presence of high levels of c-Myc (Morgenbesser et al., 1995).Furthermore, reexpression of c-Myc in terminally differentiated myogenic cells is unable to reverse their differentiated state, and c-myc expression is not sufficient to promote DNA synthesis in myotubes (Endo and Nadal-Ginard, 1986). Although posttranscriptional regulation might, at least in some cases, explain these observations (Dean et al., 1986a), it is clear that c-myc expression is not necessarily incompatible with cell cycle exit and differentiation. This suggests that terminal differentiation states might be regulated by a variety of mechanisms, only some of which respond to c-myc expression. What is known of the mechanisms by which c-myc influences differentiation? It is widely thought that c-Myc, in conjunction with its heterodimeric partner Max, is involved in the activation of genes promoting proliferation, a process that may be antagonized by the transcriptional repressors Mad1 (Ayer et al., 1993) and Mxil Zervos et al., 1993). These proteins also heterodimerize with Max and bind identical DNA sequences to Myc/Max complexes. An emerging view is that replacement of c-Myc/Max by Mad/Max represses Myc target genes, thereby facilitating the differentiation pathway. Indeed, levels of Mxil mRNA increase upon myeloid cell differentiation (Zervos et al., 1993), and Mad/Max protein complexes accumulate at the expense of Myc/Max complexes as human myeloid leukemic cell lines differentiate (Ayer and Eisenman, 1993). Similar observations have been

130

lean-Marc Lemaitre et al.

made during keratinocyte differentiation (Hurlin et al., 1995a) and in developing mouse embryos (Chin et al., 1995; Hurlin et al., 1995b). Antagonism between c-Myc and the transcription factor C/EBPa also appears important for modulating the switch between proliferation and differentiation in adipocytes. C/EBPa is both necessary and sufficient for adipogenesis, and its expression is strongly induced as 3T3-Ll preadipocytes commit to the differentiation pathway. High levels of c-Myc can block cell cycle exit by suppressing the expression of C/EBPa, while, conversely, a constitutive overexpression of C/EBPaovercomes this differentiation block (Freytag and Geddes, 1992).Transcriptional repression by c-Myc of differentiation-specific genes is also observed in MEL cells, where differentiation may be blocked by constitutive c-Myc expression. The histone H10 and H 1 variant genes are normally expressed as MEL cells are induced to differentiate, but in this instance c-Myc suppresses their transcription (Cheng and Skoultchi, 1989).

VIII. C-MYCAND APOPTOSIS During the last decade it has become increasingly apparent that programmed cell death, or apoptosis, is a fundamental process affecting both growth and development. Furthermore, and somewhat counterintuitively, numerous studies also suggest that apoptosis and cell proliferation are linked, and a variety of oncogenes have been shown to be potent inducers of both processes, suggesting that common mechanisms may be involved (Evan et al., 1995). C-myc is one such oncogene. Deregulated expression of c-myc leads to increased cell death by apoptosis in a variety of physiological situations. Increased c-myc expression has been linked to apoptosis in the regression of human mammary tumors (Kyprianou et al., 1991) and rat prostate tumors (Quarmby et al., 1987), and correlates with the induction of apoptosis in embryonic thymocytes (Riegel et al., 1990).Furthermore, increased levels of apoptosis are observed in the lymphocytes of transgenic mice constitutively expressing c-myc in the lymphoid cell lineage (Dyall and Cory, 1988; Neiman et al., 1991). Parallel to these physiological observations, it was also demonstrated that c-Myc induces apoptosis in some cultured cells lines under suboptimal growth conditions. In myeloid cells whose growth depended upon the presence of the cytokine interleukin (1L)-3, constitutive c-myc expression leads to increased levels of apoptosis following withdrawal of IL-3 (Askew et al., 1991). Furthermore, basal ectopic c-Myc expression induced apoptosis in primary or immortalized fibroblasts, provided that proliferation is blocked

c-Myc in Cell Cycle a n d Embryonic Development

131

by serum starvation or the addition of antiproliferative drugs or cytokines (Evan et al., 1992). C-myc expression is also required for receptor-mediated apoptosis in T-cell hybridomas (Shi et al., 1992). In this study, the inhibition of c-myc expression by antisense mRNA blocked both apoptosis and proliferation, whereas a similar inhibition of c-fos prevented only proliferation, indicating a specific role for c-myc in the apoptotic process.

A. Induction of Apoptosis or Proliferation Appears Mechanistically Related Clues to the mechanism by which c-myc induces apoptosis are supplied by mapping the regions of the c-Myc protein required for this activity. Interestingly, they correspond to the N-terminal transactivation and C-terminal DNA-binding and protein dimerization domains, which are also those required for transformation by c-Myc (Evan et al., 1992). Dimerization with its heterodimeric partner Max is also essential for apoptosis (Amati et al., 1993b), suggesting that transcriptional activation may be involved. Thus Myc/Max dimers may activate specific or overlapping sets of genes to promote either proliferation or apoptosis. Mechanisms common to both proliferation and apoptosis are suggested by studies showing that c-Myc-induced apoptosis is associated with an increase in cyclin A transcription (Hoang et al., 1994); transcription of the cyclin A gene is also induced by c-Myc during cellular proliferation (JansenDurr et al., 1993). Another target gene for c-Myc, the ODC gene, is also implicated in both processes. Although ODC is normally required for S-phase progression, overexpression of ODC leads to an increased sensitivity to apoptosis. Furthermore, in cells overexpressing c-myc, inhibition of ODC transcription results in a decrease in apoptosis (Packham and Cleveland, 1994). One important difference in the regulation of proliferation and apoptosis by c-Myc is indicated by the observation that c-Myc-induced apoptosis is independent of cell cycle position (Harrington et al., 1994). Apoptosis may occur in G1, after the induction of c-myc in quiescent (GO) cells (Evan et al., 1992) or in the S-G2 period of the cell cycle after an ectopic expression of c-myc in S phase (Harrington et al., 1994).

B. c-Myc and p 5 3 in Apoptosis lnduction of apoptosis by c-Myc appears to function in a p53-dependent manner, although recent data suggest p53-independent pathways may also exist. The tumor suppressor protein p53 is a key regulator of the G1-S

132

Jean-MarcLemaitre et al.

checkpoint, and functions to prevent the amplification of cells containing abberant genomes. This is effected by either triggering the apoptotic pathway or arresting the cell cycle to allow DNA repair (reviewed in Cox and Lane, 1995). Evidence supporting a requirement for p53 in c-myc-induced apoptosis is provided by studies using a homozygous p53-deficient cell line, where ectopic expression of c-Myc induced serum-starved cells to reenter the cell cycle but did not trigger apoptosis (Hermeking and Eick, 1994). The mechanism by which p53 mediates c-Myc-induced apoptosis in this case is independent of its ability to arrest the cell cycle or induce transcription of the cell cycle inhibitor p21 (Wagner et al., 1994). However, a recent analysis of cellular proliferation and apoptosis during lymphomagenesis in p53 knockout mice suggests that c-myc-induced apoptosis is unaltered in the absence of p53 (Hsu et al., 1995). The notion of a p53-independent mechanism is also supported by the finding that c-myc can induce apoptosis in S phase (Harrington et al., 1994), after the G1-S checkpoint where p53 is thought to act.

C. c-Myc

and bcl-2 in Apoptosls

If c-Myc can induce both of these processes, how could they be modulated independently? A key regulator is the proto-oncogene bcl-2, an inhibitor of apoptosis. Bcl-2 exhibits oncogenic synergy with c-myc, allowing cells aberrantly expressing c-myc to proliferate unchecked (Vaux et al., 1988; Strasser et al., 1990; Nunez et al., 1990). The mechanism of bcl-2 action is unclear, although bcl-2 does not cooperate with c-myc in the classical cotransformation assay as shown by ras (Reed et al., 1993). Direct evidence for the role of bcl-2 is provided by the finding that bcl-2 can block c-Myc-induced apoptosis in growth-suppressed fibroblasts (Bissonette et a/., 1992; Fanidi et al., 1992). Bcl-2 can also block apoptosis induced by tumor necrosis factor-a (TNF-a) (Klefstrom et al., 1994). c-Myc is a downstream effector of TNFinduced apoptosis, as indicated by the increased sensitivity of c-myc-overexpressing cell lines to TNF-induced apoptosis (Klefstrom et al., 1994), and the ability of antisense c-myc oligonucleotides to make cell lines TNF resistant (Janicke et al., 1994). The oncogenic cooperation of c-myc with bcl-2 is highly analogous to that observed with the adenovirus proteins E1A and E1B. This is of particular interest since E1A is responsible for promoting cellular proliferation upon adenovirus infection, and appears to function by using mechanisms similar to those of the host c-Myc protein. ElA is also a strong inducer of apoptosis (White et al., 1992), and ElB functions to suppress this activity, allowing the host cell to survive and thereby promoting viral proliferation (Debbas and White, 1993). Intriguingly, Bcl-2 also restricts E l A-induced apoptosis, and can directly replace E1B function (Rao et al., 1992).

c-Myc in Cell Cycle and Embryonic Development

I33

D. Apoptosis or Proliferation? An attractive model to explain how cellular fate is chosen after the simultaneous induction of the apoptotic and proliferative pathways by c-Myc has been proposed by Evan and Littlewood (1993; see Fig. 8). Achievement of one or the other of these cellular programs is dependent on the secondary action of survival factors such as bcl-2 or cytostatic factors such as transforming growth factor-p and interferon. The requirement for dimerization with Max in both these processes (Amati et al., 1993b) suggests that, once established, regulation is dependent upon downstream sets of target genes. Such Myc-Max activated genes might be common to the two processes or totally independent. It is also possible that Myc-induced regulation of these two programs might be achieved by as yet unidentified nontranscriptional mechanisms. This model further suggests how such a system might safeguard the development of an organism. Since all cells undergoing proliferation would also be primed for apoptosis, only those receiving the appropriate survival signals would continue to grow. Cells undergoing aberrant proliferation due to a single genetic lesion would not receive these signals, activating the apoptotic pathway and resulting in their elimination. For example, cells proliferating due to deregulated c-myc expression would normally undergo apoptosis, unless a survival factor such as bcl-2 was activated due to a specific developmental program or, in the case of carcinogenesis, by a secondary genetic mutation.

Mitogenic agents

I Survival factors Fig. 8 Model for the induction of apoptosis and proliferation mediated by c-Myc. Induction of c-Myc expression by mitogenic agents seems to be responsible for the simultaneous induction of both the apoptotic and proliferative pathways. Achievement of one or other of these cellular programs is dependent upon the secondary action of survival factors or cytostatic factors, which block the apoptotic or proliferative pathways, respectively.

I34

Jean-MarcLemaitre et at.

An alternative viewpoint is the so-called conflict of interest model, whereby apoptosis is induced by contradictory cell cycle signals. In this scenario, activation of the proliferative pathway by mutation in a gene such as c-myc might lead to cell cycle progression beyond the G1-S restriction point. However, unless the appropriate signals for the continuation of S phase were present, the conflicting signals within the cell would lead to activation of cellular suicide. Secondary mutations would thus be required to escape the resulting apoptosis and allow uncontrolled proliferation. However, an argument against this second model is the observation that c-Myc-inducedapoptosis is independent of cell cycle position (Harrington et al., 1994). The characterization of c-Myc as a key component of the apoptotic process suggests that c-Myc functions as more than simply an inducer of cellular proliferation. Thus c-Myc might have a more fundamental role as an essential regulator of cell fate, being at the crossroads of the proliferation and differentiation programs.

REFERENCES Adnane, J., and Robbins, P. D. (1995). Oncogene 10,381-387. Afar, D. E., Goga, A., McLaughlin, J., Wine, 0. N., and Sawyers, C. L. (1994). Science 264, 424-426. Albert, T., Urlbauer, B., Kohlhuber, F., Hammersen, B., and Eick, D. (1994). Oncogene 9,759763. Alex, R., Sozeri, O., Mayer, S., and Dildrop, R. (1992). Nucleic Acids Res. 20, 2257-2263. Alexandrova, N., Niklinski, J., Blikovsky, V., Otterson, G. A., Blake, M., Kaye, F., J., and ZajacKaye, M. (1995). Mol. Cell. Biol. 15, 5188-5195. Alvarez, E., Northwood, 1. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991). J. Biol. Chem. 266, 15277-15285. Amati, B., Dalton, S., Brooks, M. W., Littlewood, T. D., Evan, G. I., and Land, E. H. (1992). Nature (London) 359,423-426. Amati, B., Brooks, M. W., Levy, N., Littlewood, T. D., Evan, G. I., and Land, H. (1993a). Cell 72,233-245. Amati, B., Littlewood, T. D., Evan, G. I., and Land, H. (1993b). EMBOJ. 12, 5083-5087. Amin, C., Wagner, A. J., and Hay, N. (1993). Mol. Cell. Biol. 13, 383-390. Ariga, H., Imamura, Y., and Iguchi-Ariga, S. M. (1989). E M B O J. 8,4273-4279. Armelin, H. A., Armelin, M. C., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984). Nature (London) 310,655-660, Arsura, M., Deshpande, A., Ham, S. R., and Sonenshein, G. E. (1995). Mol. Cell. Biol. 15, 6702-6709. Askew, D., Ashman, R., Simmons, B., and Cleveland, K. (1991). Oncogene 6, 1915-1922. Auvinen, M., Paasinen-Sohns, A,, Hirai, H., Anderson, L. C., and Holtta, E. (1995).Mol. Cell. Biol. 15, 6513-6525. Ayer, D. E., and Eisenman, R. N. (1993). Genes Dev. 7,2110-2119. Ayer, D. E., Kretzner, L., and Eisenman, R. N. (1993). Cell 72,211-222. Ayer, D. E., Lawrence, Q. A., and Eisenman, R. N. (1995). Cell 80, 767-776. Balasundaram, D., Tabor, C. W., andTabor, H. (1991).Proc. Natl. Acad. Sci. U.S.A. 88,58725877.

c-Myc in Cell Cycle and Embryonic Development

135

Barone, M. V., and Courtneidge, S. A. (1995). Nature (London) 378,509-512. Beckman, H., and Kadesch, T. (1991). Genes Dev. 5, 1057-1066. Beckman, H., Su, L. K., and Kadesch, T. (1990). Genes Dev. 4, 167-179. Beijersbergen, R. L., Hijmans, E. M., Zhu, L., and Bernards, R. (1994). EMBOJ. 13,40804086. Beimling, P., Benter, T., Sander, T., and Moelling, K. (1985). Biochemistry 24, 6349-6355. Bello-Fernandez, C., Packham, G., and Cleveland, J. L. (1993).Proc. Natl. Acad. Sci. U.S.A. 90, 7804-7808. Bentley, D. L., and Groudine, M. (1986a).Mol. Cell. Biol. 6, 3481-3489. Bentley, D. L., and Groudine, M. (1986b). Nature 321, 702-6. Benvenisty, N., Leder, A., Kuo, A., and Leder, P. (1992). Genes Dev.6,2513-2523. Berberich, S. J., and Cole, M. D. (1992). Genes Dev. 6, 166-176. Berberich, S., Trivedi, A., Daniel, D. C., Johnson, E. M., and Leffak, M. (1995).J. Mol. Biol. 245,92-109. Bernard, O., Cory, S., Gerondakis, S., Webb, E., and Adams, J. A. (1983). EMBO J. 2,23752383. Bernard, O., Drago, J., and Sheng, H. (1992). Neuron 9, 1217-1224. Bhatia, K., Huppi, K., Spangler, G., Siwarski, D., Iyer, R., and Magrath, 1. (1993). Genet 5,5661. Bianchi-Scarra, G. L., Romani, M., Coviello, D. A., Garre, C., Ravazzolo, R., Vidali, G., and Ajmar, F. (1986). Cancer Res. 46, 6327-6332. Bissonnette, R., Echeverri, F., Mahboubi, A., and Green, D. (1992). Nature (London) 359, 552-554. Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., and Weintraub, H. (1990). Science 250, 1149-1151. Blackwell, T. K., Huang, J., Ma, A., Kretzner, L., Aly, F. W., Eisenman, R. N., and Weintraub, H. (1993).Mol. CeN. Biol. 13,5216-5224. Blackwood, E. M., and Eisenman, R. N. (1991). Science 251, 1211-1217. Blackwood, E. M., Liischer, B., and Eisenman, R. N. (1992).Genes Dev. 6, 71-80. Blackwood, E. M., Gross Lugo, T., Kretzner, L., King, M. W., Street, A. J., Witte, 0. N., and Eisenman, R. N. (1994). Mol. Biol. Cell 5 , 597-609. Blanchard, J., Piechaczyk, M., Dani, C., Chambard, J. C., Franchi, A., Pouyssegur, J., and Jeanteur, P. (1985).Nature (London) 317,443-445. Born, T. L., Frost, J. A., Schonthal, A., Prendergast, G. C., and Feramisco, J. R. (1994). Mol. Cell. Biol. 14, 5710-5718. Bourbon, H. M., Prudhomme, M., and Almaric, F. (1988). Gene 68, 73-84. Bousset, K., Henriksson, M., Liischer-Firzlaff,J. M., Litchfield, D. W., and Liischer, B. (1993). Oncogene 8, 3211-3220. Buckle, R. S., and Mechali, M. (1995). Oncogene 10, 1249-1255. Busch, H., Reddy, R., Rothblum, L., and Choi, Y. C. (1982). Annu. Rev. Biochem. 51, 617654. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. Z. (1984). Cell 36,241247. Carr, C. S., and Sharp, P. A. (1990).Mol. Cell. Biol. 10, 4384-4388. Cerni, C., Bousset, K., Seelos, C., Burkhardt, H., Henriksson, M., and Liischer, B. (1995). Oncogene 11,587-596. Charron, J., Malynn, B. A., Fisher, P., Stewart, V., Jeanotte, L., Goff, S. P., Robertson, G. J., and Alt, F. W. (1992).Genes Deu. 6, 2248-2257. Cheng, G., and Skoultchi, A. I. (1989). Mol. Cell. Biol. 9, 2332-2340. Chin, L., Schreiber-Agus, N., Pellicer, I., Chen, K., Lee, H. W., Dudast, M., Cordon, C. C., and Depinho, R. A. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 8488-8492. Chisholm, O., Stapleton, P., and Symonds, G. (1992). Oncogene 7, 1827-1836.

I36

Jean-Marc Lemaitre el al.

Chou, T. Y., Hart, G. W., and Dang, C. V. (1995).J. Biol. Cbern. 270, 18961-18965. Churchill, M. E., and Suzuki, M. (1989).E M B O I. 8, 4189-4195. Classon, M., Henriksson, M., Klein, G., and Symegi, J. (1990). Oncogene 5, 1371-1376. Classon, M., Wennborg, A., Henriksson, M., and Klein, G. (1993). Gene 133, 153-161. Cleveland, J. L., Dean, M., Rosenberg, N., Wang, J.Y., and Rapp, U. R. (1989). Mol. Cell. Biol. 9,5685-5695. Cochran, B. H., Reffel, A. C., and Stiles, C. D. (1983). Cell 33, 939-947. Coffey, R. J., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E., and Moses, H. L. (1988).Mol. Cell. Biol. 8, 3088-3093. Coppola, J. A., and Cole, M. D. (1986).Nature (London) 320,760-763. Coppola, J. A., Parker, J. M., Schuler, G. D., and Cole, M. D. (1989).Mol. Cell. Biol. 9, 17141720. Coughlin, S. R., Lee, W. M., Williams, P. W., Giels, G. M., and Williams, L. T. (1985).CeN43, 243-25 1. Courtneidge, S. A., Dhand, R., Pilat, D., Twamley, G. M., Waterfield, M. D., and Roussel, M. F. (1993). E M B O 1. 12,943-950. Cox, L. S., and Lane, D. P. (1995). BioEssays 17, 501-508. Craig, R. W., Buchan, H. L., Civin, C. L., and Kastan, M. B. (1993). Cell Growth Differ.4, 349-357. Cutry, A. F., Kinniburgh, A. J., Krabak, M. J., Hiu, S.-W., and Wenner, C. E. (1989).1. Biol. Cbem. 264,19700-19705. Daksis, J. I., Lu, R. Y., Facchini, L. M., Martin, W. W., and Penn, L. J. (1994). Oncogene 9, 3635-3645. Dang, C. V., and Lee, W. M. (1988). Mol. Cell. Biol. 8,4084-4054. Dang, C. V., and Lee, W. M. (1989).J.Biol. Cbem. 264, 18019-18023. Dang, C. V., van Dam, H., Buckmire, M., and Lee, W. M. (1989). Mol. Cell. Biol. 9, 24772486. Davidson, E. H. (1986). “Gene Activity in Early Development,” 3rd ed. Academic Press, Orlando, FL. Davis, A. C., Wims, M., Spotts, G. D., Hann, S. R., and Bradley, A. (1993). Genes Dev. 7,671682. Dean, M., Levine, R. A., and Campisi, J. (1986a). Mol. Cell. Biol. 6, 518-524. Dean, M., Levine, R. A., Ran, W., Kindy, M. S., Sonenshein, G. E., and Campisi, J. (1986b).J. Biol. Cbern. 261, 9161-9166. Debbas, M., and White, E. (1993). Genes Dev. 7, 546-554. DePamphilis, M. L. (1988). Cell 52, 635-638. DePinho, R. A., Hatton, K. S., Tefaye, A., Yancopoulos, G. D., and Alt, F. W. (1987).Genes Dev. 1, 1321-1326. Dmitrovsky, E., Kuehl, W. M., Hollis, G. F., Kirsh, I. R., Bender, T. P., and Segal, S. (1986). Nature (London) 322,748-750. Dony, C., Kessel, M., and Gruss, P. (1985).Nature (London) 317, 636-639. Dotto, G. P., Gilman, M. Z., Maruyama, M., and Weinberg, R. A. (1986). EMBOJ. 5,28532857. Downs, K. M., Martin, G. R., and Bishop, J. M. (1989).Genes Dev. 3, 860-869. Du, H., Roy, A. L., and Roeder, R. G. (1993). EMBO J. 12,501-511. Dyall, S. D., and Cory, S. (1988). Oncogene Res. 2,403-409. Eilers, M., Schirm, S., and Bishop, J. M. (1991). E M B O J. 10, 133-141. Einat, M., Resnitzky, D., and Kimchi, A. (1985). Nature (London) 313,597-600. Eisenman, R. N., Tachibana, C. Y., Abrams, H. D., and Ham, S. R. (1985).Mol. Cell. Biol. 5, 114-126. El-Deiry, W. S., Harper, J. W., O’Connor, P. M., Velculescu, V. E., Canman, C. E., Jackman, J., Pietenpol, J. A., Burrell, M., Hill, D. E., and Wang, Y. (1994). Cancer Res. 54, 1169-1174.

c-Myc in Cell Cycle and Embryonic Development

,

137

Endo, T., and Nadal-Ginard, B. (1986).Mol. Cell. Biol. 6, 1412-1421. Eschenfeldt, W. H., and Berger, L. S. (1986).Proc. Natl. Acad. Sci. U.S.A. 83, 9403-9407. Evan, G. I., and Hancock, D. (1985). Cell 43,253-261. Evan, G. I., and Littlewood, T. D. (1993).Curr. Opin. Genet. Dev. 3,44-9. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, A., Brooks, M., Penn, L. Z., and Hancock, D. C. (1992). Cell 69, 119-128. Evan, G. I., Brown, L., White, M., and Harrington, E. (1995).Curr. Opin. Cell Biol. 7 , 825834. Fanidi, A., Harrington, E., and Evan, G. (1992), Nature (London)359, 554-556. Fisher, F., Crouch, D. H., Jayaraman, P., Clark, W., Gillespie, D. A., and Goding, C. R. (1993). E M B O 1. 12,2075-2082. Freytag, S. 0. (1988).Mol. Cell. Biol. 8, 1614-1624. Freytag, S. O., and Geddes, T. J. (1992).Science 256, 379-382. Freytag, S. O., Dang, C. V., and Lee, W. M. (1990). Cell Growth Differ. 1, 339-343. Frick, K. K., and Scher, C. D. (1990).Mol. Cell. Biol. 10, 184-192. Fryberg, L., Graf, T., and Vennstrom, B. (1987). Oacogene 1, 415-421. Garcia-Burtos, J., Heitman, J., and Hall, M. N. (1991).Biochim. Biophys. Acta 1071,83-101. Gaubatz, S., Imhof, A., Dosch, R., Werner, O., Mitchell, P., Buettner, R., and Eilers, M. (1995). E M B O J. 14,1508-1519. Gazin, C., Rigolet, M., Briand, J. P., van Regenmortel, M. H., and Galibert, F. (1986).E M B O J . 5,2241-2250, Geraudie, J., Hourdry, J., Vriz, S., Singer, M., and Mechali, M. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 3797-801. Gillespie, D. A., and Eisenman, R. N. (1989). Mol. Cell. Biol. 9, 865-868. Gomez-Marquez, J., and Sega, F. (1988).FEBS Lett. 226, 217-219. Gonda, T. J., and Metcalf, D. (1984).Nature (London)310, 345-347. Goodrich, D. W., and Lee, W. H. (1992).Nature (London)360, 177-179. Greenberg, M. E., and Ziff, E. B. (1984).Nature 311, 433-438. Greenberg, M. E., Greene, L. A., and Ziff, E. B. (1985). J. Biol. Chem. 260, 14101-14110. Gregor, P. D., Sanadogo, M., and Roeder, R. G. (1990).Genes Dev. 4, 1730-1740. Griep, A. E., and Westphal, H. (1988).Proc. Natl. Acad. Sci. U.S.A. 85, 6806-6810. Grignani, F., Lombardi, L., Inghirami, G., Sternas, L., Cechova, K., and Dalla-Favera, R. (1990).E M B O J. 9,3913-3922. Gu, W., Cechova, K., Tassi, V., and Dalla-Favera, R. (1993).Proc. Natl. Acad. Sci. U.S.A. 90, 2935-2939. Gu, W., Bhatia, K., Magrath, I. T., Dang, C. V., and Dalla-Favera, R. (1994).Science, 264,251254. Gualberto, A., Le Page, D., Pons, G., Mader, S. L., Park, K., Atchison, M. L., and Walsh, K. (1992).Mol. Cell. Biol. 12, 4209-4214. Gupta, S., and Davis, R. J. (1994).FEBS Lett. 353, 281-285. Gupta, S., Seth, A., and Davis, R. J. (1993). Proc. Nutl. Acad. Sci. U.S.A. 90, 3216-3220. Gusse, M., Ghysdael, J., Evan, G., Soussi, T., and Mechali, M. (1989).Mol. Cell. Biol. 9,53955403. Gutierrez, C., Guo, Z. S., Farrell-Towt, J., Ju, G., and DePamphilis, M. L. (1987).Mol. Cell. Biol. 7, 4594-4598. Gutierrez, C., Guo, 2. S., Burhans, W., and DePamphilis, M. L. (1988). Science 240, 12021203. Hamel, P. A., Gill, R. M., Phillips, R. A., and Gallie, B. L. (1992). Mol. Cell. Biol. 12,34313438. Hann, S. R., and Eisenman, R. N. (1984).Mol. Cell. Biol. 4, 2486-2497. Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985). Nature (London) 314, 366369.

I38

lean-Marc Lemaitre et al.

Hann, S. R., King, M. W., Bentley, D. L., Anderson, C . W., and Eisenman, R. N. (1988). Cell 52, 185-195. H a m , S. R., Sloan-Brown, K., and Spotts, G. (1992). Genes Dev. 6, 1229-1240. Hann, S. R., Dixit, M., Sears, R. C., and Sealy, L. (1994). Genes Dev. 8,2441-2452. Hanson, K. D., Shichiri, M., Follansbee, M. R., and Sedivy, J. M. (1994). Mol. Cell. Biol. 14, 5748-5 755. Harrington, E., Fanidi, A., Bennet, M., and Evan, G. (1994). EMBO ]. 13, 3286-3295. Harris, L. L., Talian, J. C., and Zelenka, P. S. (1992). Development 115, 813-820. Hateboer, G. H., Timmers, M., Rustgi, A. K., Billaud, M., van’t Veer, L. J., and Bernards, R. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 8489-8493. Heaney, M. L., Pierce, J., and Parson, J. T. (1986).]. Virol. 60, 167-176. Heby, O., and Persson, L. (1990). Trends Biochem. Sci. 172, 153-158. Heikkila, R., Schwab, G., Wickstrom, E., Loke S. L., Pluznik, D. H., Watt, R., and Neckers, L. M. (1987).Nature (London) 328, 445-449. Heintz, N. H. (1992). CUYY.Opin. Cell Biol. 4,459-467. Henriksson, M., Bakardjiev, A., Klein, G., and Lyscher, B. (1993). Oncogene 8, 3199-3209. Hermeking, H., and Eick, D. (1994). Science 265, 2091-2093. Hermeking, H., Funk, J. O., Reichert, M., Ellwart, J. W., and Eick, D. (1995). Oncogene 11, 1409-1415. Hershey, J. W. B. (1991). Annu. Rev. Biochern. 60,717-755. Hibshoosh, H., Johnson, M., and Weinstein, I. B. (1991). Oncogene 6, 739-743. Hoang, A. T., Cohen, K. J., Barratt, J. F., Bergtrom, D. A,, and Dang, C. V. (1994).Proc. Natl. Acad. Sci. U.S.A. 91, 6875-6879. Hoang, A. T., Lutterbach, B., Lewis, B. C., Yano, T., Chou, T. Y., Barett, J. F., Raffeld, M., Hann, S. R., and Dang, C . V. (1995). Mol. Cell. Biol. 15, 4031-4042. Hoffman-Libermann, B., and Libermann, D. A. (1991). Mol. Cell. Biol. 11, 3375-3381. Holt, J. T., Redner, R. L., and Nienhuis, A. W. (1988). Mol. Cell. Biol. 8, 963-973. Hopewell, R., and Ziff, E. B. (1995). Mol. Cell. Biol. 15, 3470-3478. Hourdry, J., Brulfert, A., Gusse, M., Schoevaert, D., Taylor, M. V., and Mechali, M. (1988). Development 104,631-641. Hsu, B., Marin, M. C., El-Naggar, A. K., Stephens, L. C., Brisbary, S., and McDonnell, T. J. (1995). Oncogene 11, 175-179. Hu, Q., Dyson, N., and Harlow, E. (1990).E M B O ] . 9, 1147-1155. Hu, Y.F., Luscher, B., Admon, A., Mermod, N., and Tijan, R. (1990). Genes Deu. 4, 17411752. Hurlin, P. J., Foley, K. P., Ayer, D. E., Eisenman, R. N., Hanahan, D., and Arbeit, J. M. (1995a). Oncogene 11,2487-2501. Hurlin, P. J., Queva, C., Koskinen, P. J., Steingrimsson, E., Ayer, D. E., Copeland, N. G., Jenkins, N. A., and Eisenman, R. N. (1995b). EMBO J. 14, 5646-5659. Ingvarsson, S., Asker, C., Axelson, H., Klein, G., and Sumegi, J. (1988). Mol. Cell. Biol. 8, 3168-3174. Jackson, T., Allard, M. F., Sreenan, C. M., Doss, L. K., Bishop, S. P., and Swain, J. L. (1990). Mol. Cell. Biol. 10, 3709-3716. Jaffredo, T., Vandenbunder, B., and Dieterlen-Lievre, F. (1989). Development 105, 679-695. Janicke, R. U., Lee, F. H. H., and Porter, A. G. (1994). Mol. Cell. Biol. 14, 5661-5670. Jansen-Durr, P., Meichle, A., Steiner, P., Pagano, M., Finke, K., Botz, J., Wessbecher,J., Draetta, G., and Eilers, M. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 3685-3690. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984). Cell 39,499-509. Karn, J., Watson, J. V., Lowe, A. D., Green, S. M., and Vedeckis, W. (1989). Oncogene 4,773787. Kato, G. J., and Dang, C . V. (1992). Fuseb]. 6, 3065-3072.

c-Myc in Cell Cycle and Embryonic Development

139

Kato, G. J., Barret, J., Villa-Garcia, M., and Dang, C. V. (1990).Mol. Cell. Biol. 10, 59145920. Kato, G. J., Lee, W. M., Chen, L., and Dang, C. V. (1992). Genes Dev. 6, 81-92. Katzav, S., Packlam, G., Sutherland, M., Aroca, P., Santos, E., and Cleveland, J. L. (1995). Onogene 11, 1079-1088. Kelly, K., and Siebenlist, U. (1986). Annu. Rev. Immunol. 4, 317-338. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983).Cell 35, 603-610. King, M. W., Roberts, J. M., and Eisenman, R. N. (1986). Mol. Cell. Biol. 6, 4499-4508. Klefstrom, J., Vastrik, I., Saksela, E., Valle, J., Eilers, M., and Alitalo, K. (1994). EMBO J. 13, 5442-5450. Kohl, N. E., Legouy, E., DePinho, R. A., Nisen, P. D., Smith, R. K., Gee, C. E., and Alt, F. W. (1986).Nature (London) 319, 73-77. Koskinen, P. J., Vastrik, I., Makela, T. P., Eisenman, R. N., and Alitalo, K. (1994). Cell Growth Differ. 5, 313-320. Kretzner, L., Blackwood, E. M., and Eisenman, R. N. (1992).Nature (London) 359,426-429. Kyprianou, N., English, H. F., Davidson, N. E., and Isaacs, J. T. (1991). Cancer Res. 51, 162166. Lachman, H. M., and Skoultchi, A. I. (1984). Nature (London) 310,593-594. Lachman, H. M., Cheng, G., and Skoultchi, A. I. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 6480-6484. Land, H., Parada, L. F., and Weinberg, R. A. (1983).Nature (London) 304,596-602. Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F., and Weinberg, R. A. (1986).Mol. Cell. Biol. 6, 1917-1925. Langdon, W. Y., Harris, A. W., Cory, S., and Adams, J. M. (1986). Cell 47, 11-18. Langdon, W. Y., Harris, A. W., and Cory, S. (1988). Oncogene Res. 3, 271-279. Larsson, L. G., Ivhed, I., Gidlund, M., Pettersson, U., Vennstrom, B., and Nilsson, K. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 2638-2642. Larsson, L. G., Petterson, M., Oberg, F., Nilsson, K., and Liischer, B. (1994). Oncogene 9, 1247-1252. La Thangue, N. B. (1994). Trends Biochem. Sci. 19, 108-114. Lau, L. F., and Nathans, D. (1987).Proc. Natl. Acad. Sci. U.S.A. 84, 1182-1186. Leder, A., Pattengale, P. K., Kuo, A., Stewart, T. A., and Leder, P. (1986). Cell 45, 485-495. Lee, W. M., Schwab, M., Westaway, D., and Varmus, H. E. (1985).Mol. Cell. Biol. 5, 33453356. Lemaitre, J. M., Michali, M., and Giraudie, J. (1992).1st. J. Biol. 36, 483-489. Lemaitre, J. M., Bocquet, S., Thierry, N., Buckle, R. S., and Mechali, M. (1994). Gene 150, 325-330. Lemaitre, J. M., Bocquet, S., Buckle, R. S., and Mechali, M. (1995).Mol. Cell. Biol. 15,50545062. Li, L.-H., Nevlov, C., Prendergast, G., Macgregor, D., and Ziff, E. B. (1994).EMBO I. 13, 4070-4079. Littlewood, T. D., Amati, B., Land, H., and Evan, G. I. (1992). Oncogene 7, 1783-1792. Loke, S. L., Stein, C., Zhang, X., Avigan, M., Cohen, J., and Neckers, L. M. (1988). Curr. Top. Microbiol. Immunol. 141,282-289. Lukas, L., Muller, H., Bartkova, J., Spitkovsky, D., Kjerulff, A. A., Jansen-Durr, P., Strauss, M., and Bartek, J. (1994).J. Cell Biol. 125, 625-638. Liischer, B., Kuenzel, E. A., Krebs, E. G., andEisenman, R. N. (1989).EMBOJ.8,1111-1119. Lutterbach, B., and Ham, S. R. (1994).Mol. Cell. Biol. 14, 5510-5522. Ma., A., Moroy, T., Collum, R., Weintraub, H., Alt, F. W., and Blackwell, T. K. (1993). Oncogene 8, 1093-1098. Maheswaran, S., Lee, H., and Sonenshein, G. E. (1994).Mol. Cell. Biol. 14, 1147-1152.

140

Jean-MarcLemaitre eta/.

Mai, S., and Jalava, A. (1994).Nucleic Acids Res. 22, 2264-2273. Makela, T., Koskinen, P., Vastrik, I., and Alitalo, K. (1992). Science 256, 373-377. Makino, R., Hagashi, K., and Sugimura, T. (1984). Nature (London) 310, 697-698. Marcu, K. B. (1987).BioEssays 6,28-82. Marsh, A. G., and Chen, T. T. (1995). Mol. Mar. Biol. Biotechnol. 4, 185-192. Mazan, S., and Bachellerie, J.-P. (1988).J. Biol. Chem. 263, 19461-19467. McWhinney, C., and Leffak, M. (1990). Nucleic Acids Res. 5, 1233-1242. Mechti, N., Piechaczyk, M., Blanchard, J. M., Marty, L., Bonnieu, A., Jeanteur, P., and Lebleu, B. (1986). Nucleic Acids Res. 14, 9653-9666. Miltenberger, R. J., Sukow, K. A., and Farnham, P. J. (1995). Mol. Cell. Biol. 15,2527-2535. Miner, J. H., and Wold, B. J. (1991).M o l . Cell. Biol. 11, 2842-2851. Mitchell, P. J., and Tijan, R. (1989).Science 245, 371-378. Mol, P. C., Wang, R. H., Batey, D. W., Lee, L. A., Dang, C. V., and Berger, S. L. (1995).Mol. Cell. Biol. 15, 6999-7009. Morello, D., Asselin, C., Lavenu, A., Marcu, K. B., and Babinet, C. (1989).Oncogene 4,955961. Morgenbesser, S. D., Schreiber-Agus,N., Bidder, M., Mahon, K. A., Overbeek, P. A., Homer, J., and DePinho, R. A. (1995). E M B O J. 14,743-756. Mori, M., Barnard, G. F., Staniunas, R. J., Jessup, J. M., Steele, G. D., and Chen, L. B. (1993). Oncogene 8,2821-2828. Moshier, J. A., Dosescu, J., Skunca, M., and Luk, G. D. (1993). Cancer Res. 53, 2618-2622. Mudryj, M., Hiebert, S. W., and Nevins, J. R. (1990). EMBO J. 9, 2179-2184. Mukherjee, B., Morgenbesser, S. D., and De Pinho, R. D. (1992). Genes Deu. 6, 1480-1492. Murakami, H., Sanderson, N. D., Nagy, P., Marino, P. A., Merlino, G., and Thorgeirsson, S. S. (1993). Cancer Res. 53, 1719-1723. Nath, P., Getzenberg,, R., Beebe, D., Pallansch, L., and Zelenka, P. (1987).Exp. Cell Res. 169, 215-222. Nau, M. M., Brooks, B. J., Battey, J., Suasville, E., and Gazdar, A. F. (1985). Nature (London) 318, 69-73. Negishi, Y., Nishita, Y., Saegusa, Y., Kakizaki, t., Galli, I., Kihara, F., Tamai, K., Miyajima, N., Iguchi-Ariga, S. M., and Ariga, H. (1994). Oncogene 9, 1133-1143. Neiman, P. E., Thomas, S. J., and Loring, G. (1991). Proc. Nutl. Acud. Sci. U.S.A. 88, 58575861. Nepveu, A., Marcu, K. B., Skoultchi, A. I., and Lachman, H. M. (1987). Genes Dev. 1,938945. Nevins, J. R. (1992). Nature (London) 358, 375-376. Nishikura, K. (1987). Oncol. Res. 1, 179-191. Nunez, G., London, L., Hockenbery, D., Alexander, M., McKearn, J. P., and Korsmeyer, S. J. (1990).J. Immunol. 144,3602-3610. Oswald, F., Lovec, H., Moroy, T., and Lipp, M. (1994). Oncogene 9,2029-2036. Packham, G., and Cleveland, J. L. (1994).Mol. Cell. Biol. 14,5741-5747. Pal, S. K., Crowell, R., Kiessling, A. A., and Cooper, G. M. (1993). Mol. Reprod. Dev. 35,815. Palmieri, S., Kahn, P., and Graf, T. (1983).EMBO J. 2, 2385-2389. Papas, T. S., and Lautenberger, J. A. (1985).Nature (London) 318,237. Papoulas, O., Williams, N. G., and Kingston, R. E. (1992).J. Biol. Chem. 267,10470-10480. Pardee, A. B. (1989). Science 246, 603-608. Paria, B. C., Dey, S. K., and Andrews, G. K. (1992).Proc. Natl. Acud. Sci. U.S.A. 89, 1005110055. Pegg, A. E. (1986).Biochem. 1. 234, 249-262. Penn, L. J. Z., Brooks, M. W., Laufer, E. M., and Land, H. (1990a), E M E O J . , 9, 1113-1121.

c-Myc in Cell Cycle and Embryonic Development

141

Penn, L. J. Z., Brooks, M. W., Laufer, E. M., Littlewood, T. D., Morgenstern, J. P., Evan, G. I., Lee, W. M., and Land, H. (1990b). Mol. Cell. Biol. 10, 4961-4966. Persson, H., and Leder, P. (1984).Science 225, 718-721. Pfeifer-Ohlsson, S., Rydnert, J., Goostin, A. S., Larsson, E., Betsholz, C., and Ohlsson, R. (1985).Proc. Nutl. Acad. Sci. U.S.A. 82, 5050-5054. Philipp, A., Schneider, A., Vasrik, I., Finke, K., Xiong, Y., Beach, D., Alitalo, K., and Eilers, M. (1994). Mol. Cell. Biol. 14, 4032-4043. Piechaczyk, M., Blanchard, J.-M., Bonnieu, A., Fort, P., Mechti, N., Rech, J., Cuny, M., Marty, L., Ferre, F., LeBleu, B., and Jeanteur, P. (1988). Gene 72, 287-295. Pietenpol, J. A., Stein, R. W., Moran, E., Yachik, P., Schelgel, R., Lyons, R. M., Pittlekow, M. R., Munger, K., and Howley, P. M. (1990). Cell 61, 777-785. Pohjanpelto, P., Virtanen, I., and Holtta, E. (1981). Nature (London) 293, 475-477. Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994). Genes Dev. 8, 9-22. Prendergast, G. C., and Ziff, E. B. (1991).Science 251, 186-189. Prendergast, G. C., and Lawe, D., and Ziff, E. B. (1991). Cell 65, 395-407. Prendergast, G. C., Hopewell, R., Gorham, B. J., and Ziff, E. B. (1992). Genes Dev. 6,24292439. Prochownik, E. V., and VanAntwerp, M. E. (1993).Proc. Natl. Acad. Sci. U.S.A. 90,960-964. Prochownik, E. V., Kukowska, J., and Rodgers, C. (1988).Mol. Cell. Biol. 8, 3683-3695. Pulverer, B. J., Fischer, C., Vousden, K., Littlewood, T., Evan, G. I., and Woodgett, J. R. (1994). Oncogene 9, 59-70. Qin, X. Q., Chittenden, T., Livingston, D., and Kaelin, W. G. (1992).Genes Dev. 6,953-964. Quarrnby, V., Beekman, W., Wilson, E., and French, F. (1987). Mol. Endocrinol. 1, 865-874. Rabbitts, T. H., Harnlyn, P. H., and Baer, R. (1983).Nature (London) 306, 760-765. Rabbitts, P., Watson, J. V., Lamond, A., Forster, A., Stinson, M. A., Evan, G., Fisher, W., Atherton, E., Sheppard, R., and Rabbitts, T. H. (1985). EMBO J. 4, 2009-2015. Ramsay, G., Evans, G., and Bishop. J. M. (1984).Proc. Nutl. Acad. Sci. U.S.A. 81,7742-7746. Ran, W., Dean, M., Levine, R. A., Henkle, C., and Campisi, J. (1986). Proc. Nutl. Acad. Sci. U.S.A. 83, 8216-8220. Rao, C. D., Pech, M., Robbins, K. C., and Aaronson, S. A. (1988).Mol. Cell. Biol. 8,284-292. Rao, L., Debbas, M., Sabbatini, P., Hockenborg, D., Korsmeyer, S., and White, E. (1992).Proc. Nutl. Acad. Sci. U.S.A. 89, 7742-7746. Ramer, L. (1989).Nucleic Acids Res. 17, 4101-4115. Reddy, C. D., Dasgupta, P., Saikumar, P., Dudek, H., Rauscher, F. J., and Reddy, E. P. (1992). Oncogene 7,2085-2092. Reed, J. C., Haldar, S., Croce, C. M., and Cuddy, M. P. (1993).Mol. Cell. Biol. 10,4370-4374. Reisman, D., Elkind, N. B., Roy, B., Beamon, J., and Rotter, V. (1993). Cell Growth Differ. 4, 57-65. Reitsma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Bar-shavit, Z., Hall, A., Tertelbaum, S. L., and Kahn, A. 1. (1983). Nature (London) 306, 492-494. Renshaw, M. W., Kipreks, E. T., Albrecht, M. R., and Wang, J. Y. (1992).EMBO J. 11,39413951. Resar, L. M., Dolde, C., Barrett, J. F., andDang, C. V. (1993).Mol. Cell. Biol. 13, 1130-1136. Resnitzky, D., Yarden, A., Zipori, D., and Kimchi, A. (1986).Cell 46, 31-40. Richardson, W. D., Roberts, B. L., and Smith, A. E. (1986).Cell 44, 77-85. Riegel, J. S., Richie, E. R., and Allison, J. P. (1990).J. Immunol. 144, 3611-3618. Riggs, K. J., Saleque, S., Wong, K. K., Merrell, K. T., Lee, J.-S., Shi, Y., and Calame, K. (1993). Mol. Cell. Biol. 13, 7487-7495. Rosenwald, 1. B., Rhoads, D. B., Callanan, L. D., Isselbacher, K. F., and Schmidt, E. V. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 6175-6178.

142

lean-Marc Lemaitre et al.

Roussel, M., Saule, S., Lagrou, C., Rommens, C., Beug, H., Graf, T., and Stehelin, D. (1979). Nature 281, 452-455. Roussel, M. F., Cleveland, J. L., Shurtleff, S. A., and Sherr, C. J. (1991). Nature (London) 353, 361-363. Roy, A. L., Meisterernst, M., Pognonec, P., and Roeder, R. G. (1991). Nature (London) 354, 245-248. Roy, A. L., Carruthers, C., Gutjahr, T., and Roeder, R. G. (1993).Nature (London)365,359361. Roy, B., Beamon, J., B a h t , E., and Reisman, D. (1994). Mol. Cell. Biol. 14, 7805-7815. Rustgi, A. K., Dyson, N., and Bernards, B. (1991). Nature (London) 352,541-544. St. Arnaud, R., Nepveu, A., Marcu, K. B., and McBurney, M. W. (1988). Oncogene 3,553559. Sarid, J., Halazonetis, T. D., Murphy, W., and Leder, P. (1987).Proc. Natl. Acad. Sci. U.S.A. 84, 170-173. Sawyers, C. L., Callahan, W., and Witte, 0. N. (1992). Cell 70, 901-910. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1991).Proc. Natl. A d . Sci. U.S.A. 88,253257. Schmid, P., Schulz, W. A., and Hameister, H. (1989). Science 243,226-229. Schmidt, E. V., Pattengale, P. K., Weir, L., and Leder, P. (1988).Proc. Natl. Acad. Sci. U.S.A. 85, 6047-6051. Schoenenberger, C. A., Andres, A. C., Groner, B., van der Valk, M., LeMeur, M., and Gerlinger, P. (1988). EMBO J. 7, 169-175. Schreiber-Agus, N., Horner, J., Torres, R., Chiu, F. C., and DePinho, R. A. (1993).Mol. Cell. Biol. 13, 2765-2775. Schreiber-Agus, N., Chin, L., Chen, K., Torres, R., Rao, G., Guida, P., Skoultchi, A., I., and DePinho, R. A. (1995). Cell 80, 777-786. Schwab, M., Alitalo, K., Klempnauer, K. H., Varmus, H. E., and Bishop, J. M. (1983).Nature (London) 305,245-248. Seth, A., Alvarez, E., Gupta, S., and Davis, R. J. (1991).J. Biol. Chem. 266, 23521-23524. Seth, A., Gupta, S., and Davis, R. J. (1993). Mol. Cell. Biol. 13, 4125-4136. Seto, E., Shi, Y., and Shenk, T. (1991). Nature (London) 354,241-245. Sheiness, D. K., and Bishop, J. M. (1979).J. Virol. 31,514-518. Sheiness, D. K., Hughes, S. H., Varmus, H. E., Stubblefield, E., and Bishop, J. M. (1980). J. Virol. 105, 415-424. Shi, Y., Glynn, J., Guilbert, L., Cotter, T., Bissonette, R., and Green, D. (1992). Science 257, 212-214. Shibuya, H., Yoneyama, M., Ninomiya-Tsuji, J., Matsumoto, K., and Taniguchi, T. (1992). Cell 70,57-67. Shrivastava, A., and Calame, K. (1995).Curr. Top. Microbiol. Immunol. 194, 273-282. Shrivastava, A., Saleque, S., Kaplana, G. V., Artandi, S., Goff, S. P., and Calame, K. (1993). Science 262, 1889-1892. Siebenlist, U., Bressler, P., and Kelly, K. (1988). Mol. Cell. Biol. 8, 867-874. Sinn, E., Muller, W., Pattengale, P., Tepler, I., Wallace, R., and Leder, P. (1987). Cell 49,465475. Solomon, D. L., Amati, B., and Land, H. (1993). Nucleic Acids Res. 21, 5372-5376. Sorrentino, V., Drozdoff, M. D., Mc Kinney, M. D., Zeitz, L., and Fleissner, E., (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 8167-8171. Spencer, C. A,, and Groudine, M. (1991). Adv. Cancer Res. 56, 1-48. Srivastava, M., Mc Bride, 0. W., Fleming, P. J., Pollard, H. B., and Burns, A. L. (1990).J. Biol. Chem. 265,14922-14931. Stanton, B. R., Perkins, A. S., Tessarolo, L., Sassoon, D. A., and Parada, L. F. (1992). Genes D ~ v .6,2235-2247.

c-Myc in Cell Cycle and Embryonic Development

143

Stanton, L. W., Fahrlander, P. D., Tesser, P. M., and Marcu, K. B. (1984).Nature (London)310, 423-425. Steiner, P., Philipp, A,, Lukas, J., Godden-Kent, D., Pagano, M., Mittnacht, S., Bartek, J., and Eilers, M. (1995).E M B O J. 14, 4814-4826. Stern, D. F., Roberts, A. B., Roche, N. S., Sporn, M. B., and Weinberg, R. A. (1986).Mol. Cell. Biol. 6, 870-877. Stone, J., De Lange, T., Ramsay, G., Jakobovits, E., Bishop, J. M., Varmus, H., and Lee, W. (1987).Mol. Cell. Biol. 7, 1697-1709. Strasser, A., Haris, A. W., and Cory, S. (1990).Cell 67, 889-899. Studzinski, G. P., Brelvi, Z. S., Feldman, S. C., and Watt, R. A. (1986). Science 234,467-470. Studzinski, G. P., Shankavaram, U. T., Moore, D. C., and Reddy, P. V. (1991).J. Cell. Physiol. 147,412-419. Sugiyama, A., Kume, A., Nemoto, K., Lee, S. Y., Asami, Y., Nemoto, F., Nishimura, S., and Kuchino, Y. (1989).Proc. Natl. Acad. Sci. U.S.A. 86, 9144-9148. Suzuki, M. (1989).J. Mol. Biol. 267, 61-84. Tabor, C. W., and Tabor, H. (1984).Annu. Rev. Biochem. 53,749-790. Tamemoto, H., Kadowaki, T., Tobe, K., Ueki, K., Izumi, T., Chatani, Y., Kohno, M., Kasuga, M., Yazaki, Y., and Akanuma, Y. (1992).J. Biol. Chem. 267, 20293-20297. Taylor, M. V., Gusse, M., Evan, G. I., Dathan, N., and Mechali, M. (1986).E M B O J. 5,35635370. Thalrneier, K., Synovzik, H., Mertz, R., Winnacker, E. L., and Lipp, M. (1989). Genes Dev. 3, 527-536. Thompson, C. B., Challoner, P. B., Neinmann, P. E., and Groudine, M. (1985).Nature (London) 314,363-366. Tobias, K. E., Shor, J., and Kahana, C. (1995).Oncogene 11, 1721-1727. Toyoshima, H., and Hunter, T. (1994). Cell 78, 67-74. Van Beneden, R. J., Watson, D. K., Chen, T. T., Laulenberger, J. A., and Papas, T. S. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 3698-3702. Vandenbunder, B., Pardanaud, L., Jaffedo, T., Mirabel, M. A., and Stehelin, D. (1989). Development 106,265-274. Van den Heuvel, S., and Harlow, E. (1993).Science 262, 2050-2054. Van den Ouweland, A. M., van Groningen, J. J., Hendriksen, P. J., Bloemers, H. P., and Van de Ven, W. J. (1987).Nucleic Acids Res. 15, 4349. Vastrik, I., Makela, T. P., Koskinen, P. J., and Alitalo, K. (1995).Oncogene 11, 553-560. Vaux, D. L., Cory, S., and Adams, J. M. (1988).Nature (London)335,440-442. Von Straaten, J. P., and Rabbitts, T. H. (1987). Oncogene Res. 1, 221-228. Vriz, S., Taylor, M. V., and Mechali, M. (1989).E M B O . J. 8, 4091-4097. Wagner, A. J., Meyers, C., Laimins, L. A., and Hay, N. (1993).Cell Growth Difer. 4,879-883. Wagner, A., Kokontis, J., and Hay, N. (1994). Genes Dev. 8, 2817-2830. Walker, C. W., Boom, J. D., and Marsh, A. G. (1992).Oncogene 7, 2007-2012. Wang, H., and Stillman, D. J. (1993).Mol. Cell. Biol. 13, 1805-1814. Waters, C. M., Littlewood, T. D., Hancock, D. C., Moore, J. P., and Evan, G. 1. (1991). Oncogene 6, 797-805. Watson, R. (1988).Mol. Cell. Biol. 8, 3938-3942. Watt, R., Shatzman, A. R., and Rosenberg, M. (1984).Mol. Cell. Biol. 5, 448-456. Watts, J. D., Cary, P. D., and Crane-Robinson, C. (1989).FEBS Lett. 245, 17-20. Weintraub, S. J., Prater, C. A., and Dean, D. C. (1992).Nature 358, 259-261. Wenzel, A., Cziepluch, C., Hamann, U., Schurmann, J., and Schwab, M. (1991).E M B O ] . 10, 3703-3712. Westin, E. H., Wong-Staal, F., Gelmann, E. P., Dalla-Favera, R., Papas, T. S., Lautenberger, J. A., Eva, A., Reddy, E. P., Tronick, S. R., Aaronson, S. A., and Gallo, R. C. (1982). PYOC. Natl. Acad. Sci. U.S.A. 79, 2490-2494.

144

Jean-Marc Lemaitre et al.

White, E., Sabbatini, P., Debbas, M., Wold, W., and Kusler, D. (1992). Mol. Cell. Biol. 12, 2570-2580. Winquist, R., Saksela, K., and Alitalo, K. (1984). EMBO 1. 3, 2947-2950. Wong, K. K., Hardin, J. D., Boast, S., Cooper, C. L., Merell, K. T., Doyle, T. G., Goff, S. P., and Calame, K. L. (1995a). Oncogene 10, 705-711. Wong, K. K., Zou, X., Merell, K. T., Patel, A. J., Marcu, K. B., Chellappan, S., and Calame, K. (1995b). Mol. Cell. Biol. 15, 6535-6544. Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993). Nature (London) 366,701-704. Yang, B. S., Gilbert, J. D., and Freytag, S. 0. (1993). Mol. Cell. Biol. 13, 3093-3102. Yano, T., Sander, C. A., Clark, H. M., Dolezal, M. V., Jaffe, E. S., and Raffeld, M. (1993). Oncogene 8,2741-2748. Yokoyama, K., and Imamoto, F. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 7363-7367. Younus, J., and Gilchrist, B. A. (1992).1. Cell. Physiol. 152, 232-239. Zervos, A. S., Cyuris, J., and Brent, R. (1993). Cell 72,223-232. Zervos, A. S., Faccio, L., Gatto, J. P., Kyriakis, J. M., and Brent, R. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 10531-10534. Zhu, L., van den Heuvel, S., Helin, K., Fattaey, A., Ewen, M., Livingston, D., Dyson, N., and Harlow, E. (1993). Genes Dev. 7, 1111-1125. Zimmermann, K., and Alt, F. W. (1990). Crit. Rev. Oncog. 2,75-95. Zimmermann, K. A., Yancopoulos, G. D., Collum, R. G., Smith, R. K., Kohl, N. E., Denis, K. A., Nau, M. M., Witte, 0. N., Toran-Allerand, D., Gee, C. E., Minna, J. D., and Alt, F. W. (1986).Nature (London) 319, 781-783.

Identification of the Genes Encoding Cancer Antigens: Implications for Cancer Immunotherapy Steven A. Rosenberg, Yutaka Kawakami, Paul F. Robbins, and Rong-fu Wang Surgery Branch, Division of Clinical Science National Cancer Institute, Bethesda, Maryland 20892

I. Introduction 11. Methodology 111. Human Melanoma Antigens Recognized by T Cells A. Melanocyte Lineage Antigens B. Tyrosinase C. MART-l/Melan-A D. gpl00 E. Tyrosinase-Related Protein-1 (gp7S) F. Melanoma Antigens Expressed in Normal Testes or Placenta C . Implications of the T-cell Response to Nonmutated Epitopes H. Tumor-Specific Antigens IV. Cancer Therapies Based on the Molecular Identification of Cancer Antigens References

1. INTRODUCTION The development of new cancer therapies based on stimulating the immune response of the host against a growing malignancy is based on the hypothesis that cancers contain, on their surface, unique antigens that can serve as targets for immune destruction. The validity of this hypothesis was bolstered by the development of immune manipulations that could mediate the rejection of growing cancers in humans and by the identification of T lymphocytes that could specifically recognize cancer cells in vituo. Interleukin (1L)-2 is a cytokine that plays an important role in immune regulation. IL-2 has no direct impact on cancer cells and yet the administration of IL-2 to patients with metastatic melanoma or metastatic kidney Advances in CANCER RESEARCH, Vol. 70

146

Steven A. Rosenberg et al.

cancer can mediate significant clinical rejection of established growing malignancy in about 20% of patients (Rosenberg et ul., 1994). The predominant role of IL-2 in stimulating the growth of activated T cells strongly suggested that the immune destruction following IL-2 administration resulted from a stimulation of an immune reaction against antigens selectively present on the surface of cancer cells. A variety of techniques have been developed that enable the identification of T lymphocytes that selectively recognize tumor antigens. Peripheral blood lymphocytes can be sensitized in vitro to tumor cells and give rise to lymphocytes with the ability to selectively lyse cancer and not normal cells from the autologous patient (Darrow et al., 1989). The development of techniques for growing tumor-infiltrating lymphocytes (TIL) provided a ready means for generating lymphocytes with antitumor reactivity (Muul et al., 1987; Topalian et al., 1987; Itoh et al., 1986). TIL can be grown from about 50% of patients with metastatic melanoma that have the ability to recognize selectively, and in a major histocompatibility complex (MHC)-restricted fashion, tumor cells from the autologous patient. Studies using tumors matched at MHC loci or tumors transfected with MHC genes have demonstrated that melanomas contain broadly expressed shared antigens that are recognized in the context of different class I MHC loci (Crowley et al., 1991; Hom et al., 1991; Schwartzentruber et al., 1991; Kawakami et al., 1992; O’Neil et al., 1993). The use of recombinant DNA expression cloning techniques has enabled the isolation of a variety of genes that encode the antigens recognized by tumor-reactive T cells. The recognition of a tumor antigen by a T cell in vitro, however, does not necessarily imply that these antigens are important in the in vivo immune response of a patient against a growing malignancy. In an attempt to identify the tumor antigens involved in in vivo tumor rejection responses in humans with cancer, we have combined in vivo human studies with in vitro laboratory efforts. TIL from resected metastatic deposits were expanded to large numbers in vitro and administered intravenously to the autologous patient along with IL-2, the requisite growth factor for these TIL (Rosenberg et al., 1988, 1995; Rosenberg, 1991). Using information from these human clinical trials, TIL were identified that were capable of mediating the rejection of established cancer in humans. These TIL, with known in vivo antitumor activity, were then used to clone the genes that encoded the antigens they recognized. A variety of both shared and unique tumor antigens have been identified utilizing this approach (Kawakami et al., 1994a,b; Robbins et al., 1994, 1995, 1996; Wang et al., 1995, 1996). The antigens thus identified have been different in many cases from the antigens identified utilizing T cells that were generated by in vitro sensitization of peripheral blood lymphocytes against autologous tumor cells. The

Genes Encoding Cancer Antigens

147

results of these studies, with an emphasis on the antigens identified by TIL with in vivo antitumor activity, serve as the basis for this review.

11. METHODOLOGY A variety of methods have been used to identify human melanoma antigens recognized by T cells. Known melanoma-associated proteins have been tested for recognition by tumor-reactive T cells. A strong correlation was found between reactivity with the monoclonal antibody HMB45 and lysis of melanoma cells by human lymphocyte antigen (HLA)-A2-restricted melanomareactive TIL (Kawakami et af., 1993). This protein was subsequently shown to be recognized by melanoma-reactive cytotoxic T lymphocytes (CTL) by evaluating the recognition of cells transfected with the gpl00 gene, which encoded the HMB45 antigen (Kawakami et al., 1994b; Bakker et af., 1994). Two peptides derived from the melanoma antigen MAGE-3, EVDPIGHLY (Cellis et af., 1994) and FLWGPRALV (van der Bruggen et al., 1994) were identified as melanoma T-cell epitopes by in vitro induction of specific CTL using peptide-pulsed stimulator cells. This technique, however, may result in the induction of T cells bearing T-cell receptors with low affinity for these peptides. These T cells may not recognize tumor cells, which express relatively low levels of the peptide-MHC complexes on their surfaces. Another method that has been utilized is the direct isolation and sequencing of antigenic peptides on tumor cells. Peptides isolated by acid treatment of melanoma cells or elution from HLA-A2 molecules that had been affinity purified from melanoma cells were fractionated on reverse-phase high-performance liquid chromatography (HPLC) columns and pulsed on indicator cells, such as T2 cells, that expressed HLA-A2. Several fractions were recognized by CTL established from different melanoma patients, indicating that these represented shared or common antigens (Slingluff et al., 1993; Storkus et af., 1993). Two melanoma antigen epitopes that are recognized by HLAA2-restricted melanoma-reactive CTL have now been identified by sequencing peptides obtained from active fractions through the use of mass spectrometry: YLEPGPVTA, derived from gpl00 (Cox et al., 1994), and ILTVILGVL, derived from MART-1 (Castelli et al., 1995). This technique has the advantage that it can directly identify antigenic peptides that are naturally processed and presented on the tumor cell surface. In some cases, however, it may be difficult to obtain sufficient amounts of peptides to allow sequence determination, since many of the melanoma epitopes appear to bind MHC class I with an intermediate affinity and may be present at relatively low levels on the cell surface. The isolated peptides may also, in

I48

Steven A. Rosenberg et al.

some cases, be derived from unknown proteins, which would then require the use of other techniques such as gene cloning methods in order to carry out further characterization of the antigen. The majority of the melanoma antigens recognized by T cells have been identified through the use of DNA expression cloning systems. MAGE-1 was first isolated as a human melanoma antigen by screening a melanoma genomic DNA library (van der Bruggen et al., 1991). A melanoma antigen loss variant cell line was stably transfected with a DNA library, and transfeaed clones were screened for their ability to stimulate tumor necrosis factor-a release from a melanoma-reactive CTL clone. MART-1 and gpl00 were isolated following stable transfection of the HLA-AZpositive MDA23 1 breast cancer cell line with a melanoma cDNA library (Kawakami et al., 1994a,b). Stable transfectant clones were screened for their ability to stimulate interferon-y release from HLA-A2-restricted melanoma TIL. Alternatively, cDNA pools have been transiently transfeaed into highly transfectable cell lines such as COS or 293 cells along with genes encoding the appropriate MHC class I restriction element, and transfectants then screened for their ability to stimulate cytokine release from tumor-reactive T cells. Many melanoma antigens, including tyrosinase (Robbins et al., 1994; Brichard et al., 1993), Melan-A (Coulie et al., 1994), which is identical to MART-1, tyrosinaserelated protein-1 (TRP-1; gp75) (Wang et al., 1995), p l 5 (Robbins et al., 1995), BAGE (Boel et al., 1995), GAGE (Van Den Eynde et al., 1995), p-catenin (Robbins et al., 1996), melanoma ubiquitous mutated (MUM)-1 (Coulis et al., 1995) and cyclin-dependent kinase (cdk) 4 (Wolfel et al., 1995), have been isolated using this method. Although this method requires the identification and cloning of the genes encoding the appropriate MHC class I restriction element, large numbers of cDNA clones can readily be screened using this technique. The high levels of protein expression that are possible using these techniques may, however, result in the isolation of genes encoding cross-reactive epitopes that do not represent the naturally processed peptide. ~The T-cell epitopes derived from the isolated melanomaantigens have been identified using a number of different approaches. The regions containing T-cell epitopes have generally been identified by testing T-cell recognition of cells transfected with truncated cDNA fragments generated by sequential deletions carried out with exonuclease 111 or by the generation of gene fragments through the use of the polymerase chain reaction (PCR). In some cases, sequence comparisons between isolated genes and other members of multigene families or normal genes aided in the identification of the regions containing the epitopes. The peptide MHC-binding motifs have also been used to further identify epitopes. Candidate peptides were then synthesized and tested for their ability to sensitize target cells expressing the appropriate MHC class I products for recognition by melanoma-reactive T cells. A ~~

I49

Genes Encoding Cancer Antigens

number of melanoma epitopes presented by HLA-A1, -A2, -A24, -A31, -B44, -Cw6, or -Cw16 have been identified using these approaches.

Ill. HUMAN MELANOMA ANTIGENS RECOGNIZED BY T CELLS Melanoma antigens thus far identified can be grouped into three general categories (Table I). The first group comprises melanocyte lineage-specific antigens, including tyrosinase, gp100, MART-1/Melan-A, and TRP-1 (gp75). The second group includes antigens that are expressed in normal testis or placenta and a variety of cancer cells, comprising the MAGE, BAGE, and GAGE gene families. The third group includes antigens that appear to be Table I Human Melanoma Antigens Recognized by T Cells Melanoma

Presenting MHC

Epitopea

Melanocyte lineage proteins gPl00 HLA-A2 KTWGQYWQV HLA-A2 ITDQVPFSV HLA-A2 YLEPGPVTA HLA-A2 LLDGTATLRL HLA-A2 VLYRYGSFSV MART-l/Melan-A HLA-A2 AAGIGILTV HLA-A2 ILTVILGVL HLA-A3 1 MSLQRQFLR TRP-1 (gp75) Tyrosinase HLA-A2 MLLAVLYCL HLA-A2 YMNGTMSQV HLA-B44 SEIWRDIDF HL A-A24 AFLPWHRLF HLA-DR4 QNILLSNAPLGPQFP HLA-DR4 SYLQDSDPDSFQD Proteins expressed in testis or placenta and other cancers MACE-1 HLA-A1 EADPTGHSY HLA-Cwl6 SAYGEPRKL MAGE-3 HLA-A1 EVDPIGHLY HLA-A2 FLWGPRALV BAGE HLA-Cwl6 AARAVFLAL GAGE-1 HLA-Cw6 YRPRPRRY Tumor-specific antigens p-Catenin HLA-A24 SYLDSGIHF MUM-1 HLA-B44 EEKLIWLF cdk4 HLA-A2 ACDPHSGHFV PI5 HLA-A24 AYGLDFYIL aBold type indicates mutation.

150

Steven A. Rosenberg et a/.

expressed only on tumor cells, which appear to be composed primarily of the products of mutated genes.

A. Melanocyte Lineage Antigens Many melanoma-reactive T cells can recognize cultured normal melanocytes in vitro as well as melanoma cells (Kawakami et al., 1992, 1993, 1994a,b; O’Neil et al., 1993; Anichini et al., 1993). A correlation has been reported between vitiligo and clinical response to chemoimmunotherapy or a good prognosis in melanoma patients (Nordlund et al., 1983; Bystryn et al., 1987; Richards et al., 1992). In the Surgery Branch of the National Cancer Institute (NCI), a correlation has also been observed between the development of vitiligo and clinical responses to IL-2 based immunotherapy (Table 11) (Rosenberg and White, 1996). These results indicate that some melanoma-reactive T cells recognize normal self-peptides derived from melanocyte-specific proteins. Furthermore, these autoreactive T cells, which recognize proteins expressed on both melanoma and melanocytes, may be responsible for in vivo melanoma regression. To date, four melanocyte proteins-tyrosinase, MART-1/Melan-A, gp100, and TRP-1-have been shown to represent antigens that are recognized by melanoma-reactive T cells. These antigens have been isolated using TIL, which induced tumor regression when administered to autologous patients and thus may represent tumor rejection antigens.

B. wrosinase A cDNA clone that was nearly identical to the previously isolated tyrosinase gene was isolated by screening a cDNA library from the SK29 melanoma, which had been transiently transfected into COS cells along with HLA-A2 (Brichard et al., 1993). Tyrosinase is a 529-amino-acid melanosomal membrane protein previously shown to be required for the Table I I Vitiligo in Melanoma Patients Treated with IL-2a Diagnosis Nonresponder Responder Total

Total 27 42 69

O/O

with Vitiligo

0 (0%) 12 (29%)(p = 0.002) 12 (16%)

aAll patients assessed for vitiligo at least 1 year after receiving IL-2.

Genes Encoding Cancer Antigens

151

synthesis of melanin (Bouchard et al., 1989). The product isolated from the SK29 library represented a normal, nonmutated gene product. This sequence differed from previously isolated sequences at a few residues; however, all of these genes appeared to represent common allelic variants. Two tyrosinase-reactive clones were found to react with distinct epitopes on the tyrosinase molecule: one, MLLAVLYCL, corresponding to the sequence at the amino terminus of the signal sequence of tyrosinase, and the second, YMNGTMSQV, corresponded to amino acids 369-377. When the peptide YMNGTMSQV was used to carry out in vitro stimulations, it was observed that peptide-specific CTL could be induced from five out of six normal donor peripheral blood lymphocytes (PBL); however, only three of the five specific CTL lines were found to recognize HLA-A2-positive melanomas (Visseren et al., 1995). High concentrations of peptides were used to stimulate in vitro responses, however, which may have resulted in the stimulation of T cells with a low affinity for peptide-MHC complexes. Presumably, in some cases T cells raised in this manner may not be capable of recognizing melanoma cells, which express the relatively low levels of peptide that are naturally processed and presented on the cell surface. Similar findings were made in studies utilizing T cells derived by in vitro stimulation with mutated as well as nonmutated peptides derived from p53 and ras (Houbiers et al., 1993; Elas et al., 1995). Tyrosinase was also shown to be recognized by an HLA-A24-restricted CTL line derived from the melanoma patient 888 (Robbins et al., 1994). This TIL cell line was shown to result in the regression of multiple metastatic lesions when transferred into the autologous patient along with IL-2. For these studies, the human kidney cell line 293 was initially transfected with the HLA-A24 class I gene, and a stable cell line was derived following selection and sorting of cells that expressed this gene product at high levels. A cDNA library was produced from the 888 melanoma cell line, and pools of cDNA generated from this library were screened for their ability to stimulate cytokine release from TIL 888 following transient transfection into the HLA-A24-expressing 293 cell line. A cDNA clone isolated from a positive pool was shown to encode tyrosinase, which again was found to represent a nonmutated gene product. A second CTL line, derived from melanoma patient 1415, was also shown to recognize tyrosinase in the context of HLAA24 (Kang et al., 1995). Overlapping deletions of the 3' end of tyrosinase carried out using exonuclease 111 were then used to demonstrate that TIL 1415 and TIL 888 recognized different regions of the tyrosinase molecule. Although a peptide epitope on tyrosinase recognized by TIL 888 has not been isolated, TIL 1415 was shown to recognize the overlapping 9-mer and 1O-mer peptides AFLPWHRLF and AFLPWHRLFL, corresponding to amino acids 206-214 and 206-215 of this molecule. Although the majority of studies have focused on the role of CD8+ T cells,

152

Steven A. Rosenberg et al.

some studies suggest that MHC class 11-restricted T cells may also play a role in tumor rejection. Tyrosinase was shown to be recognized by CD4+ melanomareactive T cells (Topalian et al., 1994a). Dendritic cells, macrophages, and B cells have been shown to present antigens to MHC class 11-restrictedT cells through a pathway involving endocytosis of exogenous proteins, followed by processing in acidic vesicles and presentation of the derived peptides by class I1 molecules on the cell surface. Initially, Epstein-Barr virus (EBV)B cells were found to present extracts of melanoma cells to CD4+ T cells isolated from some cultures of TILs in a class I1 HLA-DR-restricted fashion, as demonstrated by antibody blocking experiments as well as by the use of HLA-DR matched and nonmatched EBV B cells to present the cell extracts (Topalian et al., 1994b). Although some TIL appeared to respond only to extracts made from the autologous melanoma, CD4+ TIL derived from patient 1088 were shown to recognize extracts derived from multiple melanoma and melanocyte cell lines (Topalian et al., 1994a). This observation suggested that TIL 1088 recognized a nonmutated melanosomal gene product; therefore, COS cells were transfected with tyrosinase, gp7.5, MART-1, and gpl00 (Topalianet al., 1994a). Extracts made from transfectants of COS cells that expressed tyrosinase were found to be recognized by TIL 1088. Subsequent studies have demonstrated that TIL 1088 recognizes 2 distinct tyrosinase peptides in the context of HLA-DRB1*0401 (Topalian et al., 1996). These peptides appeared to bind to this HLA class I1 allele with low affinity, but when modifications were made at the presumed anchor residues in order to conform better to the HLA-DRBl"0401 binding motif, the peptide affinities were significantly increased. In addition, the high affinity peptides were recognized by CD4+ TIL 1088 cells at significantly lower concentrations than the native peptides. These results may indicate that T cells with high affinity for non-mutated peptides derived from melanocyte proteins such as tyrosinase may be anergized, perhaps due to the expression of these proteins in normal melanocytes, whereas peptides which bind to class I1 with low affinity may not induce T cell tolerance.

C. MART- I /Melan-A A previously unknown gene, MART-1 was isolated following the screening of a melanoma cDNA library with the HLA-A2-restricted melanomareactive TIL 1235 (Kawakami et al., 1994a). Melan-A (Coulie et al., 1994), an identical gene that was independently cloned, was found to contain five exons spanning a 18.5-kb region of genomic DNA. The MART-1 cDNA isolated from melanoma cells did not contain any mutations and encoded a 118-amino-acid polypeptide. The sequence did not contain a classical signal sequence at the amino terminus, but appeared to contain an internal transmembrane domain consisting of 23 hydrophobic amino acids. This region

G e n e s Encoding Cancer Antigens

I53

was flanked by two glutamic acids at the amino-terminal side and three arginines at the carboxyl-terminal side, which may serve to anchor this segment in the membrane. Northern blot analysis carried out on a variety of cell lines and tissues indicated that MART-1 was expressed in most melanoma lines, all cultured melanocyte lines, and retinal tissue, which presumably contains retinal pigmented epithelial cells as well as melanocytes, which are present in the uvea. Other normal tissues, including brain, adrenal gland, lung, liver, kidney, spleen, thymus, testis, and fetal liver, and other types of cancer cell lines, including colon, breast, neuroblastoma, sarcoma, and renal cell lines, did not appear to express MART-1. Immunohistochemical studies using monoclonal antibody also demonstrated that MART-1 protein was expressed in melanocytes as well as most melanoma cells, but not other types of cell lines and normal tissues tested. MART-1 was found to be an immunodominant melanoma antigen recognized by the majority of HLA-A2-restricted melanoma-reactive CTL established from TIL in the Surgery Branch of the NCI (Kawakami et al., 1 9 9 4 ~ ) ~ as well as a large percentage of melanoma-reactive clones derived from the PBL of HLA-A2-positive melanoma patients (Sensi et al., 1995). Among more than 21 HLA-A2-restricted melanoma-reactive TIL that were tested, only 3 failed to recognize MART-1. By screening 28 MART-1 peptides (810 mer) that were synthesized on the basis of the HLA-A2-binding motif, one 9-mer peptide, AAGIGILTV (M9-27), and two overlapping 10-mer peptides, EAAGIGILTV and AAGIGILTVI, were found to be recognized by HLA-A2-restricted melanoma-specific CTL. The M9-27 peptide was found to have an intermediate binding affinity to HLA-A2.1, whereas both of the overlapping 10-mer peptides had low binding affinities. T2 indicator cells could be sensitized for recognition by specific CTL using lower concentrations of the M9-27 peptide than the overlapping 10-mer peptides, suggesting that M9-27 may represent the predominant peptide from MART-1 recognized on the melanoma cell surface in the context of HLA-A2. The M9-27 peptide was found to be recognized by all HLA-A2-restricted MART-1specific TIL established from different patients, suggesting that it represents an immunodominant epitope in MART-1 (Table 111). The presence of the highly immunogenic M9-27 peptide in the MART-1 protein may, at least in part, be responsible for the dominant role of HLA-A2 expression on the induction of melanoma-reactive CTL (Crowley et al., 1991). Several studies have addressed the issue of which MART-1 peptides are naturally processed and presented on the cell surface (Castelli et af., 1995; Wolfel et al., 1994). When HLA-A2-binding peptides were isolated from melanoma cells and fractionated on a reverse-phase HPLC column, three distinct fractions were found to stimulate a single MART-l-specific T-cell clone. Some of these fractions may have contained the MART-1 9-mer and 10-mer peptides described above; however, the predominant peptide found in one of these fractions was XXTVXXGVX, where X represents isoleucine

154

Steven A. Rosenberg et al.

Table Ill Recognition of lmmunodominant MART-1 Peptide by HLA-A2-Restricted Melanoma-Reactive TILa

TIL' Target 501-me1 397-me1 T2 T2

Peptide (1 pg/ml) None None None M9-27'

501

620

42 49 3 1 6 0 7 86 75

660

1074 1088

1128 1143 1200

35 6 -3 73

32 1 -6 79

19 4 -6 30

31 1 7 98

24 3 -7 36

41

3 -6

2

1235 1363 32 3 -7 92

43 4 -7 82

aData from Kawakami et al. (1994~). 'Percent specific lysis at E :an effect to target ratio of 40 to 1. 'All TIL except TIL1200 lyse T2 cells pulsed with M9-27 MART-1 peptide (AAGICILTV).

or leucine, which could not be distinguished by mass spectrometry. This peptide may correspond to the MART-1 peptide ILTVILGVL (M9-32), which overlaps with the four carboxyl-terminal residues of M9-27 and has a similar HLA-A2 binding affinity. Some T-cell lines and one T-cell clone were found to recognize both M9-27 and M9-32; however, the M9-32 peptide was only recognized when target cells were pulsed with the peptide in the presence of anti-HLA-A2 antibody and P2-microglobulin. The immunogenicity of additional MART-1 peptides was examined by stimulating PBL from melanoma patients or healthy donors in vitro with 10 MART-1 9-mer peptides that varied in their HLA-A2 binding affinities (Table IV) (Rivoltini et d., 1995). Peptide-specific CTL could only be induced with the M9-27 and M9-35 peptides, both of which possessed intermediate HLA-A2 binding affinities, but not with two high-affinity peptides. CTL that were induced with the M9-27 peptide, but not M9-35, recognized melanoma cells, indicating that M9-35 may not be naturally processed and presented on the surface of melanoma cells. One possible explanation for the failure to generate responses with peptides that bind MHC class I with high affinity may be that these peptides, which are expressed at relatively high levels on the normal melanocyte cell surface, are more efficient at inducing tolerance in vivo. The results of several experiments carried out to analyze immune response to self-antigens using normal or transgenic mice have suggested that T-cell tolerance is most efficiently induced with peptides that appear to be expressed at high densities on the cell surface, termed dominant self-determinants (Sercarz et al., 1993: Moudgil et al., 1993). The results of several studies indicate that the M9-27 peptide may represent a good candidate for peptide-based antimelanoma vaccine trials. CTL that were generated using the M9-27 peptide were found to recognize uncultured melanoma cells, indicating that this peptide is expressed at sufficient levels on the melanoma cell surface in vivo for recognition by activated

%able IV In Viiro Induction of CTL against MART-1 Peptides from PBL of a Patienta Melanoma'

T2 cells pulsed with peptide= Peptide for CTL Induction

M22 TTAEEAAGI M27 AAGIGILTV M29 GIGILTVIL M3 1 GILTVILGV M32 ILTVILGVL M33 LTVILGVLL M34 TVILGVLLL M35 VILGVLLLI M56 ALMDKSLHK M61 SLHVGTQCA

Affinityb

L M L H M L L M H L

aData from Rivoltini e t a / . (1995). 'L, low; M, medium, H, high. 'Percent specific lysis; positive ( + ) or negative (-1 result of "Cr release assay performed after 5 weeks of culture.

M35

I56

Steven A. Rosenberg et al.

CTL. Melanoma-reactive CTL could be raised from seven of eight patients’ PBL and two of four normal donors’ PBL by stimulation with the M9-27 peptide. The induction of melanoma-reactive CTL from PBL of melanoma patients occurred more rapidly than induction from normal donors, suggesting that there was an increase in the CTL precursor frequency in melanoma patients (Rivoltini et al., 1995; Marincola et al., 1996). In addition, the cytolytic activity of CTL generated in vitro with the M9-27 peptide was 50to 100-fold greater, on a per cell basis, than conventional TIL expanded with IL-2 alone, suggesting that these CT may be more effective in adoptive immunotherapy (Rivoltini et al., 1995). Although MART-1-specific CTL efficiently lyse melanoma cells in vitro, their role in the in vivo regression of tumors is not clear. Tumor regression was not observed in several patients who received autologous TIL reactive to MART-1, and no significant correlation was observed between MART-1 recognition by TIL and clinical responses to TIL therapy among 10 patients who received HLA-AZrestricted melanoma-reactive TIL (Kawakami et al., 1995) (Table V). The MART-1 peptide may not be recognized efficiently by administered CTL because it is expressed at only a low density on the melanoma cell surface. The structure of T-cell receptors (TCR) of three CTL clones that recognize the M9-27 peptide in association with HLA-A2 has also been analyzed (Cole et al., 1994). No common TCR variable gene regions were found to be shared by these clones, with the exception of Vp7, which was utilized by two of the CTL clones. Thus, different TCR are capable of recognizing a single melanoma peptide that is presented in the context of HLA-A2.1. Similar findings were reported in studies of CTL specific for the MAGE-1 peptide in the context of HLA-A1 (Romero et al., 1995). However, it has been reported that Va2, Vp7, and Vp14, and particularly the combination of Va2 and Vp14, may be frequently used by TCR recognizing MART-1 in the context

Table V Clinical Response and Antigen Recognition by HLA-A2-Restricted Melanoma-Reactive TILa

TIL 1200 620 Clinical responseb Antigen reactivityC

gPl00 MART-1

660

1143 1074 1088

1235

1318 1363 1399

PR

PR

PR

PR

NR

NR

NR

NR

+

+

+

+

-

-

-

-

-

-

+

+

+

+

-

+

+

YData from Kawakami et al. (1995). ~ P R partial , response; NR, no response. t , reactive; -, nonreactive.

+

+

+

NR

NR

G e n e s Encoding Cancer Antigens

157

of HLA-A2, although the precise epitope recognized by those T-cell clones was not determined (Sensi et al., 1995). If preferential usage of particular TCRs by melanoma-reactive T cells can be demonstrated, it may be possible to monitor the immune response to tumor in vivo by evaluating the frequency and distribution of T cells expressing these TCR in vivo. These studies may lead to a better understanding of the mechanisms that regulate in vivo antitumor immune responses. In addition, it may be possible to generate therapeutic cells by the transfer of TCR genes from tumor-reactive T-cell clones to alternative effector cells or to hematopoietic stem cells (Cole et al., 1995).

D. gpl00 Screening assays carried out with the same cDNA expression cloning system used to isolate MART-1 resulted in the isolation of a gene recognized by the HLA-A2-restricted TIL 1200 (Kawakami et al., 1994b). The cDNA that was isolated was almost identical to two genes, g p l 0 0 and Pmell7, that had previously been cloned using anti-melanoma antibodies (Kwon et al., 1991; Adema et al., 1994). The gpl00 glycoprotein was also identified as a melanoma antigen by testing for the recognition by TIL 1200 of HLA-A2positive cell lines transfected with a previously isolated gpl00 cDNA clone (Bakker et al., 1994). In addition, the gpl00 peptide YLEPGPVTA was isolated by HPLC fractionation of peptides eluted from HLA-A2 molecules on melanoma cells and shown to be recognized by HLA-A2-restricted melanoma-specific CTL (Cox et al., 1994). The gpl00 gene is homologous to RPE1, which is expressed in bovine retinal pigmented epithelium (Kim and Wistow, 1991), and MMP115, which is a melanosomal matrix protein expressed in chicken pigmented epithelial cells (Mochii et al., 1991) and has been localized to chromosome 12p ter-q21 (Kwon et al., 1991). The 661amino-acid gpl00 glycoprotein appears to contain a signal peptide as well as a single transmembrane domain. Consistent with the previous immunohistochemical studies using anti-gp100 antibodies (Colombari et al., 1988; Vennegoor et al., 1988; Gown et al., 1986), Northern blot analysis demonstrated that gpl00 was expressed in neonatal cultured melanocyte lines, most melanoma cell lines, and retina but was not expressed in nonmelanoma cancer cell lines, including breast, colon, lung, and cancers of neuroectodermal origin. Ultrastructural studies using either HMB45 or NKI/betab anti-gp100 antibody demonstrated that gpl00 was mainly located in the membrane and filamentous matrix of premelanosomes where melanin was synthesized (Schaumberg-Lever et al., 1991). Supernatants of melanoma cell culture lines also contained gp100, suggesting that this molecule may be shed or secreted from melanoma cells (Vogel and Esclamado, 1988). The function of gpl00 has not been definitively established, but it

I58

Steven A. Rosenberg et al.

may represent one of the structural components of the melanosomal membrane and may be involved in the late steps of melanin synthesis (Kobayashi et al., 1994). The gpl00 molecule appears to represent a highly immunogenic antigen, since it was found to be recognized by 8 out of 21 HLA-A2-restricted melanoma-reactive TIL established from different patients in the Surgery Branch of the NCI (Kawakami et al., 1994~).Five of the 8 gp100-reactive TIL also recognized MART-1, demonstrating that many TIL lines contained T-cell populations that recognized distinct tumor antigens. Five distinct gpl00 epitopes have been identified using 8 different gp100-reactive CTL lines (Kawakami et al., 1995) (Table VI). Some CTL lines were found to recognize multiple gpl00 epitopes, and 3 of the identified gpl00 epitopes were recognized by CTL from different patients, suggesting that these represented immunodominant common epitopes. One of the peptides, G9-280 (YLEPGPVTA),was recognized by 6 of 8 of the gp100-reactive TIL derived from different patients. This peptide was also isolated from the HLA-A2 molecule on melanoma cells and was reported to be recognized by 5 of 5 CTL raised from PBLs (Cox et al., 1994). Two additional epitopes, G9-154 (KTWGQYWQV) and G9-209 (ITDQVPFSV),were recognized by 5 of 8 and by 4 of 8 gp100-reactive TIL, respectively. Melanoma-reactive CTL could be induced by in vitro stimulation with these gpl00 peptides, but not as efficiently as with the MART-1 M9-27 peptide (Salgalleret al., 1995). The in vitro induction of melanoma-reactive CTL with gpl00 peptides required more restimulations than were needed with the MART-1 M9-27 peptide. One possible explanation for this finding is that the precursor frequency of CTL that are specific for these gpl00 epitopes is lower than the frequency of MART-1-specificT cells. When the gp100-reactive TIL 1200 was administered to the autologous

Table VI g p l 0 0 Epitopes Recognized by HLA-A2-Restricted Melanoma-Reactive TILa TILb

Peptide M9-12 G9-154' G9-209' (29-280' G 10-457 G10-476

771

1200

1520

620

660

697

-

-

+ + +

+

-

+ + + +

+ + + +

+

-

-

-

+ -

+ + -

-

OData from Kawakami et a/. (1995). '+, epitope recognized by TIL; -, epitope not recognized by TIL. 'Recognized by TIL derived from different patients.

-

+ +

1143

1495

+

+ +

-

-

+

-

-

-

-

-

-

-

G e n e s Encoding Cancer Antigens

159

patient, the injected TIL accumulated at tumor sites and caused a dramatic regression of multiple metastases (.Kawakami et al., 1994b). In addition, clinical responses to TIL therapy in HLA-A2 patients appeared to correlate with recognition of gpl00 (Kawakami et al., 1995) (Table V). There did not appear to be any correlation between the specificity of the gp100-reactive TIL for particular epitopes in this molecule and clinical response to TIL therapy. The gpl00 epitopes have higher HLA-A2 binding affinities and may be expressed at higher densities on the melanoma cell surface than the MART-1 M9-27 epitope. Thus, these peptides may represent better targets for tumor-reactive T cells. A clinical protocol is now being carried out in the Surgery Branch of the NCI, involving the injection of the G9-154, G9-209, or G9-280 peptides in incomplete Freund’s adjuvant. The presence of multiple HLA-A2-binding epitopes in gpl00 may be related to its relatively large size (661 amino acids) and suggests that gpl00 may contain CTL epitopes presented by additional class I molecules. In addition, gpl00 may contain helper T-cell epitopes presented by class I1 MHC molecules. Thus, gpl00 represents an attractive candidate antigen for the development of effective antitumor vaccines.

E. vrosinase-Related Protein-I (gp75) The infusion of TIL586 plus IL-2 into the autologous patient with melanoma resulted in the objective regression of tumor (Topalian et al., 1988), suggesting that this TIL line recognized a tumor rejection antigen. Studies carried out to analyze tumor recognition by TIL 586 demonstrated that this TIL lysed the autologous melanoma cell line and several allogeneic melanoma target cells derived from different patients that shared HLA-A31, -A29, -Cw7, or -B44 (Wang et al., 1995; Hom et al., 1993a,b). A subsequent study demonstrated that this TIL predominantly recognized one or more antigens in the context of HLA-A31 (Wang et af., 1995). When a melanoma cDNA expression library was screened by transient transfection into COS cells along with the gene encoding HLA-A31, a partial cDNA clone was isolated that was identical to that of the previously reported TRP-1 gene (Cohen et al., 1990; Vijayasyradhi et al., 1990). The protein encoded by TRP-1, gp75, was originally identified as an antigen recognized by immunoglobulin G antibodies found in the serum of a patient with melanoma (Mattes et al., 1993).The results of Northern blot analysis indicated that the TRP-1 gene was expressed in melanoma, normal melanocyte cell lines, and retina but not in other normal tissues tested (Wang et al., 1995). This was consistent with results obtained with monoclonal antibodies, which indicated that the gp75 protein was detected in pigmented human melanocytes and melanomas but not in nonmelanocytic cell types (Thomson et al., 1985, 1988). The gp75 molecule is involved in melanin synthesis, and has recently

160

Steven A. Rosenberg et al.

been shown to have DHI-2-carboxylic acid oxidase activity (JimenezCervantes et al., 1994). This molecule also appears to be one of the most abundant intracellular glycoproteins in melanocyte-lineage cells (Thomson et al., 1985, 1988). In order to identify the epitope on gp75 recognized by TIL 586, a set of nested deletions were created using exonuclease 111 as well as by generating fragments of the cDNA clone by PCR (Wang et al., 1996). Using this approach, the epitope recognized by TIL 586 was narrowed down to a small region from nucleotides 247 to 442. To determine the identity of the epitope in this region, a number of synthetic peptides were made based upon the previously identified HLA-A3 1 peptide-binding motif, which consists of a hydrophobic residue at position 2 and an arginine at the carboxyl terminus (Falk et al., 1993).However, the peptides that were derived from the normal gp75 protein failed to stimulate cytokine release by TIL 586 when pulsed onto 586EBV B cells (Wang et al., 1996). One possible explanation for this result was that TIL 586 recognized the product of an alternative reading frame of gp75, since this fragment (nucleotides 247-442) did not contain any ATG codons in the normal gp75 open reading frame (ORFl). However, two ATG codons in a relatively good context were present in different ORFs relative to the normal gp75 ORFl (Fig. 1). Three peptides derived from ORF2 and two peptides from ORF3 were selected and synthesized on the basis of the HLA-A3l-binding motif (Wang et al., 1996). Surprisingly, the peptide MSLQRQFLR, which was derived from ORF3, was capable of stimulating cytokine release from TIL 586 when pulsed onto 586EBV B cells. Other reports have demonstrated that protein translation can be initiated at more than one AUG codon (Ossipow et al., 1993; Descombes and Schibler, 1991). In addition, in rare cases, initiation can occur at both AUG and non-AUG codons, such as CUG; however, in all of these examples, the same reading frame is utilized for translation of the different gene products (Hann, 1995; Muralidhar et al., 1994). This appears to represent one of the first direct demonstrations that overlapping ORFs can be utilized to translate two distinct polypeptides from a single eukaryotic cellular mRNA. Overlapping reading frames, however, are commonly translated in viral mRNAs (Shaw et al., 1983; Spiropoulou and Nichol, 1993; Schwartz et al., 1992; Fajardo and Shatkin, 1990). Houghton and his coworkers have demonstrated that passive immunization with a mouse monoclonal antibody (TA99) against gp75 induced protection against and rejection of the gp75-positive B16F10 melanoma in syngeneic mice (Hara et al., 1995). There was no evidence of decrease in pigmentation, inflammation, or changes in cellular morphology or tissue architecture in the eyes of mice treated with antibody (Hara et al., 1995), suggesting that the threshold required for antitumor treatment is lower than that

161

Genes Encoding Cancer Antigens

OW1

ORFl ORFl

ORFl

OW1 ow2 ORF3

ORFl ow2 ORF3

OW1

oRF2 ORF3

ORFl

~ G n ; C T C C T A A A C T C C T T C T C ~ % T A T C ~ ~ C C ~ 60 T A C ~ G M S A P K L L S L G C I F F P L L L F Q C A G G C C C G G G C T C A A ' I T C C C ~ ~ C A G ~ ~ C A C ~ ~ G 1G20C ~ ~ G ~ T A ~ Q A R A Q F P R Q C A T V E A L R S G M TGTn;cCCAGACCTGTCCCCTGTGTCTGGGCCTGGGACAGACC~~TA~A!KA 180 C C P D L S P V S G P G T D R C G S S S ~ G A % T G A G G C A G T G A C T G C A G A C T C C C C G C C 240 G R G R C E A V T A D S R P H S P Q Y P CATGA-TCGCCTTGCGCTTC-amAC 300 H D G R D D R E V W P L R F F N R T C H M I G R S G P C A S S I G H V T

YSL n ; C ~ C ~ ~ T A ~ ~ 60 C N G N F S G H N C G T C R P G W R G A A T A I S Q D T T V G R A V L A G E E L 9 x 0 F L R T Q L W D V P S W L E R S C GCCTGTGACCAGAGGG'ITCTCATAGTPAGAGAUTTTC'IGGACTTAAGTAAAGA?.W 420 A C D Q R V L I V R R N L L D L S K E E P V T R G F S STOP L SMP ApaI AAGAACCAC?TX;TCCGGCACAAC!KACCCT. . .ATATGA 1584 K N H F V R A L D M A K R T T H P

Fig. 1 The nucleotide, amino acid sequence, and ORFs of the gp75 gene. The partial nucleotide and amino acid sequences of the first 157 amino acids was shown from the start codon for translation of ORFl (gp75).The DNA fragment that conferred the ability to stimulate granulocyte-macrophage colony-stimulating factor release from TIL 586 is underlined. Two putative start codons, ATG (254-256) and ATG (294-296), (in bold type) may result in the translation of ORF2 and ORF3, respectively. The peptide sequence recognized by TIL 586 from ORF3 is in bold type and underlined.

which results in autoimmunity. These results further demonstrate that gp75 may represent a good target for the development of antitumor vaccines.

F. Melanoma Antigens Expressed in Normal Testes or Placenta A number of T cells have been isolated from the melanoma patient MZ2, who was repeatedly immunized with mutagenized autologous tumor. Initially, a genomic library isolated from the MZ2 parental tumor cell line was transfected into a resistant variant that was not recognized by a CTL clone derived from this patient (Van Den Eynde et al., 1989). Stable transfectants that were recognized by the CTL clone were isolated, and the gene that was isolated from this transfectant, termed MAGE-1, was shown to belong to a previously undescribed multigene family (van der Bruggen et al., 1991). The

~

C

162

Steven A. Rosenberg et al.

peptide epitope recognized in the context of HLA-A1, EADPTGHSY, was then identified in this molecule (Traversari et al., 1992). Expression of the MAGE-2 gene product was found in approximately 40% of melanomas, as well as a smaller percentage of breast, lung, and laryngeal cancers as well as sarcomas. Another member of the MAGE-1 gene family, MAGE-3, was subsequently shown to be recognized by a HLA-A1-restricted clone isolated from patient MZ2 (Gaugler et al., 1994). MAGE-3 may represent a better target for vaccination protocols than MAGE-1, since almost 70% of melanomas expressed MAGE-3. The MZ2 cDNA library was subsequently screened using a class I HLACw*Z 61 0-restricted T-cell clone, which resulted in the isolation of a previously undescribed gene, termed BAGE (Boel et al., 1995). This gene product appeared to be expressed in a pattern similar to that of members of the MAGE family. Using the same approach, another gene encoding an antigen recognized by a class I HLA-Cw*060l -restricted T-cell clone was derived from patient MZ2. This gene, termed GAGE, was found to have the same expression pattern as the MAGE and BAGE genes, as the only normal tissue where expression was found was the testis (Van Den Eynde et al., 1995). This gene was found to be a member of a multigene family, and two of the members of the family were shown to encode a peptide that was recognized in association with HLA-Cw*0601. Expression of the two GAGE-1 and GAGE-2 gene products was observed in 25% of sarcomas, 24% of melanomas, and a variety of additional tumor types. Studies have examined whether or not it was possible to stimulate responses in vitro to epitopes from MAGE-1 or MAGE-3 using cells from HLA-A1 individuals. In one study, the previously identified MAGE-3 peptide was shown to stimulate responses in vitro from 1 of 2 normal donors’ PBL (Cellis et al., 1994). In another study, in vitro stimulation of PBL from a melanoma patient with the MAGE-1 peptide resulted in generation of a peptide-specific T-cell line (Salgaller et al., 1994). Subsequent studies further demonstrated that a response to the MAGE-1 peptide could be generated from PBL of only 1 of 6 melanoma patients (M. Salgaller, unpublished observations). In another study, an HLA-A2-binding peptide-FLWGPRALV, derived from MAGE-3-was found to stimulate peptide-specific responses in vitro (van der Bruggen et al., 1994). The T cells that were induced with this peptide were found to lyse tumor cell lines that had been treated with interferon-y but not untreated cells, which may have resulted from an increase in HLA class I gene expression following treatment. This peptide appears to bind MHC with high affinity, which may result in induction of tolerance in T cells expressing TCR with high affinity for the complex of this peptide plus HLA-A2 in vivo. The T cells that can be induced in vitro using stimulators that express these peptides at relatively high levels on the cell surface may express TCR with a relatively low affinity for this peptideMHC complex.

Genes Encoding Cancer Antigens

I63

Clinical trials using HLA-A1 binding MAGE-1 or MAGE-3 peptide has begun in patients with melanoma as well as other tumors. Partial tumor repression was observed in 3 out of 12 patients with the MAGE-3 peptide although CTL specific for immunized peptide was not detected in PBL in the responding patients (Marchand et al., 1996).

G. Implications of the T-cell Response to Nonmutated Epitopes The melanoma epitopes identified in melanocyte lineage proteins, as well as those in the MAGE, BAGE, and GAGE proteins, were shown to represent the products of nonmutated genes (Table I). It is not clear why these proteins, in particular the melanocyte lineage proteins, are immunogenic, since the expression of these proteins in normal melanocytes would be expected to induce tolerance to these molecules. Melanosomes appear to be related to endosomes and lysosomes, however, and it is possible that intracellular expression through this pathway renders these molecules more immunogenic than other self-peptides. In addition, these organelles may be transferred to keratinocytes as well as professional antigen-presenting cells, which may increase the immunogenicity of melanosomal proteins. A number of additional studies have been carried out to further characterize the epitopes identified in these melanoma antigens. The majority of peptides eluted from purified HLA-A2 molecules, as well as HLA-A2-binding viral peptides, have been found to contain predominantly leucine or methionine at the second position (P2) and valine or leucine at the carboxyl terminus (P9). In addition, the majority of these peptides have been shown to bind HLA-A2 with high affinity (Falk et al., 1993; Hunt et al., 1992; Sette et al., 1994; Parker et al., 1995). In contrast, the epitopes identified in melanoma antigens generally contained nondominant amino acids at the primary anchor positions. M9-27, G9-154, and G9-209 contained either alanine or threonine at the P2 anchor position, and G9-280 contained an alanine residue at P9. These results demonstrate that it may be important to utilize an extended binding motif that includes nondominant anchor residues when attempting to characterize the epitopes recognized in self-proteins. The majority of the peptides identified in these melanoma antigens did not appear to bind to HLA-A2 with a high affinity (Kawakami et al., 1995) (Table VII). Binding affinity appears to be one of the major factors influencing the density of peptides bound to MHC class I on the cell surface, although it may be influenced by other factors such as the protein degradation rate, the efficiency of enzyme cleavage in the region of the protein containing the peptide epitope, and the efficiency of peptide transport to the endoplasmic reticulum. The self-peptides derived from these melanosomal pro-

I64 Table VII

Steven A. Rosenberg et al. HLA-A2-Binding Melanoma Epitopesa

Protein

Peptide

gP 100

G9-154 G9-209 G9-280 Gl O-457 G10-467 M9-27 M9-32 T9- 1 T9-369

MART-I Tyrosinase

Sequenceb KTWGQYWQV ITDQVPFSV YLEPGPVTA LLDGTATLRL VLTRYGSFSV AAGIGILTV ILTVILGVL MLLAVLYCL YMNGTMSQV

HLA-A2 binding affinityC High Medium Medium Medium High Medium Medium Medium High

aData from Kawakami ef al. (1995). bBold type indicates nondominant anchor amino acids. 500 nM) by the competitive inhibition assay.

teins that are recognized by tumor-reactive T cells may generally be subdominant or cryptic determinants that are expressed at low densities on the cell surface, resulting in a lack of complete tolerance induction (Sercarz et al., 1993; Kawakami and Rosenberg, 1996a,b). Self-peptides that bind to MHC class I with high affinity should be more effective at inducing T-cell tolerance. It is not clear what leads to the recognition of subdominant or cryptic epitopes. Overexpression of the antigen or the associated MHC-presenting molecules in tumor cells could result in the activation of T cells responsive to these normally silent epitopes. Tissue destruction, as well as nonspecific inflammatory responses present at the tumor site, might cause the release of cytokines that up-regulate the expression of MHC, antigen, or accessory molecules, leading to the activation of antigen-specific T cells. It has been reported that T cells from patients with breast or ovarian cancers can recognize nonmutated peptides derived from Her2/neu protein on tumor cells (Peoples et al., 1995; Fisk et al., 1995; Cheever et al., 1995), and tissuespecific proteins expressed in these and other cancers could also potentially serve as tumor rejection antigens. Stimulation of immune responses to subdominant or cryptic epitopes that bind poorly to MHC may require the use of methods that result in a high level of expression of these epitopes on the surfaces of antigen-presenting cells (Table VIII) (Kawakami and Rosenberg, 1996a,b). This may be accomplished through the use of recombinant viral vectors that express tumor antigen gene products at high levels, or by immunization with peptide-

Genes Encoding Cancer Antigens

165

'bble Vlll Methods for Induction of T Cells Specific for Subdominant or Cryptic Self-Determinants 1. 2. 3. 4.

Use of high-protein-expressionsystem (recombinant virus vectors) Exogenous loading of epitopes (epitope-pulseddendritic cells) Use of modified epitopes (high-MHC-bindingmodified peptides) Use of epitope-linked MHC molecules.

pulsed antigen-presenting cells. The sequences of some of these peptides have also been altered in an attempt to increase their MHC binding affinity, since immunogenicity has been generally shown to correlate with the MHC binding affinity of T-cell epitopes (Chen et al., 1994). The low binding affinity of the M9-27, G9-209, and G9-280 peptides to HLA-A2 molecules may be related to the presence of nondominant amino acids at the primary anchor positions; therefore, these peptides were modified to contain the dominant anchor residues at these positions (P2 and P9) (Parkhurst et al., 1996). Following the screening of a number of modified peptides, several peptides were identified that possessed a significantly better HLA-A2 binding affinity than the natural peptides but were still recognized by melanomareactive TIL specific for the native epitopes. When tested for their ability to stimulate CTL responses in vitro, some of the modified epitopes were found to be more efficient than the natural epitopes (Parkhurst et al., 1996). The results of a study carried out with the G9-280-9V peptide, which contained a substitution of valine for alanine at P9, indicated that this peptide was capable of inducing potent melanoma-reactive CTL from PBL derived from 6 of 6 patients. Stimulations carried out with the natural G9-280 peptide resulted in induction of weak melanoma reactivity in only 2 of 6 patients. In addition, the G9-209-2M peptide, which contained a methionine substitution for threonine at P2, was found to induce melanoma-reactive CTL more efficiently than the native G9-209 peptide. These results indicated that some of the modified peptides were more immunogenic in vitro than the native peptides, which may also be evident in patient protocols involving active immunization with these peptides. These amino acid changes could also be incorporated into gpl00 cDNA constructs, which may result in more effective immunization with recombinant viruses or naked DNA. Another approach that may enhance the immunogenicity of subdominant or cryptic epitopes involves the covalent attachment of peptides to HLA-A2 molecules to prevent their dissociation from the MHC-binding groove. Preliminary results indicate that melanoma-reactive CTL can be generated by in vitro stimulation of PBL with B cells that had been transfected with a gene construct containing a fusion of the M9-27 epitope with HLA-A2 (X. Kang et al., unpublished data). It may also be possible to improve the recognition of epitopes by CTL at

I66

Steven A. Rosenberg et al.

the effector phase by increasing the surface expression of peptide-MHC complexes on tumor cells by augmenting the expression of MHC and/or antigen molecules, or by increasing the levels of antigen processing within cells. The administration of interferon-y may increase the number of complexes containing subdominant or cryptic epitopes bound to class I MHC molecules on the tumor cell surface and, as a consequence, augment tumor rejection. A potential outcome of active immunization with melanoma peptides is the development of autoimmune reactions. An autoimmune disease unrelated to melanoma involving the destruction of melanocytes, the VogtKoyanagi-Harada syndrome, has been shown to result in ocular and auditory impairment as well as lymphocytosis in the spinal fluid. Activation of T cells that are reactive with normal self-peptides, particularly as a result of active immunization, could potentially result in similar side effects. A positive correlation between the development of vitiligo and good prognosis or clinical responses to immunochemotherapy in melanoma patients has been reported (Nordlund et al., 1983; Bystryn et al., 1987; Richards et al., 1992), and in the Surgery Branch of the NCI, a correlation was observe between the development of vitiligo and tumor regression following IL-2-based immunotherapy (Table 11) (Rosenberg and White (1996). These observations indicate that autoreactive T cells that respond to epitopes present on both melanoma and melanocytes may be involved in tumor rejection. The adoptive transfer of TIL specific for MART-1 or gpl00 epitopes resulted in dramatic tumor regression in patients, but only occasionally led to the development of vitiligo, however, which indicates that autoreactive T cells may be used for the treatment of melanoma patients without severe adverse effects on normal melanocytes (Kawakami and Rosenberg, 1996a,b; Moudgil and Sercarz, 1994; Nanda and Sercarz, 1995). However, it is possible that the use of modified peptides that possess a higher binding affinity for MHC class I than the natural peptides may lead to the induction of autoimmunity, since viral and bacterial peptides that mimic self-peptides may in some cases initiate autoimmunity (Wucherpfennig and Strominger, 1995).

H. Tumor-Specific Antigens Cancer cells have been shown to acquire many mutations, and altered peptides thus have the potential to be recognized as truly tumor-specific antigens. Mutated peptides have been identified in several mouse tumors as T-cell antigens (Boon, 1992; Mandelbolm et al., 1994; Uenaka et al., 1994). but the majority of the human melanoma antigens described to this point appear to represent normal gene products. Research results indicate, however, that some melanoma-specific T cells can recognize mutated gene products.

G e n e s Encoding Cancer Antigens

167

In one study, antigen loss variants of the LB33 melanoma cell line were derived by immunoselection with autologous CTL clones (Coulie et al., 1995). One of the CTL clones was found to recognize a resistant melanoma variant following the transfection of HLA-B44. Following the transient transfection of COS cells with HLA-B44 and pools of cDNAs, a positive cDNA clone was isolated. The sequence of this clone showed no significant similarity to any sequences in the GenBank or EMBL data bases, and the gene product appeared to be expressed in a variety of normal tissues, such as liver, colon, muscle, and heart. The peptide epitope identified in this molecule, EEKLIWLF, conformed to the recently described HLA-B44-binding motif (Fleischhauer et al., 1995). When the gene was isolated from the normal cells of this patient, a single nucleotide substitution was found between this sequence and the sequence of the cDNA isolated from the tumor, which was designated MUM-1. The normal product contained a serine residue in place of the isoleucine found at position 5 in the mutated sequence, and the mutated peptide could sensitize targets for recognition with a half-maximal level of 0.4 nM, whereas the normal peptide was not recognized at a concentration of 300 nM. This mutation appeared to have an effect on T-cell recognition but not MHC class I binding, since both the normal and mutated peptides bound efficiently to the HLA-B44 molecule. Evidence was obtained that the cDNA that was initially isolated represented an incompletely spliced message, and the antigenic peptide appeared to span the intron-exon boundary. A mutated gene product derived from the cdk4 gene was recently shown to be recognized by three class I HLA-A2-restricted T-cell clones isolated from melanoma patient SK29 (Wolfel et al., 1995). The cDNA clone that was isolated from the SK29 melanoma contained a single mutation in the cdk4 gene. This mutation, a C-to-T transition, may have resulted from ultraviolet (W)-induced DNA damage (Brash et al., 1991), and resulted in substitution of a cysteine for an arginine residue at codon 24. When fragments of the mutated cdk4 cDNA produced by PCR were cotransfected into COS cells along with HLA-A2, the fragment from amino acids 22 to 32 of cdk4 was shown to be recognized by the T-cell clones. Sensitization of targets with the mutated (KACDPHSGHFV) and normal (KARDPHSGHFV) peptides resulted in equivalent lysis. These peptides were 11-mers, however, and optimal HLA-A2-binding peptides have generally been found to be 9 and 10 amino acids long. When the 10-mer peptides ACDPHSGHFV and ARDPHSGHFV were tested for their ability to sensitize targets, the mutated peptide was found to induce half-maximal lysis at a concentration of about 10 nM, whereas 100- to 1000-fold higher concentrations of the normal peptide were required to sensitize targets for an equivalent level of lysis. The effect of this mutation on cdk4 function was then examined. This protein normally forms a complex with cyclin D1 that acts to promote cell

1 68

Steven A. Rosenberg et al.

cycle progression from G1 to S phase. When the mutated protein was expressed in insect cells along with cyclin D1 and a variety of inhibitors, it was observed that the mutated protein failed to bind to two of the inhibitors that were tested, p16INK4a and p16INK4b. Inactivation of p16 is commonly found in a variety of tumors and may be responsible for the genetic predisposition to melanoma. Thus the mutated cdk4 gene product may play a role in the malignant transformation of some melanomas. A mutated product of the p-catenin gene has also been shown to be recognized by the TIL 1290 line, which was derived from a recurrent tumor in melanoma patient 888 (Robbins et al., 1996). Two partial cDNA clones corresponding to p-catenin were isolated through the use of transient transfection of cDNA pools into class I HLA-A24-expressing 293 cells. The sequences of the two cDNA clones that were isolated differed from the previously published p-catenin sequence at a single nucleotide in the coding region, resulting in a change in the encoded amino acid sequence from serine to phenylalanine. The sequence isolated from the normal cells of the autologous patient, as well as from 12 melanoma cell lines derived from additional patients, appeared to correspond to the previously published sequence. Thus it appears that the melanoma cell lines from patient 888 express a mutated form of the p-catenin gene. This mutation involved a C-to-T transition at a dipyrimidine site, as was found for the cdk4 mutation, and may represent another example of a UV-induced mutational event (Brash et al., 1991). The p-catenin peptide SYLDSGIHF, which contained the mutated phenylalanine residue at the carboxyl terminus, was found to induce half-maximal lysis of target cells at a concentration of 1pM, whereas a l-p,M concentration of the normal peptide (SYLDSGIHS)was required for an equivalent level of sensitization. Thus, it appeared that the mutated peptide was recognized by TIL 1290 about 106-fold more efficiently than the normal peptide. Phenylalanine has previously been shown to be an anchor residue at position 2 or 9 in HLA-A24-binding peptides, whereas serine has not been observed at these positions in peptides that bind to this HLA allele (Kondo et al., 1995). This substitution thus appears to have resulted in the generation of an immunogenic T-cell epitope by enhancing HLA-A24 binding, and the results of a competitive binding assay were consistent with this hypothesis (Robbins et al., 1996). The original 888 melanoma cell line derived from this patient, as well as the 1290 melanoma cell line derived from the recurrence in the patient, both appeared to express the mutant and the normal p-catenin gene product. Thus there was no evidence that immunoselection of variants that failed to express the mutated gene product had occurred in vivo. One possible explanation for this observation is that cells that express the mutated p-catenin gene product may have a selective advantage in vivo. The fbcatenin protein has been shown to be a cytoplasmic protein that interacts with the cellular

Genes Encoding Cancer Antigens

169

adhesion molecule E-cadherin. A number of tumors have been found to contain mutations in these products that appear to affect cell adhesion, and some studies have suggested that the loss of these cell adhesion molecules may be associated with metastasis (Kawanishi et al., 1995; Doki et al., 1993; Vleminckx et al., 1991; Morton et al., 1993). Antigens that appear to have unexpected expression patterns have also been isolated. When the class I HLA-A24-restricted TIL 1290 cell line was used to screen pools of cDNAs generated from the autologous melanoma, a previously undescribed gene, termed p15, was isolated (Robbins et al., 1995). Northern blot analysis indicated that this gene was expressed in all of the normal tissues examined; however, this TIL failed to recognize cells that appeared to express this gene product, such as autologous fibroblasts and EBV B cells. A peptide derived from this molecule, AYGLDFYIL, was shown to be recognized by TIL 1290. The region of this gene that encoded the T-cell epitope was then isolated from autologous EBV B cells and was found to be identical to the corresponding region in the melanoma cDNA clone, demonstrating that this represented a nonmutated product. It is unclear what accounts for the apparent tumor specificity of this product, but there are several possible explanations. Posttranscriptional mechanisms may be involved in regulating expression of this gene product in melanomas. Alternatively, the peptide epitope expressed in the p15 gene product may crossreact with another peptide derived from a distinct protein. In that case, the p15 epitope would not represent the peptide, which is naturally processed and presented in the context of HLA-A24 on the tumor cell surface, and may only be recognized when overexpressed. Similarly, a T-cell clone that appeared to specifically recognize an antigen expressed in lung tumors also recognized a gene that appeared to be widely expressed in a variety of normal cells (Coulie et al., 1993).

IV. CANCER THERAPIES BASED ON

THE MOLECULAR IDENTIFICATION OF CANCER ANTIGENS Approaches to cancer immunotherapy can be divided into active or passive categories. Active immunotherapy involves the direct immunization of cancer patients with cancer antigens in an attempt to boost immune responses against the tumor. Passive immunotherapy refers to the administration of immune reagents, such as immune cells or antibodies with antitumor reactivity, with the goal of directly mediating antitumor responses. Most prior attempts at active immunotherapy utilized either intact cancer cells or cancer cell extracts with the expectation that these materials con-

I70

Steven A. Rosenberg et al.

Table I X Cancer Therapies Based on the Molecular Identification of Cancer Antigens ~

1. Active immunotherapy with:

A. Immunodominant peptides 1. alone 2. combined with adjuvants 3. linked to helper peptides, lipids, or liposomes 4. pulsed onto antigen-presenting cells B. Immunodominant peptides with amino acids substitutions to increase binding to MHC molecules C. Proteins alone or combined with adjuvants D. “Naked” DNA-encoding cancer antigens 1. “gene gun” for intradermal injection 2. intramuscular injection 3. linked to lipids E. Recombinant viruses such as vaccinia, fowlpox, or adenovirus encoding 1. cancer antigens alone 2. cancer antigens plus genes encoding cytokines, costimulatory molecules, or other genes to enhance the immune response F. Recombinant bacteria such as bacillus Calmette-Guerin, Salmonella or Listeriu encoding- cancer antigens - alone or in combination with other molecules 11. Active immunotherapy (above) followed by the administration of immunostimulatory cytokines A. IL-2 B. IL-6 c. IL-10 D. IL-12 E. 1L-15 111. Passive immunotherapy with antitumor lymphocytes raised by in vitro sensitization of TIL or PBL to A. Immunodominant peptides pulsed onto antigen-presenting cells (raise CD8 cells) B. Antigenic proteins coincubated with antigen-presenting cells (exogenous antigenpresenting pathway to raise CD4 cells)

tained tumor antigens in an amount and form capable of stimulating immune responses. The molecular identification of cancer antigens, however, has opened new possibilities for developing immunotherapies for the treatment of human cancer. A summary of some of these approaches is presented in Table IX. The insertion of the gene encoding cancer antigens into high-efficiency expression systems such as Escherichia coli, yeast, or baculovirus provided the opportunity to obtain large amounts of purified tumor antigen for use in immunization. Alternatively, the immunodominant peptides from these tumor antigens could readily be synthesized in vitro and purified in large amounts for immunization either alone or in a form intended to improve their immunogenicity, such as in combination with adjuvant, linked to lip-

G e n e s Encoding Cancer Antigens

171

ids-Iiposomes or helper peptides, or pulsed onto antigen-presenting cells. Modification of individual amino acids of the immunodominant peptides to improve binding efficiency to MHC antigens can potentially increase immunogenicity compared to the native peptide. Techniques utilizing “naked” DNA injected directly into muscle or into the skin have been shown to raise both cellular and humoral immune reactions to encoded antigens (Fynan et af., 1993; Irvine et af., 1995). Techniques using nonviable DNA vectors have the advantage of ease of preparation and safety of administration, and a variety of approaches utilizing this technique are being explored. The most effective forms of immunization involve the incorporation of genes encoding immunogenic molecules into recombinant bacteria such as bacillus Calmette-Guerin, Salmonella, or Listeria or into recombinant viruses such as vaccinia, fowlpox, or adenovirus. The genes encoding cancer antigens can be expressed either alone or in combination with genes encoding cytokines, costimulatory molecules or other genes that can enhance the immune response following infection. Studies with model tumor antigens in murine models have shown that incorporation of the gene for 1L-2 or B7.1 can increase the immunogenicity of model tumor antigens and even mediate the regression of established lung metastases bearing these antigens (Bronte et af., 1995). Active immunotherapy followed by the exogenous administration of immunostimulatory cytokines such as IL-2, IL-6, IL-10, IL-12, or IL-15 may also be used to improve immune responses (Bronte et al., 1995; Rao et al., 1996; Chamberlain et af., 1996). Many of these approaches to active immunization are being pursued in experimental animal models using model tumor antigens. The availability of the genes encoding human tumor antigens has led to the development of clinical trials in humans with cancer. The Surgery Branch of the NCI is conducting clinical trials utilizing immunization with the immunodominant peptides from the MART-1 and gpl00 molecules, and clinical trials immunizing patients with the genes encoding MART-1 and gpl00 in recombinant viruses are about to begin. A listing of approved clinical trials in the Surgery Branch of the NCI is presented in Table X. Passive immunotherapy with immune cells (commonly referred to as adoptive immunotherapy) capable of recognizing human tumor antigens is effective in mediating the regression of cancer in selected patients with metastatic melanoma. In vitro techniques have been developed in which human lymphocytes are sensitized in vitro to tumor antigen-immunodominant peptides presented on antigen-presenting cells (Rivoltini et al., 1995; Salgaller et al., 1995). By repetitive in vitro stimulation, cells can be derived with a far greater capacity to recognize human tumor antigens than the TIL that were used to clone the genes encoding these antigens. Thus, by repeated in vitro sensitization with the MART-1 peptide, lymphocytes could be derived with

172

Steven A. Rosenberg et al.

Table X Clinical Protocols in Surgery Branch of the NCI: Immunization Against Cancer Antigens Immunization

Date Started

Protocols initiated February 2, 1995 MART-1 immunodominant peptide (M9-27) in adjuvant June 20, 1995 gpl00 immunodominant peptides (G9-154, G9-209, G9-280) in adjuvant gpl00 immunodominant peptides modified to November 21, 1995 increase MHC binding (G9-209-2M, G9-290-9V).in adjuvant December 8, 1995 Recombinant adenovirus encoding MART-1 (alone or with systemic IL-2) MART-1 immunodominant peptide January 11, 1996 (M9-27) in adjuvant plus systemic IL-12 Recombinant adenovirus encoding gp100 April 23, 1996 (alone or with systemic IL-2) Protocols approved by NCI-IRB (due to start in 1996) Recombinant vaccinia virus encoding MART-1 or gpl00 (alone or with systemic IL-2) Recombinant fowlpox virus encoding MART-1 or gpl00 (alone or with systemic IL-2) Adoptive transfer of PBL sensitized in vitro to gpl00 peptides

50-100 times the potency of TIL (Rivoltini et al., 1995). The adoptive transfer of these cells may be more effective in mediating tumor regression in vim than are conventionally grown TIL. Techniques are currently being developed to utilize repeated in uitro sensitization to generate large numbers of cells with specific antitumor reactivity for use in the adoptive immunotherapy of human cancer.

REFERENCES Adema, G. J., de Boer, A. J., Vogel, A. M., Loenen, W. A. M., and Figdor, C. G. (1994).J. Biol. Chem. 269,20126-20133. Anichini, A., Maccalli, C., Mortarini, R., Salvi, S., Mazzocchi, A., Squarcina, P., Herlyn, M., and Parmiani, G. (1993). /. Exp. Med. 177, 989-998. Bakker, A. B. H., Schreurs, M. W. J., de Boer, A. J., Kawakami, Y.,Rosenberg, S. A., Adema, G. J., and Figdor, C. G. (1994). J. Exp. Med. 179, 1005-1009. Boel, P., Wildmann, C., Sensi, M. L., Brasseur, R., Renauld, J.-C., Coulie, P., Boon, T., and Van der Bruggen, P. (1995). Immunity 2, 167-175.

Genes Encoding Cancer Antigens

173

Boon, T. (1992).Adv. Cancer Res. 58, 177-210. Bouchard, B., Fuller, B. B., Vijayasaradhi, S., and Houghton, A. N. (1989).J. Exp. Med. 169, 2029-2042. Brash, D. E., Rudolph, J. A., Lin, A., McKenna, G. J., Baden, H. P., Halperin, A. J., and Ponten, J. (1991).Proc. Natl. Acad. Sci. U.S.A. 88, 10124-10128. Brichard, V., Van Pel, A., Wolfel, T., Wolfel, C., De Plaen, E., Lethe, B., Coulie, P., and Boon, T. (1993).J. E x p . Med. 178, 489-495. Bronte, V., Tsung, K., Rao, J. B., Chen, P. W., Wang, M., Rosenberg, S. A., and Restifo, N. P. (1995).J. Immunol. 154, 5282-5292. Bystryn, J.-C., Rigel, D., Friedman, R. J., and Kopf, A. (1987). Arch. Dermatol. 123, 10531055. Castelli, C., Storkus, W. J., Maeurer, M. J., Martin, D. M., Huang, E. C., Pramanik, B. N., Nagabhushan, T. L., Parmiani, G., and Lotze, M. T. (1995). J. Exp. Med. 181, 363-368. Cellis, E., Tsai, V., Crimi, C., DeMars, R., Wentworth, P. A., Chestnut, R. W., Grey, H. M., Sette, A., and Serra, H. M. (1994). Proc. Natl. Acad. Sci. U.S.A. 91, 2105-2109. Chamberlain, R. S., Carroll, M. W., Bronte, V., Hwu, P., Warren, S., Yang, J. C., Nishimura, M., Moss, B., Rosenberg, S. A., and Restifo, N. P. (1996). Cancer Res. 56, 2832-2836. Cheever, M. A., Disis, M. L., Bernhard, H., Gralow, J. R., Hand, S. L., Huseby, E. S., Qin, H. L., Takahashi, M., and Chen, W. (1995). Immunol. Ra? 145,33-59. Chen, W., Khilko, S., Fecondo, J., Margulies, D. H., and McClusky, J. (1994).]. Exp. Med. 180, 1471-1483. Cohen, T., Muller, R. M., Tomita, Y.,and Shibahara, S. (1990).Nucleic Acids Res. 18,28072808. Cole, D. J., Weil, D. P., Shamamian, P., Rivoltini, L., Kawakami, Y., Topalian, S., Jennings, C., Eliyafu, S., Rosenberg, S. A., and Nishimura, M. I. (1994). Cancer Res. 54, 5265-5268. Cole, D. J., Weil, D. P., Shilyansky, J., Custer, M., Kawakami, Y., Rosenberg, S. A., and Nishimura, M. I. (1995). Cancer Res. 55, 748-752. Colombari, R., Bonetti, F., Zamboni, G., Scarpa, A., Marino, F., Tomezzoli, A., Capelli, P., Menestrina, F., Chilosi, M., and Fiore-Donati, L. (1988). Virchows Arch. A Pathol. Anat. 413,17-24. Coulie, P. G., Weynants, P., Lehmann, F., Herman, J., Brichard, V., Wolfel, T., Van Pel, A., De Plaen, E., Brasseur, F., and Boon, B. (1993).J. Immunother. 14, 104-109. Coulie, P. G., Brichard, V., Van Pel, A., Wolfel, T., Schneider, J., Traversari, C., Mattei, S., De Plaen, E. D., Lurquin, C., Szikora, J.-P., Reauld, J.-C., and Boon, T. (1994).J. Exp. Med. 180, 35-42. Coulie, P. G., Lehmann, F., Lethe, B., Herman, J., Lurquin, C., Andrawiss, M., and Boon, T. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 7976-7980. Cox, A. L., Skipper, J., Cehn, Y., Henderson, R. A., Darrow, T. L., Shabanowitz, J., Engelhard, V. H., Hunt, D. F., and Slingluff, C. L. (1994).Science 264, 716-719. Crowley, N. J., Darrow, T. L., Quinn-Allen, M. A., and Seigler, H. F. (1991).J. lmmunol. 146, 1692-1699. Darrow, T. L., Slingluff, C. L., and Seigler, H. F. (1989).J. Immunol. 142, 3329-3335. Descombes, P., and Schibler, U. (1991).Cell 67, 569-579. Doki, Y.,Shiozaki, H., Tahara, H., Inoue, M., Oka, H., Ihara, K., Kadowaki, T., Takeichi, M., and Mori, T. (1993). Cancer Res. 53, 3421-3426. Elas, A. V., Nijman, H. W., Van der Minne, C. E., Mourer, J. S., Kast, W. M., Melief, C. J. M., and Schrier, P. I. (1995). Int. J. Cancer 61,389-396. Fajardo, J. E., and Shatkin, A. (1990).Proc. Natl. Acad. Sci. U.S.A. 87, 328-332. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G. (1993). Nature (London) 351,290-296. Fisk, B., Blevins, T. L., Wharton, J., and loannides, C. (1995).J. Exp. Med. 181,2109-2117.

I74

Steven A. Rosenberg et al.

Fleischhauer, K., Avila, D., Vilbois, F., Traversari, C., Bordignon, C., and Wallny, H. J. (1995). Tissue Antigens 44, 311-317. Fynan, E., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., and Robinson, H. L. (1993). Proc. Nutl. Acud. Sci. U.S.A. 90, 11478-11482. Gaugler, B., Van Den Eynde, B., Van der Bruggen, P., Romero, P., Gaforio, J. J., De Plaen, E., Lethe, B., Brasseur, F., and Boon, T. (1994).J. Exp. Med. 179, 921-930. Gown, A. M., Vogel, A. M., Hoak, D., Cough, F., and McNutt, M. A. (1986). Am. J. Pathol. 123,195-203. H a m , S. R. (1995).Biochtmie 76, 880-886. Hara, I., Takechi, Y., and Houghton, A. N. (1995).J. Exp. Med. 182, 1609-1614. Hom, S. S., Topalian, S. L., Simoni, S. T., Mancini, M. J., and Rosenberg, S. A. (1991). J. Immunother. 10, 153-164. Hom, S. S., Rosenberg, S. A., and Topalian, S. L. (1993). Cancer lmmunol. Immunother. 36, 1-8. Hom, S. S., Schwartzentruber, D. J., Rosenberg, S. A., and Topalian, S. L. (1993).J. Immunother. 13, 18-30. Houbiers, J. G. A., Nijman, H. W., van der Burg, S. H., Drijfhout, J. W., Kenemans, P., van de Velde, C. J. H., Brand, A., Momburg, F., Kast, W. M., and Melief, C. J. M. (1993). Eur. J. Immunol. 23,2072-2077. Hunt, D. F., Henderson, R. A., Shabanowitz, J., Sakaguchi, K., Michel, H., Sevilir, N., Cox, A. L., Appella, E., and Engelhard, V. H. (1992). Science 255, 1261-1263. Irvine, K., Rao, J., Rosenberg, S., and Restifo, N. (1995).J. lmmunol. 156,238-245. Itoh, K., Tilden, A. B., and Balch, C. M. (1986). Cancer Res. 46,3011-3017. Jimenez-Cervantes, C., Solano, F., Kobayashi, T., Urabe, V. J., Hearing, J. A., Lozano, J. A., and Garcia-Borron, J. C. (1994).J. Biol. Chem. 269, 17993-18001. Kang, X.-Q., Kawakami, Y., Sakaguchi, K., El-Gamil, M., Wang, R.-F., Yannelli, J. R., Appella, E., Rosenberg, S. A., and Robbins, P. F. (1995).J. Immunol. 155, 1343-1348. Kawakami, Y., and Rosenberg, S. (1996a). lnt. Rev. lmmunol. (in press). Kawakami, Y., and Rosenberg, S. (1996b). lmmunol Res. 15, 179-190. Kawakami, Y., Zakut, R., Topalian, S. L., Stotter, H., and Rosenberg, S. A. (1992).J.lmmunol. 148,638-643. Kawakami, Y., Nishimura, I., Restifo, N. P., Topalian, S. L., O’Neil, B. H., Shilyansky, J., Yannelli, J. R., and Rosenberg, S. A. (1993).J. Immunother. 14, 88-93. Kawakami, Y., Eliyahu, S., Delgaldo, C. H., Robbins, P. F., Rivoltini, L., Topalian, S. L., Miki, T., and Rosenberg, S. A. (1994a). Proc. Nutl. Acud. Sci. U.S.A. 91, 3515-3519. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Sakaguchi, K., Appella, E., Yannelli, J. R., Adema, G. J., Miki, T., and Rosenberg, S. A. (1994b). Proc. Natl. Acud. Sci. U.S.A. 91, 6458-6462. Kawakami, Y., Eliyahu, S., Sakaguchi, K., Robbins, P. F., Rivoltini, L., Yannelli, J. B., Appella, E., and Rosenberg, S. A. (1994~). J. Exp. Med. 180, 347-352. Kawakami, Y., Eliyahu, S., Jennings, C., Sakaguchi, K., Kang, X.-Q., Southwood, S., Robbins, P. F., Sene, A., Appella, E., and Rosenberg, S. A. (1995).J. Zmmunol. 154, 3961-3968. Kawanishi, J., Kato, J., Sasaki, K., Fujii, S., Watanabe, M., and Niitsu, Y. (1995).Mol. Cell. Biol. 15, 1175-1181. Kim, R. Y., and Wistow, G. (1991).Exp. Eye Res. 55, 657-662. Kobayashi, T., Urabe, K., Orlow, S. J., Higashi, K., Imokawa, G., Kwon, B. S., Potterf, B., and Hearing, V. J. (1994). J. Biol. Chem. 269, 29198-29205. Kondo, A., Sidney, J., Southwood, S., del Guercio, M.-F., Appella, E., Sakamoto, H., Celis, E., Grey, H. M., Chestnut, R. W., and Kubo, R. T. (1995).J. Immunol. 155, 4307-4312. Kwon, B. S., Chintamaneni, C., Kozak, C. A., Copeland, N. G., Gilbert, D. J., Jenkins, N., Barton, D., Francke, U., Kobayashi, Y., and Kim, K. K. (1991).PYOC.Natl. Acad. Sci. U.S.A. 88,9228-9232.

Genes Encoding Cancer Antigens

I75

Mandelbolm, O., Berke, G., Fridkin, M., Feldman, M., Eisenstein, M., and Eisenbach, L. (1994). Nature (London) 369, 67-71. Marchand, M., Weynants, P., Rankin, E., Arienti, F., Belli, F., Parmiani, G., Cascinelli, N., Bouriond, A., Vanwijck, R., Humblet, Y., Cannon, J. L., Laurent, C., Naryaert, J. M., Plagne, R., Deraemaeker, R., Knuth, A., Jaeger, E., Brasseur, F., Herman, J., C o n k , P. G., and Boon, T. (1996). Int. J. Cancer 63, 883-885. Marincola, F. M., Rivoltini, L., Salgaller, M. L., Player, M., and Rosenberg, S. A. (1996). J. Immunother. (in press). Mattes, M. J., Thomson, T. M., Old, L. J., and Lloyd, K. 0. (1983).Int. J. Cancer 32,717-721. Mochii, M., Agata, K., and Eguchi, G. (1991). Pigm. Cell Res. 4,41-47. Morton, R. A., Ewing, C. M., Nagafuchi, A., Tsukita, S., and Isaacs, W. B. (1993). Cancer Res. 53,3585-3590. Moudgil, K. D., and Sercarz, E. E. (1994). Immunol. Today 15,353-355. Moudgil, K. D., Ametani, A., Grewal, I. S., Kumar, V., and Sercarz, E. E. (1993). Int. Rev. Immunol. 10,365-377. Muralidhar, S., Becerra, S. P., and Rose, J. A. (1994).J. Virol. 68, 170-176. Muul, L., Spiess, P., Director, E., and Rosenberg, S. A. (1987). J. Immunol. 138, 989-995. Nanda, N. K., and Sercarz, E. E. (1995). Cell 82, 13-17. Nordlund, J. J., Kirkwood, J. M., Forget, B. M., Milton, G., Albert, D. M., and Lerner, A. B. (1983).I. Am. Acad. Dermatol. 9, 689-695. O’Neil, B. H., Kawakarni, Y., Restifo, N. P., Bennink, J. R., Yewdell, J. W., and Rosenberg, S. A. (1993).J. Immunol. 151, 1410-1418. Ossipow, V., Descombes, P., and Schibler, U. (1993). Proc. Natl. Acad. Sci. U.S.A. 90,82198223. Parker, K. C., Shields, M., DiBrino, M., Brooks, A., and Coligan, J. E. (1995). Immunol. Res. 14,34-57. Parkhurst, M. R., Salgaller, M., Southwood, S., Robbins, P., Sette, A., Rosenberg, S. A., and Kawakami, Y. (1996).J. Imrnunol. (in press). Peoples, G. E., Goedegebuure, P. S., Smith, R., Linehan, D. C., Yoshino, I., and Eberlein, T. J. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 432-436. Rao, J. B., Bronte, V., Irvine, K. R., Carroll, M. W., Rosenberg, S. A., and Restofo, N. P. (1996). J. Immunol. (in press). Richards, J. M., Mehta, N., Ramming, K., and Skosey, P. (1992). J. Clin. Oncol. 10, 13381343. Rivoltini, L., Kawakami, Y., Sakaguchi, K., Southwood, S., Sette, A., Robbins, P. F., Marincola, F. M., Salgaller, M. L., Yannelli, J. R., Appella, E., and Rosenberg, S. A. (1995).J. Immunol. 154,22S7-2265. Robbins, P. F., El-Garnil, M., Kawakami, Y., Stevens, E., Yannelli, J., and Rosenberg, S. A. (1994). Cancer Res. 54, 3124-3126. Robbins, P. F., El-Garnil, M., Li, Y. F., Topalian, S. L., Rivoltini, L., Sakaguchi, K., Appella, E., Kawakami, Y., and Rosenberg, S. A. (1995).J. Immunol. 154, 5944-5950. Robbins, P. F., El-Gatnil, M., Li, Y. F., Kawakami, Y., Loftus, D., Appella, E., and Rosenberg, S. A. (1996).J. Exp. Med. 183, 1185-1192. Romero, P., Pannetier, C., Herman, J., Jongeneel, C. V., Cerottini, J.-C., and Coulie, P. G. (1995).J. Exp. Med. 182, 1019-1028. Rosenberg, S. A. (1991). Cancer Res. 51, 5074s-5079s. Rosenberg, S. A., Packard, B. S., Aebersold, P. M., Solomon, D., Topalian, S. L., Toy, S. T., Simon, P., Lotze, M. T., Yang, J. C., Seipp, C. A., Simpson, C., Carter, C., Bock, S., Schwartzentruber, D., Wei, J. P., and White, D. E. (1988). New Engl. J. Med. 319 1676-1680. Rosenberg, S. A., Yang, J. C., Topalian, S. L., Schwartzentruber, D. J., Weber, J. S., Parkinson, D. R., Seipp, C. A., Einhorn, J. H., and White, D. E. (1994).JAMA 271, 907-913. Rosenberg, S. A., Yannelli, J. R., Yang, J. C., Topalian, S. L., Schwartzentruber, D. J., Weber,

I76

Steven A. Rosenberg et al.

J. S., Parkinson, D. R., Seipp, C. A., Einhorn, J. H., and White, D. E. (1995).J. Natl. Cancer Inst. 86, 1159-1166. Rosenberg, S. A., and White, D. E. (1996).J. Immunother. 19, 81-84. Salgaller, M. L., Afshar, A., Marincola, F. M., Rivoltini, L., Kawakami, Y., and Rosenberg, S. A. (1995). Cancer Res. 55, 4972-4979. Salgaller, M. L., Weber, J. S., Koenig, S., Yanelli, J. R., and Rosenberg, S. A. (1994). Cancer Immunol. Immunother. 39, 105-116. Schaumburg-Lever, G., Metzler, G., and Kaiserling, E. (1991).J. Cutaneous Pathol. 18,432435. Schwartz, S., Felber, B. K., and Pavlakis, G. N. (1992). Mol. Cell. Biol. 12, 207-219. Schwartzentruber, D. J., Topalian, S. L., Mancini, M. J., and Rosenberg, S. A. (1991). J. Immunol. 146,3674-3681. Sensi, M., Traversari, C., Radrizzani, M., Salvi, S., Maccalli, C., Mortarini, R., Rivoltini, L., Farina, C., Nicolini;G., Wolfel, T., Brichard, V., Boon, T., Bordignon, C., Anichini, A., and Parmiani, G. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, 5674-5678. Sercarz, E. E., Lehmann, P. V., Ametani, A., Benichou, G., Miller, A., and Moudgil, K. (1993). Annu. Rev. Immunol. 11,729-766. Sette, A., Vitiello, A., Reherman, B., Fowler, P., Nayersina, R., Kast, W. M., Melief, C. J., Oseroff, C., Yuan, L., and Ruppert, J. (1994).J. Immunol. 153,5586-5592. Shaw, M. W., Choppin, P. W., and Lamb, R. A. (1983).Proc. Natl. Acad. Sci. U.S.A. 80,48794883. Slingluff, C. L. Jr., Cox, A. L., Henderson, R. A., Hunt, D. F., and Engelhard, V. H. (1993).J. lmmunol. 150,2955-2963. Spiropoulou, C. F., and Nichol, S. T. (1993).J. Virol. 67, 3103-3110. Storkus, W. J., Zeh, H. J., Maeurer, M. J., Salter, R. D., and Lotze, M. T. (1993).J. Immunol. 151,3719-3727. Thomson, T. M., Mattes, J. M., Roux, L., Old, L. J., and Lloyd, K. 0. (1985).J. Invest. DennatoI. 85, 169-174. Thomson, T. M., Real, F. X., Murakami, S., Cordon-Cardo, C., Old, L. J., and Houghton, A. N. (1988).J. Invest. Dermatol. 90, 459-466. Topalian, S. L., Muul, L. M., Solomon, D., and Rosenberg, S. A. (1987).J. Immunol. Meth. 102, 127-141. Topalian, S., Solomon, D., Avis, F. P., Chang, A. E., Freeksen, D. L., Linehan, W. M., Lotze, M. T., Robertson, C. N., Seipp, C. A., Simon, P., Simpson, C. G., and Rosenberg, S. A. (1988).J. Clin. Oncol. 6, 839-853. Topalian, S. L., Rivoltini, L., Mancini, M., Markus, N. R., Robbins, P. F., Kawakami, Y., and Rosenberg, S. A. (1994a). Proc. Natl. Acad. Sci. U.S.A. 91, 9461-9465. Topalian, S. L., Rivoltini, L., Mancini, M., Ng, J., Hartzman, R. J., and Rosenberg, S. A. (1994b). Int. J. Cancer, 58, 69-79. Topalian, S. L., Gonzales, M. I., Parkhurst, M., Li, Y. F., Southwood, S., Sette, A., Rosenberg, S. A., and Robbins, P. F. (1996).J. Exp. Med. 183, 1965-1971. Traversari, C., Van der Bruggen, P., Luescher, I. F., Lurquin, C., Chomez, P., Van Pel, A., De Plaen, E., Amar-Costesec, A., and Boon, T. (1992).J. Exp. Med. 176, 1453-1457. Uenaka, A., Ono, T., Akisawa, T., Wada, H., Yasuda, T., and Nakayama, E. (1994).J. Exp. Med. 180,1599-1607. Van Den Eynde, B., Hainaut, P., Herin, M., Knuth, A., Lemoine, C., Weynants, P., Van der Bruggen, P., Fauchet, R., and Boon, T. (1989). Int. J. Cancer 44,634-640. Van Den Eynde, B., Peters, O., De Backer, O., Gaugler, B., Lucas, S., and Boon, T. (1995).J. Exp. Med. 182,689-698. Van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., DePlaen, E., Van Den Eynde, B., Knuth, A., and Boon, T. (1991). Science 254, 1643-1647.

Genes Encoding Cancer Antigens

I77

Van der Bruggen, P., Bastin, J., Gajewski, T., Coulie, P. G., Boel, P., de Smet, C., Traversari, C., Townsend, A., and Boon, T. (1994).Eur. J. Imrnunol. 24, 3038-3043. Vennegoor, C., Hageman, P., Van Nouhuijs, H., Ruiter, D. J., Calafat, J., Ringens, P. J., and Rumke, P. (1988).Am. J. Puthol. 130, 179-192. Vijayasyradhi, S., Bouchard, B. B., and Houghton, A. N. (1990)./. Exp. Med. 171,1375-1380. Visseren, M. J. W., van Elsas, A., van der Voort, E. I. H., Ressing, M. E., Kast, W. M., Schrier, P. I., and Melief, C. J.M. (1995).J. Immunol. 154, 3991-3998. Vleminckx, K., Vakaet, L. J., Mareel, M., Fiers, W., and Van Roy, F. (1991).Cell66, 107-119. Vogel, A. M., and Esclamado, R. M. (1988).Cancer Res. 48, 1286-1294. Wang, R.-F., Robbins, P. F., Kawakami, Y., Kang, X.-Q., and Rosenberg, S. A. (1995).J. Exp. Med. 181,799-804. Wang, R. F., Parkhurst, M. R., Kawakami, Y., Robbins, P. F., and Rosenberg, S. A. (1996).J. Exp. Med. 183, 1131-1140. Wolfel, T., Schneider, J., Meyer Zum Buschenfelde, K.-H., Rammensee, H.-G., Rotzschke, O., and Falk, K. (1994). Int. J. Cancer 57, 413-418. Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., Klehmann-Hieb, E., De Plaen, E., Hankeln, T., Meyer Zum Buschenfelde, K.-H., and Beach, D. (1995). Science 269, 12811284. Wucherpfennig, K. W., and Strominger, J. L. (1995). Cell 80, 695-705.

This Page Intentionally Left Blank

The M E N I1 Syndromes and the Role of the vet ProtoHoncogene Bruce A. 1. Ponder and Darrin Smith CRC Human Cancer Genetics Research Group, Addenbrookei Hospital, University of Cambridge, Cambridge CB2 2QQ, England

I. Introduction 11. The MEN I1 Syndromes A. Clinical Varieties of MEN 11 B. Tumor Types in MEN I1 C. The Development of Tumors in MEN 11: Multifocal Hyperplasia and Screening D. The Clinical Varieties of MEN I1 E. Hirschsprung Disease 111. The ret Proto-oncogene A. Identification of ret as an Oncogene B. Ret Structure C. Mutations in ret in the MEN I1 Syndromes D. Re# Mutations in Sporadic MTC, Pheochromocytoma, and Parathyroid Tumors E. The Effects of ret Mutations in MEN I1 F. Downstream Signaling from ret 1V. Development of the Tissues Involved in MEN 11, and Patterns of ret Expression A. Thyroid “C” Cells B. Parathyroid C. Adrenal Medulla D. Enteric Nervous System E. Other Sites of ret Expression V. Speculations on How Different ret Mutations Result in the Associated Phenotypes and in Tumor Formation A. The Extracellular Cysteine Mutations B. The M918T Mutation of MEN IIB VI. Other Events in Tumor Progression VII. Animal Models of MEN 11 VIII. Clinical Implications of the Identification of ret Mutations in MEN I1 IX. Future Prospects References

Advances in CANCER RESEARCH, Vol. 70 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

180

Bruce A. 1. Ponder and Damn Smith

I. INTRODUCTION The multiple endocrine neoplasia type I1 (MEN 11) syndromes are dominantly inherited syndromes of tumor formation and disordered development that involve principally four tissues: the "C" cells of the thyroid, the adrenal medulla, the parathyroid, and the intestinal autonomic nerve plexuses. There are distinct clinical subtypes of the syndromes, depending on the combination of tissues affected and the presence or absence of specific developmental abnormalities. The yet proto-oncogene encodes a receptor tyrosine kinase. The phenotypes associated with ret mutations in humans, as well as studies of yet expression in development and the effects of absent yet activity in mouse knockout experiments, indicate that ret has a role in the normal development of a diverse set of tissues. These include derivatives of neural ectoderm. Mutations of ret in MEN I1 syndromes are of particular interest for several reasons:

1. The mutations confer an alteration or gain of function that is dominant at the cellular level. This contrasts with the general model for the mechanism of susceptibility in the inherited cancer syndromes, which has been based on retinoblastoma, in which the inherited defect is a loss-of-function mutation, recessive at the cellular level. 2. As might be expected, since they cause specific alterations of function, the mutations in yet that predispose to the MEN I1 syndromes affect specific regions of the gene. Different mutations are highly correlated with different clinical varieties of the MEN I1 syndrome, and it is now an interesting challenge to explain these effects in terms of altered cellular signaling and its effect on developmental mechanisms. 3. Loss of function mutations of yet also occur, resulting in a different set of developmental abnormalities of which the most prominent is the absence of intestinal autonomic nerve plexuses seen in Hirschsprung disease. 4. However, a few families have been described in which both MEN I1 (presumed gain of function mutation) and Hirschsprung disease (loss of function mutation) coexist within single individuals; each of these families appears to have as its sole genetic abnormality one of two similar specific mutations in the ret gene. This rather surprising observation is not yet explained in any detail, but it illustrates a general point: the effects of yet mutations are likely to differ in specific cell types and to be influenced by genetic background. This is true of many inherited diseases, but the combination of a limited set of mutations and of distinct phenotypes based on the involvement of four cell types-thyroid C cells, adrenal medulla, parathyroid, and autonomic nerve plexuses-suggests that the MEN I1 syn-

181

The MEN 11 Syndromes a n d ret

dromes may provide a good model in which to study this type of genetic modification. This review focuses on genetic and biological aspects of the MEN I1 syndromes and of ret mutations. Discussion of clinical and pathological aspects is limited to providing the background to the biological problem and indicating the application of the genetic results.

11. THE MEN I1 SYNDROMES A. Clinical Varieties of MEN I1 There are three distinct clinical varieties of MEN I1 (MEN IIA, MEN IIB, and familial medullary thyroid carcinoma [FMTC]) that differ in the patterns of tissues involved (Farndon et al., 1986; Khairi et al., 1975; Schimke et al., 1968) (Table I).

B. Tumor vpes in M E N I1 Each of the tumors that occurs in MEN I1 has a histologically similar nonhereditary or “sporadic” counterpart, as is the case with most tumors at other sites. Pheochromocytoma and parathyroid tumors also occur as part of other hereditary syndromes (Table 11). The tumor derived from the thyroid C cells is the medullary thyroid carcinoma (MTC) (Saad et al., 1984). These tumors are malignant, metastaTable I Patterns of Tissue Involvement in the M E N 11 Syndromes

Thyroid C cells Adrenal medulla Parathyroid Enteric ganglia Other developmental abnormalities

MEN IIA

MEN IIB

FMTC

Tumor Tumor Hyperplasia/benign tumor Normala None

Tumor Tumor Not involved

Tumor Not involved Not involved

Hyperplasia

Normal None

Various6

UUsually there is no abnormality of enteric ganglia in MEN IIA, but a few families are described in which there is absence of ganglia from a variable length of the intestine in some individuals. Olncludes musculoskeletal and others (see text).

182

Bruce A. 1. Ponder and Damn Smith

Table II Occurrence of Pheochromocytoma and Parathyroid Tumors in Other Inherited Syndromes Pheochromocytoma

Parathyroid tumor

Von Hippel-Lindau (VHL) syndrome Neurofibromatosis type 1 (Probably) site-specific pheochromocytomaa Multiple endocrine neoplasia type 1

aMany, but probably not all, of these families are due to mutations in the VHL gene.

sizing generally at a stage when the primary tumor is 5-10 mm in diameter, first within the thyroid and locally within the neck, and then more widely. The rate of progression of the tumor is very variable, and survivals of 10 years or more with metastatic disease are not uncommon (Chong et al., 1975; Hill et al., 1973). The C cells, and the tumors derived from them, secrete the hormone calcitonin. This provides a valuable tumor marker for early diagnosis in family members at risk (Gage1 et al., 1988; see later), and also to follow the course of advanced disease, but there is no obvious clinical consequence of calcitonin overproduction. The tumor derived from adrenal medulla is the pheochromocytoma (Howe et al., 1993). Generally these are nonmalignant (Dralle et al., 1989). However, the tumors, like the adrenal medulla, commonly secrete adrenaline and noradrenaline. If unsuspected, this may lead to fatal hypertensive episodes, especially in situations such as childbirth or under general anesthesia. The parathyroid abnormalities in MEN I1 are benign, sometimes hyperplasia and sometimes adenoma, the latter implying true (benign) tumor formation. The parathyroid glands secrete parathormone, which mobilizes calcium from bone and raises blood calcium levels. Although parathyroid involvement in MEN I1 is often clinically silent, it may lead to symptomatic hypercalcemia or to renal stones.

C. The Development of Tumors in MEN 11: Multlfocal Hyperplasia and Screening Just as familial cancers at any site are commonly multiple and associated with multiple preneoplastic changes in the target tissue, so in the MEN I1 syndromes, in contrast to cases of nonhereditary disease, there are multiple foci of hyperplasia in the target tissues preceding the development of overt tumors (Block et al., 1980; Emmertsen et al., 1983; Wolfe et al., 1973) (Fig. 1).The multiple foci of C cell hyperplasia in the thyroid of a MEN I1 gene carrier can be detected before the stage of tumor formation by biochemical

The MEN II Syndromes and ret

183

Fig. I C Cell hyperplasia in MEN IIA thyroid. Prominent groups of “C” cells are demonstrated by immunochemistry for calcitonin, among the thyroid follicles which are unstained. Courtesy of Dr. G . Thomas.

screening based on the secretion in the C cells of calcitonin (Gagel et al., 1988). A stimulus, usually of intravenous pentagastrin and/or calcium, will cause the C cells to release their stored calcitonin into the bloodstream. Measurement of circulating calcitonin levels before and after the stimulus provides an indication of C cell mass, and hence an early recognition of those among the members of a family at risk who have inherited the predisposition. The test is sufficiently sensitive to detect C cell hyperplasia before progression to invasive tumor, and surgery based on presymptomatic biochemical screening is likely to be curative (Dralle et al., 1989; Gagel et al., 1988). Biochemical testing for adrenaline and its metabolites and for parathormone can similarly be used to detect clinically occult adrenal and parathyroid involvement. The details of the clinical use of these tests are beyond the scope of this review, but there are two points that are relevant to studies of the biology of MEN 11. The first is that there are now several reports of individuals in MEN I1 families who have had thyroidectomy on the basis of abnormal calcitonin screening and who were reported to have C cell hyperplasia, but who were subsequently shown not to have inherited the predisposing ret mutation present in the family (Lips et al., 1994; Wolfe et al., 1992). This suggests the possibility that there may be other genetic or nongenetic influences on C cell mass that might influence the expression of the

184

Bruce A. 1. Pbnder and Darrin Smith

MEN I1 mutation (Gibson et al., 1981, 1982; O’Toole et al., 1985). The second point relates to epidemiology. In studies of the penetrance of MEN I1 mutations, of genotype-phenotype correlations (such as those presented in Section IILC), or of clinical matters such as survival or the relative frequency of familial and sporadic disease, it is clearly very important to define precisely the phenotype that is being used: symptomatic tumor or biochemically detectable abnormality. Unhappily, especially in the earlier MEN I1 literature, screen-diagnosed and clinically presenting cases are often combined in single series, which makes interpretation difficult. More recently, efforts to collect data for phenotype-genotype correlation by the International RET Mutation Consortium1 (Mulligan et al., 1995) have shown that complete documentation of phenotype within a family at the level of biochemical screening is difficult to achieve even in centers that have a research interest. Critical appraisal of the quality of the phenotype data is important for the interpretation of most studies that relate to MEN 11.

D. The Clinical hrieties of MEN I1 1. MEN 11A

a. incidence The incidence of MEN IIA has not been accurately documented. An attempt to identify all new cases of MTC (hereditary and nonhereditary) in a 2-year period in England and Wales (population 45 million), based on cancer registry data and a review of requests for calcitonin estimations from regional assay centers, suggested an incidence of MTC overall of about 1per 1 million population per year (unpublished data). However, large discrepancies in numbers of registrations between cancer registries over several years suggest the data might not be entirely accurate. It is generally assumed that about 25% of all MTC are heritable. This figure is not derived from any systematic population-based study, but from two observations. In early clinical series (Chong et al., 1975; Saad et al., 1984; Sizemore et al., 1977), about 15% of cases had an evident family history; in later studies in which family members of apparently isolated cases were offered biochemical screening, as many as 10% of apparently nonfamilial cases were found to have affected relatives (Ponder et al., 1986). If these estimates are correct, they would translate to about 50 new cases of MTC per year in England and 1 The International RET Mutation Consortium was convened at the Fifth International MEN Workshop in Stockholm in 1994. The aim of the Consortium is to collate data on mutations in ret and phenotypes in the MEN I1 syndromes. All are welcome to contribute. For details, or to submit data, please contact Charis Eng at [email protected] (FAX 1-617-632-4280)or Lois Mulligan at [email protected](FAX 1-613-548-1348).

185

The MEN II Syndromes and ret

Wales, of which perhaps 12 would be new cases of MEN IIA or FMTC and an unknown but almost certainly smaller number would be MEN IIB (see later).

b. Frequency of New Mutations The frequency is not precisely known. A few documented cases of new mutations, based on normal stimulated calcitonin screening of the parents of a proven familial case, and more recently on mutation testing, have been reported (e.g., Mulligan et al., 1 9 9 4 ~ )The . common MEN IIA mutations occur on several different haplotypes, implying independent origin (Gardner et al., 1994; Narod et al., 1992). However, several very extensive MEN IIA families have been traced back 200 years and more (Narod et al., 1992; Ponder et al., 1988; Telenius-Berg et al., 1984), suggesting that founder mutations may not be uncommon. This contrasts with MEN IIB, wherein new mutations are more usual.

c. T h e Pattern of Disease within a n d between Families About 70% of individuals who carry one of the ret mutations that predispose to MEN IIA will develop clinically significant thyroid C cell tumor in their lifetime (Easton et al., 1989) (Fig. 2). Specific figures are not available, 100 1

90

I

80 I

al

70 0

20

10

0

r--

I

I

I

I

0

10

20

30

I

40

I

I

I

50

60

70

Age

Fig. 2 Age-related probability of detection of disease in MEN IIA. Probability that an individual with the MEN IIA gene will have presented to medical attention (dashed line) or be detectable by a pentagastrin stimulation test (solid line) by a given age. From Easton, D. F., Ponder, M. A., Cummings, T., et al. (1989). Am. /. Hum. Genet. 44, 208-215. Published by University of Chicago Press.

I86

Bruce A. I. Ponder and Darrin Smith

but probably about 50% will develop pheochromocytoma, and perhaps 510% symptomatic parathyroid disease (Narod et al., 1989; Schuffenecker et al., 1994). It is unusual for pheochromocytoma to be present without evidence of C cell abnormality at least by biochemical testing in the same individual (Gagel et al., 1988). However, the pattern of disease varies from family to family; some MEN IIA families have many cases of pheochromocytoma and some only one-and the FMTC families described later have none. We now know (see Section 1I.C) that the probability of occurrence of pheochromocytomas and also perhaps of parathyroid disease is influenced by the precise yet mutation involved (Mulligan et al., 1994b). Different germline mutations therefore account for some of the difference between families. However, there appears also to be variation between individuals within the same family. This could in principle be due to chance or environmental or modifying gene effects. These can be distinguished by an analysis of the pattern of phenotypes within the family-for example, modifying genes would be predicted to result in diminishing degrees of similarity between relatives with increasing distance of relationship (Easton et d., 1993). In practice, any analysis of this type in MEN I1 is likely to be difficult using current clinical material (Easton et al., 1989). This is because of the comparative rarity of the disease, but also because of the mixture of diagnostic criteria for “affected” status that will have been applied to different individuals in most families (see Section 1I.C)-clinical onset of disease, prevalence biochemical screens, and incidence screens-and the difficulty of combining this information. The best prospects for such an analysis probably lie with the phenotypes of C cell hyperplasia in noncarriers of the ret mutation (mentioned earlier) and with the combined MEN II-Hirschsprung disease families described in Section 1I.D.l.d. In the case of Hirschsprung disease, candidate modifier loci have already been suggested (Puffenberger et al., 1994).

d. Variants of MEN IIA Syndrome and Associated Phenotypes There are no developmental abnormalities that are known to be consistently associated with MEN IIA or FMTC. There are, however, two clinical variants, each of which has been described in a small number of families. i. MEN IIA with cutaneous lichen amyloidosis. Itchy skin lesions in the scapular area, with histological appearances of cutaneous lichen amyloidosis, have been described in several MEN IIA families (Chabre et al., 1992; Gagel et al., 1989; Nunziata et al., 1989; Robinson et al., 1992). In each family, only MEN IIA carriers have been affected. A dermatome distribution of the lesion in one French family (Chabre et al., 1992) suggested a neurological basis for the lesion, related to the expression of ret in dorsal

The MEN I1 Syndromes and ret

187

Fig. 3 Pedigree of a family in which individuals have features of both MEN I1 and Hirschsprung disease. Light stippling, MEN I1 tumors; heavy stippling, Hirschsprung disease.

root ganglia (see Section 1V.E); however, there is currently no evidence to support this speculation. All families in which MEN IIA has been associated with this lesion have so far been found to have the typical MEN IIA yet mutation, in codon 634 (Schuffenecker et af., 1994). ii. MEN I1 with Hirschsprung disease. Six families have been reported in which MEN I1 (MEN IIA or FMTC) and Hirschsprung disease are coinherited, some family members having both phenotypes (Angrist et af., 1995; Borst et af., 1995; Mulligan et af., 1994a; Verdy et al., 1982) (Fig. 3). In the larger families (Borst et af., 1995; Verdy et af., 1982) there is possibly the impression that the Hirschsprung phenotype is less highly penetrant than in “classical” Hirschsprung families and that it clusters in some branches, in a pattern suggestive of the effect of modifier genes, though there are no reports of a formal analysis. As is discussed in Section III.C, each of these families has a similar mutation in yet, and the conclusion at present is that this single mutation somehow causes the apparently contrasting phenotypes in the same individual. 2. FAMlLIAL MTC

Evidence for a separate category of “site-specific” familial MTC, distinct from MEN IIA, came originally from the report of two extensive families by Farndon et al. (1986). A particular feature of these families was the late onset and low mortality of the thyroid tumors. Subsequently other such

188

Bruce A. J. Ponder and Damn Smith

families have been recognized (e.g., Lairmore et al., 1991), and the results of yet mutation analysis support the idea of distinct categories of FMTC and MEN IIA, although with some overlap. Clinical impressions and mutation analysis both suggest that FMTC is less common than MEN IIA. There is a difficulty in categorizing small families with MTC only as FMTC or MEN IIA, because the inconstant occurrence of adrenal and parathyroid disease in MEN IIA means that such a family might be a MEN IIA family in which by chance these other components of the syndrome are not yet manifest. Because of this, in the classification used in the analysis of phenotypegenotype correlations given in Section III.C, FMTC is arbitrarily defined as a family with at least four cases of MTC and no evidence of adrenal or parathyroid abnormality from biochemical screening of available family members at risk. There is then a rather large set of unclassified small families with MTC only, which might belong in either group. Ret mutation analysis (see Section 1II.C) suggests that some of these families do in fact belong in the MEN IIA group, and some to FMTC, but in about 25% no yet mutation has been found. This raises the possibility of the existence of another locus that predisposes only to MTC, possibly with low penetrance (Nelkin et al., 1989). There have been occasional accounts of MTC families in which there was said to be evidence against genetic linkage to the yet locus, implying heterogeneity, but the phenotype used for linkage in at least some of these has been a mixture of MTC and C cell hyperplasia. Because C cell hyperplasia is such a difficult phenotype to define, it is difficult to know how to interpret these results. They may be completely misleading, or they may indeed indicate the existence of another locus that is involved in C cell hyperplasia, perhaps including the C cell hyperplasia seen in individuals in MEN IIA families who do not themselves carry a yet mutation (described earlier) (Lips et al., 1994; Wolfe et al., 1992).

3. MEN IIB a. Developmental Abnormalities in MEN IIB MEN IIB is recognized by developmental abnormalities, which are not seen at all in MEN IIA or FMTC (Dyck et al., 1979; Khairi et al., 1975). Disorganized peripheral nerve tissue gives rise to “neuromas,” which give a characteristic fullness to the lips and are visible as discrete lumps on the tongue (Fig. 4); and to thickened corneal nerve fibers, which are recognizable on ophthalmological examination (Khalil and Lorenzetti, 1980; Robertson et al., 1973). The corneal nerve phenotype, however, may be difficult to score (Kinoshita et al., 1991). Musculoskeletal abnormalities result in a long, thin body type resembling that of Marfan syndrome (though without the lens and palatal abnormalities). Most characteristic of all, an increase in

The MEN II Syndromes and ret

189

Fig. 4 (a) “Neuromas” of the tongue in a patient with MEN IIB. (b) Typical facies of MEN IIB showing the prominent “blubbery” lips due to neuroma tissue.

I90

Bruce A. 1. Ponder and Damn Smith

the number of intrinsic autonomic ganglia and a hyperplasia of extrinsic autonomic nerve fibers in the wall of the intestine lead to disturbances of intestinal motility, which commonly present as diarrhea or constipation and failure to thrive in infancy, and can be recognized on rectal biopsy (Carney et al., 1976). There may be musculoskeletal abnormalities, in particular pes cavus, pectus excavatum, bifid ribs, and slipped femoral epiphyses (Dodd, 1985). Males are commonly impotent because of neurological problems, and a number of females have been reported to have delayed puberty (Euro MEN Collaboration, unpublished data) for reasons that are still unclear. Occasional infants have been reported to have severe muscular hypotonia (Cunliffe et al., 1970). Because physicians are alert for the MEN IIB phenotype as an early warning of thyroid and adrenal tumors, it is probably overdiagnosed. Families have been reported in the past to have mixed MEN IIA-IIB phenotype (Lacroix et al., 1992), which is puzzling since the mutation analysis now available would lead one to expect these phenotypes to be distinct. A search for the characteristic MEN IIB mutation in adults reported to have components of the MEN IIB phenotype, but without calcitonin abnormalities suggestive of thyroid C cell involvement, has so far been negative (unpublished data). It remains an open question whether partial or modified MEN IIB phenotypes associated with yet mutation exist.

b. Spectrum of Tumors and Age at Onset in MEN I1 MEN IIB differs from MEN IIA in that, on average, the clinical presentation of the tumors is at a younger age, and the parathyroids are rarely if ever involved (Carney et al., 1980; Sizemore et al., 1992). In a combined series of patients with MEN IIB (Euro MEN Collaboration, unpublished), the average age of clinical presentation of MTC was around 18 years, and that of pheochromocytoma 24 years, compared with 38 years (Vasen et al., 1987) for MTC in MEN IIA. Similar data come from an earlier series and review of the literature (Carney et al., 1979). Early onset is not, however, invariable; as in MEN IIA, there may be a wide variation (Sizemore et al., 1992; Euro MEN Collaboration, unpublished).

c. New Mutations in MEN IIB The developmental abnormalities presumably confer a considerable reproductive disadvantage; as a result, perhaps one-half of all MEN IIB cases are the result of new mutation (Norum et al., 1990). (Large MEN IIA families are much more common, so the proportion of new mutation cases in MEN IIA is much lower; however, the absolute frequency of new mutations may not be very different.) As in retinoblastoma and neurofibromatosis type 1, new mutations in MEN IIB occur predominantly or exclusively on the chromosome derived from the male parent (Carlson et al., 1994a). It has also been suggested that there is an effect of the sex of the parent who

The MEN II Syndromes and ret

191

transmits the mutation or in whom it arose de novo on the probability that a male or female child would manifest the disease (Carlson et al., 1994a). In 25 de novo patients in whom the mutation had been shown to originate on the paternal chromosome, and 17 cases who inherited the disease from an affected father, there were 28 affected female children compared to 14 male ( p < 0.05). Data from Japan (Takarni et al., 1994) may support these observations. If they can be confirmed, a number of intriguing explanations can be considered, which have been summarized by Sapienza (1994).

E. Hirschsprung Disease A brief summary of Hirschsprung disease is given here as background for the discussion of ret mutations that follows. The genetics and phenotypes are described in Badner et al. (1990). Hirschsprung disease (Hirschsprung disease of the colon and rectum [HSCR]) is a common developmental disorder, affecting 1 in 5000 live births. It is associated with the lack of intrinsic autonomic ganglion cells in the myenteric and submucosal plexuses along variable lengths of the gastrointestinal tract, which results in constipation or bowel obstruction of varying severity, depending upon the length of bowel affected. About 3% of children with Hirschsprung disease have developmental abnormalities of the kidneys, and an association with Ondine curse (disturbance of the involuntary control of breathing; Gaisie, 1989; O’Dell, 1987) has been described. Both of these fit with the presumed role of the ret gene in development (see Section 1V.D and 1V.E). A subset of Hirschsprung disease occurs in families. The best fitting model is of autonomic dominant inheritance with variable expressivity and penetrance (Badner et al., 1990). A minority of families showing a pattern consistent with dominant inheritance have been shown to have mutations in ret, predicted to result in inactivation of the protein (Angrist et al., 1995; Attie et al., 1995; Edery et al., 1994; Pasini et al., 1995; Romeo et al., 1994). Other genetic loci have been implicated in other HSCR families: mutations in the G protein-coupled endothelin B receptor (EDNRB) were reported in a large Mennonite kindred showing a recessive pattern of inheritance (Puffenberger et al., 1994); and linkage disequilibrium analysis in this kindred suggested that ret, a locus on chromosome 21, and probably at least one further locus, were acting as modifiers. Notably, there were individuals in the Mennonite pedigree who had the HSCR phenotype but no mutation in the EDNRB gene, and others who were homozygous for the mutation but did not express the phenotype. Moreover, whereas heterozygosity for an inactivating mutation of ret can result in HSCR in humans, in the mouse knockout model, heterozygotes appears to be phenotypically normal (Schuchardt et al., 1994). Taken together, these observations indicate that the genetics of HSCR

192

Bruce A. J. Ponder and Darrin Smith

is complex, almost certainly involving interactions between several genetic loci and the effects of environmental factors.

Ill. THE ret PROTO-ONCOGENE A. Identification of ret as an Oncogene The yet proto-oncogene encodes a cell surface glycoprotein that is a member of the receptor tyrosine kinase (RTK) family (Hanks et al., 1988). The name yet is an acronym of rearranged during transfection, reflecting the orignal identification of yet as a chimeric oncogene formed by rearrangement during NIH 3T3 transformation assays of DNA from lymphoma, colonic, and gastric tumors (Takahashi and Cooper, 1987). Subsequently, three different rearranged versions of yet have been described in vivo, specifically in papillary thyroid carcinoma. (Although these are thyroid tumors, they arise from the thyroid epithelial cells that synthesize thyroid hormone, which are thought to be developmentally quite distinct from the “C” cells, which are involved in MEN 11; see Section 1V.A.) The rearranged versions of yet are designated ret-PTC1 (Grieco et al., 1990), yet-PTC2 (Bongarzone et al., 1993), and ret-PTC3 (Santoro et al., 1994a). In each case, the effect of the rearrangement is to fuse the genomic region encoding the intracellular domain of yet, which has tyrosine kinase activity, to different activating genes that are constitutively expressed in the thyroid epithelial cells. The fused activating genes contribute a novel amino-terminal portion to the Ret protein that is capable of dimerization, resulting in activation of the tyrosine kinase domain in the cytoplasm, independent of any requirement for ligand. Ret-PTC constructs have been extensively used in transfection experiments to study downstream signaling from the yet tyrosine kinase (Bongarzone et al., 1993; see later); however, mutations of the ret-PTC type are not found as part of the MEN I1 syndrome or in MEN II-associated tumors. ’

B. Ret Structure The yet proto-oncogene is located on chromosome 10q11.2. The coding sequence consists of 21 exons in a genomic region of approximately 55 kb. The protein, which occurs in three main 3’ splice isoforms of 1072-1114 amino acids, contains a typical cleavable signal sequence of 28 amino acids, an extracellular domain, a transmembrane domain, and a tyrosine kinase domain with a short interkinase region of 27 amino acids (Ceccherini et al., 1994; Kwok et al., 1993; Myers et al., 1995) (Fig. 5 ) .

Ret protein

cadherin like

cysteine rich cell membrane

r

Mutations

Clinical phenotype of mutation

4- codons 609 611 618 620 (exon 10)

634

4-

(exon 11)

codon 768 804

tyrosine kinase (exon 16)

MEN 28

3’ alternate splices 9 43 51 aminoacids

Fig. 5 The main features of the protein encoded by the ret proto-oncogene, and the sites of the mutations in the different clinical varieties of MEN 11.

194

Bruce A. 1. Fonder and Damn Smith

I . THE EXTRACELLULAR DOMAIN OF ret The extracellular domain of ret is not closely similar to that of other RTKs. The main features are a cysteine-rich region close to the cellular membrane (also seen in other RTKs), which is conserved in mouse (27 of 28 residues) (Iwamoto et al., 1993) and Drosophila ret (Sugaya et al., 1994), and a more distal region that shows homology to members of the cadherin superfamily (Schneider, 1992). The cysteine-rich region is presumably important in determining the three-dimensional structure of the extracellular domain, but no details of this are known. The role of cadherins in mediating cell adhesion by homophilic (and possibly heterophilic) interactions raises the possibility that the Ret protein interacts either with a second Ret molecule on another cell or, by heterophilic interaction, with a cadherin. However, no clear homophilic binding activity of Ret protein has been demonstrated in cell aggregation assays (Takahashi et a/., 1993), and the putative Ret ligand is still to be identified. * The extracellular domain of the Ret protein also contains several glycosylation sites. Two glycosylated isoforms of the protein are detected on Western blotting of extracts of neuroblastoma cells or of cells transfected with wild-type ret, of molecular masses 170 and 150 kDa. The fully glycosylated 170-kDa protein is present at the cell surface, while the 150-kDa protein appears to be an incompletely processed form that is localized to the endoplasmic reticulum (Asai et al., 1995). Interestingly, when a mutation (D 300 K) was made in a putative calcium-binding site in the cadherin-like domain of ret, cells transfected with the mutant construct expressed very little 170-kDa isoform, while expression of the 150-kDa isoform present in the endoplasmic reticulum was not affected (Asai et al., 1995). This suggests that the cadherin-like domain may be needed for intracellular transport of the Ret protein tc the cell membrane. 2. ALTERNATIVELY SPLICED FORMS OF ret Alternative splicing and polyadenylation site usage at the 3’ end of ret generates mRNAs predicted to encode Ret proteins with three distinct carboxyl-terminal ends of 51, 43, or 9 amino acids (ret51, ret43, or ret9) (Myers et al., 1995) (Fig. 6). The possible functional significance of these isoforms, and the different possible 3’ UTRs associated with them, is not clear. There are as yet no data about their relative abundance in different tissues or at different stages of development. However, it may be of note that r e d l , but not ret43 or ret9, contains two tyrosine residues each within a context (pYXNX) that could provide a binding site for the Grb2-src homology region 2 (SH2) domain. There is also a tyrosine (1062) immediately “See Note added in proof.

The MEN I1 Syndromes and ret

ret 9; exon 19

+ ret 43.exon 19

-+

195

intron 19 GRISHAFTRF

exon 21

DAQHSSSLVGAAFGKSQQLFWLCCQHCNFAEKSRITKT LPALQT

ret 51: exon 19

exon 20

+ GMSDPNWPGESPVPLTRADGTNTGFPRYPNDSVYANW MLSPSAAKLMDTFDS

Fig. 6 Amino acid sequences of the alternative spliced 3’ forms of the Ret protein. Adapted from Myers et al. (1995).

5’ to the splice site that lies within a putative Shc-PTB consensus-binding site, and it is of interest to speculate whether the alternatively spliced forms might differ in either phosphorylation of that tyrosine or affinity of PTB binding. Several different alternative splices near the 5’ end of ret have also been identified by polymerase chain reaction and confirmed by RNAse protection (Lorenzo et d.,1995; Xing et d., 1994). These would result in transcripts lacking exon 3, exons 3 and 4, or exons 3,4, and 5. Transcripts lacking exons 3 and 4 would encode a short, soluble form of the protein. Whether or not these alternative forms have any physiological significance is also unclear.

C. Mutations in ret in the MEN I1 Syndromes A summary of the mutations is shown in Figure 5 and in Table 111. 1. MUTATIONS IN M E N IIA A N D FMTC

The mutations in MEN IIA and FMTC are summarized in Figures 5 and 7 and Tables I11 and IV (Bolino et al., 1995; Donis-Keller et al., 1993; Eng et al., 1995c; Mulligan et al., 1993b, 1994a,b, 1995; Schuffenecker et al., 1994; Smith et al., 1994).Three categories of MEN I1 phenotype are shown: MEN IIA (subdivided according to the presence in the family of pheochromocytoma, parathyroid disease, or both); FMTC; and “other.” The last category is necessary because, as explained in Section II.D.2, since pheochromocytoma and parathyroid disease are inconstant features of MEN IIA,

Bruce A. I. Ponder and Damn Smith

I96

Table 111 Percentages of MEN I1 Families of Different Phenotypes in which ref Mutations Have Been Detectedu No. of families

Phenotype

Mutation positive (Yo)

MEN IIA MTC pheo PTH MTC pheo no PTH MTC PTH no pheo MEN 11B FMTC~ Other MTC6

94 96 13 79 34 161

91 95 13 75 30 136

Total

477

440 (92)

(97) (99) (100) (95) (88) (85)

Mutation negative (Yo)

3 1 0 4 4 25

(3) (1) (0) (5) (12) (15)

37 (8)

uData from the International RET Mutation Consortium (Eng et al., 1996b). ‘see text.

Exon 10

609

611

618

Exon11

620

634

I Exon13 768

Exon14

804

2A MTC, P, PTH

MTC, P

MTC, PTH

FMTC Other MTC Fig. 7 Proportion of mutations in different codons of ref in different phenotypic subtypes of MEN IIA, FMTC, and “other MTC” families. Based on data from the International RET Mutation Consortium (Eng et al., 1996b). (summarized in Table Ill).

The MEN II Syndromes and ret

197

Table IV

Occurrence of Pheochromocytoma in MEN IIA Families in Relation t o Mutation at ret Codon 634a Number of families

Mutation Codon 634 No mutation

MTC with

MTC without

pheo6

pheo‘

26

2s

160

18

CGC

82

6

Other

78

12

p < 0.0001 Any mutation Mutations in codon 634

p

= 0.21

aData from the International RET Mutation Consortium (Eng et a!., 1996b). 6At least one individual in the family with histologically proven abnormality. ‘Biochemical screening evidence against abnormality in all available affected family members and unaffected close relatives.

a small family with (say) two cases of MTC only might be either a MEN IIA family in which by chance adrenal and parathyroid disease had not yet manifested, or a small true site-specific MTC family. An arbitrary definition has therefore been made for the purposes of genotype-phenotype comparison that “FMTC” includes only families with at least four proven cases of MTC, and with biochemical screening data for available affected and at-risk family members that show no evidence of adrenal or parathyroid involvement (Mulligan et d., 1994b). Families in which there are fewer than four MTCs, or in which screening data are incomplete, are deemed unclassifiable as MEN IIA or FMTC and are placed in the “other” category. Table III shows that mutations have been identified in the great majority of families of all types, although, especially in the “other” category, there are still a few families in which no mutation has been found (Mulligan et al., 1995). Most mutations are in one of the six cysteine codons closest to the cell membrane, and all are missense mutations leading to amino acid substitution. Tables I11 and IV and Figures 5 and 7 show that the position of mutations within the gene is related to phenotype (Mulligan et al., 1994b, 1995; Schuffenecker et al., 1994). Codon 634 mutations are the most frequent in MEN IIA families and overall, but are present in only 30% of FMTC families, the remainder being either in the cysteine codons further from the cell membrane or (a minority) in the intracellular domain (Bolino et al., 1995; . analysis confirms that there is a strong correlaEng et al., 1 9 9 5 ~ )Statistical

I98

Bruce A. 1. Ponder and Damn Smith

tion between mutation in codon 634 and the MEN IIA phenotype, for both pheochromocytoma and parathyroid disease (Table IV). There may also be an effect, not only of the position of the mutation within the gene, but of the particular amino acid substitution (Mulligan et al., 1994b). The most common mutations at codon 634 are cysteine to arginine (C634R: TGC + CGC) and cysteine to tyrosine (C634Y: TGC + TAC), but all the possible mutations allowed by the coding sequence are seen. The greater frequency of the CGC and TAC mutations is consistent with the known frequency of T + C and G + A changes, and presumably should not imply a particular biological significance of these amino acid substitutions. Nevertheless, some surprising observations have been reported. In their original report of genotype-phenotype correlations in MEN 11, in which assessment of the phenotypes was made independently of the mutation results, Mulligan et al. (1994b) noted a strong association between the C634R mutation and the presence of parathyroid disease in the family. This correlation is still present (p = 0.0016) in the much larger cumulative data of the International RET Mutation Consortium (Eng et al., 1996b) if the original families of Mulligan et al. are included, but fails to reach significance if the original families are excluded. The International RET Mutation Consortium data also show that, of 169 MEN IIA families with a codon 634 mutation, 88 had the common mutation C634R; this particular mutation was present in none of 9 FMTC families with a codon 634 mutation (p = 0.003) (Mulligan et al., 1995, unpublished). It therefore remains an intriguing but unresolved question whether or not all amino acid substitutions resulting from codon 634 mutations are equivalent in terms of phenotype. Mutations in FMTC are not confined to the cysteine-rich region. To date, four families (three with FMTC and one with MTC only but not meeting the criteria for FMTC) have been reported to have Glu 4 Asp mutations in codon 768 (Bolino et al., 1995; Eng et al., 1995c), and two families with MTC only have been reported with Leu + Val mutations in codon 804 (Bolino et al., 1995). Thus it seems that mutations in this region of the gene are associated with MTC rather than pheochromocytoma or parathyroid disease, for reasons that are still unclear.

2. MUTATIONS IN MEN IIB (Cadson et a/., 1994b; Eng et al., 1994; Hofstra et a/., 1994) Almost all the MEN IIB families so far reported (82 of 86; Rossel et al., 1995) have the same mutation: ATG to ACG at codon 918, resulting in substitution of threonine for methionine. The significance of this for function is discussed in Section 1II.D. Each of the four well-documented MEN IIB individuals or families in which no mutation has been found has typical phenotypic features. In two of the four families, the remainder of the coding

The MEN II Syndromes and ret

I99

sequence of ret has been examined by sequencing as well as by mutation detection techniques based on mismatch. Either a coding mutation has been missed, or there are mutations elsewhere in the gene, or a mutation in another gene, presumably in the same signaling pathway that leads from the MEN IIB mutant tyrosine kinase (see later), must be responsible. 3. MUTATIONS IN FAMILIES WITH MEN 11 AND HSCR (Borst et al., 1995; Landsvater et al., 1995; Mulligan et al., 1994a)

Six families have so far been reported in which MEN I1 (MEN IIA or FMTC) and HSCR are coinherited, and in which some individuals express both phenotypes (Fig. 3). In each case, there is y e t mutation in either codon 618 (three families; one Cys + Arg, two Cys + Ser) or 620 (three families; all Cys Arg). The remainder of the coding region of ret has been examined by sequencing in at least five of these families, and no abnormality was found. The provisional conclusion is that the same mutation can in some circumstances cause either phenotype, or both phenotypes in one individual. Of interest, families have also been reported with mutations in the cysteine codons 609 and 620, which may be affected in MEN 11, but in these families HSCR is so far the only phenotype (Angrist et al., 1995; Mulligan et al., 1994a).

-

D. Ret Mutations In Sporadic MTC,

Pheochromocytoma,and Parathyroid nmors Each of the cancers that occurs in an inherited cancer syndrome has a nonhereditary counterpart. As a rule, the gene that carries the predisposing mutation in the hereditary cancer is the site of somatic mutations in the nonhereditary form. This is true for MTC, but with an interesting twist. Of 157 sporadic MTC, 61 (39%) are reported to have a somatic mutation in codon 918 of ret (the mutation characteristic of MEN IIB); in contrast, somatic mutations involving the cysteine codons characteristic of MEN IIA and FMTC are rare. Donis-Keller et al. (1993) reported one somatic 6-bp deletion that removed cysteine 630 (which in fact has not so far been reported to be involved in germline MEN I1 mutations) in a series of an unspecified number of sporadic MTC. Another somatic mutation has been reported in an MTC-a deletion-insertion encompassing codon 634 (Marsh et al., 1996a). Of 77 sporadic MTC studied by Eng et al. (1995b) and by Zedenius et al. (1994), only 3 had cysteine codon mutations; 2 of these proved to be germline mutations (the supposedly “sporadic” tumors were in fact occult cases of MEN 11, one a de novo mutation), and in the third, constitutional DNA was not available to test for germline mutation. Mutations of the

200

Bruce A. 1. Pbnder and Darrin Smith

intracellular portion of ret are occasionally seen as somatic events in MTC; 4 of 72 sporadic MTC had somatic mutations in codon 768 (Eng et al., 1995c, unpublished; Marsh et al., 1996b), and 4 of 111 sporadic MTC had somatic mutations in codon 883 in exon 15 (Dou et al., 1994; Eng et al., unpublished; Marsh et al., 1996b). The picture in sporadic pheochromocytoma is slightly different. Only 6 of 112 (5%) had a somatic mutation of codon 918 (Beldjord et al., 1995; Eng et al., 1995a); 2 of 112 (1.5%)had missense mutations, proven to be somatic in origin, affecting codon 634 (Eng et ul., 1995a; Komminoth et ul., 1995); and a further 3 had novel somatic mutations affecting the 3' splice acceptor site of exon 9 (Beldjord et al., 1995), codon 632-633, and codon 925 (Lindor et al., 1995) (one patient each). None of 32 non-MEN IIassociated parathyroid lesions (hyperplasia,adenoma, carcinoma) contained ret mutations in exons 10, 11, or 16 (Padberg et al., 1995).

E. The Effects of ret Mutations in MEN II I . THE EXTRACELLULAR DOMAIN CYSTEINE MUTATIONS Experiments in which constructs containing proto-ret in the wild-type, MEN IIA (Cys 634 Arg) or MEN IIB (Met 918 Thr) mutant forms have been transfected into NIH 3T3 cells and PC12 cells provide evidence that the MEN IIA mutations lead to activation of the ret tyrosine kinase (Asai et al., 1995; Borrello et al., 1995; Santoro et al., 1995). The evidence is of three types: 1. Biological. Under conditions in which the wild-type constructs have no effect, both IIA and IIB mutant ret induce transformed foci in NIH 3T3 cells and differentiation of PC12 cells. Santoro et al. (1995) tested three different codon 634 mutations-Cys 634 Arg, Cys 634 Tyr, and Cys 634 Trp-and the MEN IIB Met 918 Thr mutation, and found them to be of roughly equal effect. Other data (Borrello et al., 1995) in which the transforming effect of the Cys 634 Arg and Met 918 Thr mutations has been compared over a range of amounts of DNA transfected suggest, however, that the effect of the MEN IIB mutation (in the ret9 alternatively spliced form) in this system is considerably weaker than the MEN IIA. 2. Steady-state tyrosine phosphorylation of the Ret protein. MEN IIA and IIB, but not wild-type, Ret protein from NIH 3T3 transfectants is phosphorylated on tyrosine. 3. Tyrosine kinase activity. Assay of Ret protein in immunoprecipitates from transient transfection of HeLa cells shows that, while the wild-type protein has some activity in autokinase assays and in phosphorylation of the

20 1

The MEN II Syndromes and ret

exogenous substrate myelin basic protein, the activity of the MEN IIA mutant protein is considerably higher. The cysteine mutations activate ret by inducing covalent dimerization (Asai et al., 1995; Santoro et al., 1995; Wada et al., 1996). The consistent involvement of specific cysteines in the mutations in MEN IIA and FMTC suggested a mechanism in which these cysteines would normally be paired in intramolecular disulfide bonds, so that loss of one cysteine by mutation would result in the partner cysteine forming an intermolecular bond with the corresponding free cysteine on an adjacent yet molecule (Fig. 8). The resulting covalent dimerization would lead to constitutive activation of the ret tyrosine kinase domain, according to the model accepted for other RTKs. This proposal predicts that, in C cell tumors or transfected cells containing a MEN IIA mutant ret, yet dimers should be present, which could be demonstrated by Western blotting of nonreducing gels. This prediction has been confirmed (Asai et al., 1995; Santoro et al., 1995);moreover, aggregation of ret on the surface of a MTC cell line containing a MEN IIA mutation has been demonstrated by immunoelectron microscopy (Borrello et al., 1995). The D300K mutation in the cadherin homology domain described earlier dimerisation by ligand

A -cys

I

-cys

cys-

1

cys-

cys-cys X

constitutive dimerisation through cysteines

X-

activation

Fig. 8 Illustration of constitutive activation of ret as a result of mutation of a cysteine in the extracellular domain.

202

Bruce A. 1. Ponder and Damn Smith

(see Section III.B.l), which prevented transport of the mature, fully glycosylated form of yet to the cell surface, also greatly reduces the transforming ability of a MEN IIA mutant yet. This suggests that dimerization and activation of mutant yet only occurs once the molecules have reached the cell surface (Asai et al., 1995). In view of the observed genotype-phenotype correlations between mutations of the different cysteine codons 609,611,618,620, and 634 (Mulligan etal., 1994b), it would be of interest to compare the activity of these mutants in similar assays. No such studies have so far been reported.

2. THE MEN IIB M e t 918 Thr MUTATION This mutation is exclusively associated with MEN IIB. Residue 918 is predicted from modeling studies to lie at the base of a pocket in the protein that is involved in substrate binding (Carlson et al., 1994b; Hanks et al., 1988). The substitution of threonine for methionine would alter the dimensions of the pocket, and hence the substrate specificity. Furthermore, almost all receptor tyrosine kinases have methionine at position 918 (as does yet), whereas almost all cytoplasmic tyrosine kinases have threonine (Hanks et al., 1988) (Fig. 9). This leads to two predictions: (1)receptor and cytoplasmic tyrosine kinases will differ in their substrate specificity, and (2) the Met 918 Thr mutation in MEN IIB will alter the specificity of yet away from that of RTK and toward that of a cytoplasmic tyrosine kinase. There is experimental evidence to support both predictions. Using degenerate peptide libraries, Cantley’s group (Songyang et al., 1995a) showed that the preferred peptide substrate for RTKs has hydrophobic amino acids at both the +1 and + 3 positions downstream of the target tyrosine, whereas the cytoplasmic tyrosine kinases prefer a hydrophilic amino acid at +1 and a hydrophobic amino acid at +3. These different amino acid contexts flanking the tyrosine provide, in turn, preferred substrates for different groups of SH2 domains, and hence the possibility of different pathways of downstream signaling. When wild-type and MEN IIB mutant Ret proteins were compared for their ability to phosphorylate model substrates for a RTK (epidermal growth factor receptor [EGFR]) and two different cytoplasmic tyrosine kinases (abl and SIC), the MEN IIB mutant protein showed a clear shift in specificity toward the substrates preferred by the cytoplasmic kinases (Songyang et al., 1995a). The inference that the MEN IIB mutation has altered the pathway of downstream signaling from the yet kinase is supported by evidence that activated MEN IIB yet differs from wild-type both in the pattern of tyrosine phosphorylation of the Ret protein itself and in the patterns of tyrosine phosphoproteins (presumably involved in downstream signaling) that is seen in cell extracts (Santoro et al., 1995).

203

The MEN II Syndromes and ret

w T A P E ANLP IN K/R w M A I/P E S L -

Cytoplasmic

-

Q G A K F PIN K

Receptor

-

SQGRI

t MEN 2B M -+T Fig. 9 Amino acid sequence of consensus receptor and cytoplasmic tyrosine kinases in the substrate-binding region of Hanks domain VIII, showing the mutation characteristic of

MEN IIB.

It is an unresolved question whether the effects of the Met 918 Thr mutation are restricted to altering the substrate specificity of the yet kinase, and whether this is therefore the sole explanation for the MEN IIB phenotype. The results of transfection experiments in NIH 3T3 cells suggest that the catalytic activity of the tyrosine kinase domain may also be enhanced, but the interpretation of these experiments is uncertain. When proto-ret constructs encoding either a wild-type IIA or IIB mutant protein are transfected into NIH 3T3 cells, under conditions in which the wild-type yet gives no transformed foci, the IIB mutant yet does induce focus formation. However, the activity is variously reported to be comparable, less (using the yet9 alternatively spliced form), or more (using the yet51 form [Iwashita et al., 19961)than that obtained with the IIA mutant. As was described earlier, the MEN IIB Ret protein in these experiments is also active by the criteria of phosphorylation on tyrosine and tyrosine kinase activity toward other proteins. NIH 3T3 cells are not thought to secrete yet ligand, and Santoro et d. (1995) reported that, even with the use of cross-linking agents, they were unable to demonstrate dimers of MEN IIB mutant protein in the transfected cells. They therefore concluded that the MEN IIB mutation causes activation of the kinase through an intramolecular mechanism. Another approach has been to use PTC-ret constructs (see Section 1II.A) in the ret9 form to compare the activity of the wild-type and IIB mutant tyrosine kinase domains under conditions in which a consistent degree of activation is provided by the PTC dimerization (Borrello eta!., 1995).In these experiments the MEN IIB mutant tyrosine kinase shows a higher level of steady-state tyrosine phosphorylation, a higher tyrosine kinase activity against exogenous substrate, and a slightly higher transforming activity in NIH 3T3 cells than the corresponding wild-type form. If correct, this suggests that the Met 918 Thr mutation does indeed alter the properties of the ret tyrosine kinase domain in ways other than simply specificity of substrate binding. It remains unclear whether MEN IIB proto-yet has activity in vivo independent of ligandinduced dimerization, and, if so, whether and to what extent a further

204

Bruce A. 1. Pbnder and Damn Smith

increase in activity might follow on ligand binding. Resolution of this issue is likely to be important for understanding the mechanism by which the MEN IIA and MEN IIB mutations are involved in tumorigenesis (discussed later).

3. MUTATIONS ELSEWHERE IN THE TYROSINE KINASE DOMAlN IN FMTC

At the time of writing, only two other germline mutations have been reported to predispose to MEN 11. Each is uncommon and, so far, associated exclusively with FMTC. They are Glu 768 Asp, in Hanks domain 111 (Bolino et al., 1995; Eng et al., 1995c), and Val 804 Leu, in Hanks domain V (Bolino et al., 1995) (see III,C,l). The Glu 768 Asp mutation involves a Glu residue that is highly conserved between different RTKs in humans and across species. Modeling based on the structure of cyclic AMP-dependent kinase suggests that Glu 768 lies in a region of a-helix that stabilizes Glu and Lys residues that are involved in ATP binding (Eng et al., 1995~).The predicted effect of the mutation is to activate the kinase; our own preliminary data from transfection experiments are consistent with this (D. P. Smith et al., unpublished). Modeling also suggests a possible effect on substrate specificity; this is being tested experimentally using the peptide library screening described earlier in relation to MEN IIB (Songyang et al., 1995a). Since the great majority of families with FMTC result from activating mutations in the extracellular cysteines, one might speculate that a simple activating effect would be sufficient to explain the Glu 768 mutation, but the occurrence of this mutation in sporadic tumors also raises, equally speculatively, the question of altered substrate specificity (see later). The Val 804 Leu mutation affects a residue that is conserved between species, but no modeling of its possible effects has been reported.

F. Downstream Signaling from Ret Ligand binding to RTKs, such as Ret, induces dimerization and activation of tyrosine kinase activity, leading to autophosphorylation of the cytoplasmic domain (Pawson and Schlessinger, 1993; Pazin and Williams, 1992). Tyrosine autophosphorylation sites may occur within the catalytic kinase domains, where they probably have a role in regulating the kinase activity (e.g., in the platelet-derived growth factor receptor; Mori et al., 1993). However, most autophosphorylation sites are clustered outside the catalytic domains. Phosphorylation of these tyrosine residues leads to the recruitment of phosphotyrosine (pY)-binding proteins, their phosphorylation, and the initiation of signal transduction cascades that ultimately lead to the modulation of gene expression in the nucleus.

The MEN II Syndromes and ret

205

Many proteins known to interact with RTKs contain one or two copies of a related motif of approximately 100 amino acids known as a SH2 domain (Pawson and Schlessinger, 1993). SH2 domain-containing proteins interact, via their SH2 domain, with specific pYs on RTKs. This specificity is determined by particular SH2 domains having a preference for pY within a particular sequence context, the amino acids carboxyl-terminal to the pY being most significant in binding site selection. Recently a second pY interaction domain, the PTB or PI domain, has been identified in the protein Shc (which also contains an SH2 domain) (Blaikie et al., 1994; Bork and Margolis, 1995; van der Geer et al., 1995). In this case amino acids aminoterminal to the pY are the most significant in binding site selection. Cantley and colleagues have used degenerate peptide libraries containing a pY residue to determine the optimal binding sites for SH2 domains (Songyang et al., 1993, 1994) and the PTB-PI domain (Songyang et al., 1995b). This work enables the prediction of proteins that will interact with pY residues in RTKs. There are six tyrosine residues outside the catalytic domain and within the cytoplasmic domain of the 9- and 43-amino-acid carboxyl-terminal splice isoforms of Ret, and eight tyrosine residues in the 5 1amino-acid carboxyl-terminal splice isoform (Fig. 10). Which tyrosine residues in activated Ret become phosphorylated has not yet been determined. However, making the assumption that these residues do become phosphorylated, it is possible to make some predictions about which proteins will directly interact with activated Ret. Tyrosines 1090 and 1096 only in the 51amino-acid splice isoform are likely to interact with Grb2. Tyrosine 1015 is likely to interact with phospholipase C (PLC). The absence of a known ligand to activate Ret has been overcome by using PTC-Ret (see Section 1II.A) and an EGFR-Ret chimera that can be activated with EGF. Grb2 has been shown to interact with PTC-Ret (Borrello et al., 1994), and PLC to be activated by the EGFR-Ret chimera (Santoro et al., 1994b). The amino acid context of tyrosine 1062 suggests that it may interact with the Shc PTB-PI domain, although this has not yet been tested. This tyrosine is one amino acid amino-terminal of the carboxyl-terminal alternate splice, and it would be interesting to know if alternate splicing affects PTB-PI domain binding (see 111,B,2). The remaining tyrosines are not within a sequence context closely related to any known SH2 domain consensus-binding site. However, the SH2 domain of Shc is known to interact with activated Ret (Borrello et al., 1994), and the SH2 domain of GrblO (the consensus-binding site of which has not been determined) has been identified as binding to activated y e t in a yeast two-hybrid screen (Pandey et al., 1995). In view of the expression of Ret in cells of neuroectodermal origin and its involvement in HSCR, GrblO is of particular interest since it is homologous to a Caenorbabditis eleguns gene involved in neuronal migration (Ooi et al., 1995). Without precisely mapping the pY residues, Santoro et al. (1995) have shown that the pY map of MEN IIB Ret is different from the pY map of

206

Bruce A. I. Pbnder a n d Darrin Smith TYR

-5

-4

-3

-2

-1

660

F

C

I

H

687

A

F

P

752

K

G

791

H

V

N-C

0

+1

+2

+3

+4

C

Y

H

K

F

A

H

V

S

Y

S

S

S

G

A

R

A

G

Y

T

T

V

A

V

I-

K

L

Y

G

A

C

S

Q

+5

806

L

L

I

V

E

Y

A

K

Y

0

STK1

809

V

E

Y

A

K

Y

0

S

L

R

G

826

K

V

G

P

G

Y

L

G

S

G

G

864

S

Q

G

M

Q

Y

L

A

E

M

K

S

R

9

O

O

L

D

V

Y

E

B

D

S

Y

905

Y

E

E

D

S

Y

V

K

R

S

Q

928

L

F

D

H

I

Y

T

T

Q

S

DTK2

952

L

0

G

D

P

Y

P

G

I

P

P

981

,

C

S

E

E

M

Y

R

L

M

L

Q

1015

V

K

R

R

D

Y

L

D

L

A

Aplcy

S

D

S

L

I

Y

D

D

G

L

S

E

N

K

L

E

N

K

L

E

N

K

L

Y Y

1029 Ret 9 Ret43

-

1062

I ~

T

I I

I

iG

Y

j

1

*

S G

R

I

S

shcPTB HCshcSH2

D

A

Q

H shCpTB

M

S

D

PehcPTB

1090

T

G

F

P

R

Y

P

N

D

S

Vgrb2

1096

P

N

D

S

V

Y

A

N

W

M

Lgrb2

u

MEN IIA Ret and EGF-stimulated EGFR-Ret chimera (which are similar). It is not known whether MEN IIB Ret differs in pY residues that are regulatory, or that interact with signaling proteins, or both. However, this difference may contribute to the altered catalytic substrate specificityof the kinase domain of MEN IIB mutant Ret (see Section III.E.2), and may in addition lead to the recruitment of an altered set of pY-binding proteins. Mutation of tyrosine residues in the catalytic kinase domain (Iwashita et al., 1996) has pointed to possible regulatory tyrosine autophosphorylation sites that may differ between the MEN IIB and the wild-type (found in the MEN IIA

207

The MEN II Syndromes and ret

mutant) tyrosine kinase domains. Mutation of tyrosine 905 to phenylalanine has no effect on MEN IIB Ret activity (kinase activity and transformation capacity), whereas MEN IIA; Ret activity is abolished. Conversely, mutation of tyrosines 864 and 952 reduces MEN IIB Ret activity but has no effect on MEN IIA Ret activity. It is clear that there is still much to be learned about the proteins interacting with phosphorylated Ret, and there is equally little known about signaling events downstream of pY-binding proteins following Ret activation. This has been investigated using the EGFR-Ret chimera (Santoro et al., 1994b; van Weering et al., 1995). It should be noted that this chimera is the 9amino-acid carboxyl-terminal splice isoform of Ret, which lacks two tyrosine residues found in the 51-amino-acid form that are potential pYbinding sites for Grb2. Consequently, signaling downstream of the carboxylterminal splice isoforms may be significantly different. However, the 9-amino-acid splice isoform can probably also recruit Grb2 via its known interaction with Shc, which when tyrosine phosphorylated is able to interact with Grb2. The best analyzed downstream signaling pathway is the extracellular signalrelated kinase-2 (ERK-2) mitogen-activated protein (MAP) kinase pathway. The typical components of this pathway are: RTK (Shc +) Grb2 + SOS +, p21Ras + Raf-1 4 MEK-1 + ERK-2 + ELK-1 + modulation of gene transcription. Shc, Grb2, and SOS are noncatalytic adapter proteins. Ras is a GTPase that is active when bound to GTP. Raf, MEK, and ERK-2 are protein kinases. ELK is a transcription factor accessory protein. Given the known interaction of active Ret with Shc, and the observation that Ras is activated by the EGFR-Ret chimera in NIH 3T3 fibroblasts, it is surprising that Santoro et al. (1994b) reported that ERK-2 is not activated in these cells. However, van Weering et a/. (1995) have reported that the EGFR-Ret chimera is able to activate ERK-2 and the components of the pathway above in a neuroblastoma cell line. Neuroblastomas express Ret, and these tumors are of the same neuroectodermal origin as the thyroid C cells, adrenal medullary cells, and enteric ganglia affected in MEN I1 and HSCR. This implies tissue specificity in Ret signaling, and the signaling seen in the neuroblastoma cells may more closely reflect the normal in vivo signaling of Ret.

-

IV. DEVELOPMENT OF THE TISSUES INVOLVED IN MEN 11, AND PATTERNS OF ret EXPRESSION A. Thyroid

“C’Cells

The neural crest origin of the C cells has been demonstrated in birds, using the quail chick marker system (LeDouarin, 1982). In mammals, cells of the

208

Bruce A. 1. Ponder and Damn Smith

vagal neural crest migrate to the caudal portion of the fourth pharyngeal pouch, and from there to the developing thyroid, when they lie between and external to the epithelial cells in the thyroid follicles, predominantly in the upper-central portion of each thyroid lobe. The lineage relationships between the C cells and other cells of neuroectodermal origin are as yet unclear, but the orign of “C” cells from vagal neural crest and the biochemical similarities with enteric neurons (Tamir et al., 1989) suggest that they may share a common precursor with enteric neurons, and ultimately with the sympathoadrenal progenitor that is the precursor of chromaffin cells and sympathetic neurons (Anderson, 1993). Not all authors, however, accept that all the calcitonin-secreting cells of the thyroid are of neural ectodermal origin. They cite several observations: (1)the occurrence of thyroid tumors of mixed histology, containing both thyroid follicular cells (thyroglobulin positive) and C cells, both cell types being present not only in primary tumors but also metastases (Caillou, 1991); (2) the similarity of these tumors to a distinct type of thyroid follicle, containing follicular and C cells, and thought to be of ultimobranchial origin; (3) parallels with the endocrine cells of the gut, which are now thought to be of endodermal origin. Following these arguments, it remains possible that at least a subset of calcitonin-secreting cells have an endoderma1 orign from an ultimobranchial stem cell, which might also contribute to the thyroid follicular cell population. Studies of ret expression in C cells also suggest the possibility that the C cells are heterogeneous. Tsuzuki et al. (1999, in immunohistochemical studies of ret expression in rats, reported no staining of C cells in embryonic thyroid but expression in a “small number” of cells in the neonatal and adult gland; these cells were also stained by antibodies to calcitonin, but most calcitonin-positivecells were negative for ret. Similar results were reported in human thyroid studied by ret in situ hybridization and calcitonin immunohistochemistry (Fabien et al., 1994). It is not completely clear from these reports whether the observation of a subpopulation of ret-positive cells simply reflects a threshold phenomenon with a weak signal, or whether there are truly two populations of calcitonin-positive C cells, which differ in ret expression. The role, if indeed there is one, of ret in the development and maintenance of the thyroid “C” cell population is unclear. Homozygous ret knockout mice, in which disruption of the gene is predicted to result in total loss of ret function, die at birth as a result of either renal failure or, more probably, respiratory failure (Schuchardt et al., 1994). At the level of histological examination and calcitonin immunohistochemistry, and in a transgenic mouse that expresses LacZ in C cells, these mice appear to have a normal thyroid “C” cell population (V. Pachnis, personal communication). This suggests that ret function is not necessary for the development of C cells, at least until

The MEN I1 Syndromes a n d ret

209

the time of birth. Studies using in situ hybridization in the mouse and immunohistochemistry in the rat show, nevertheless, that yet is expressed in the neural crest cells migrating from the hindbrain and in the mesenchyme of the posterior branchial arches, consistent with expression in the population of neural crest cells that will give rise to C cells (Pachnis et al., 1993). In contrast to normal thyroid C cells, MTC tumors generally express ret mRNA and protein at high levels, judged by Northern blotting, in situ hybridization, and immunohistochemistry (Miya et al., 1992; Santoro et al., 1990; G . Thomas, personal communication). Thus it seems that ret may be expressed at high levels during the embryonic stage during which the precursors of C cells are migrating to their destination in the thyroid gland, but at low levels and possibly only in a subpopulation of cells once they have arrived. In C cell tumors, however, yet expression is once again at a high level-possibly as a result of stabilization of the message and the protein by the mutation (Santoro et al., 1995). The significance of these changes of expression in terms of yet signaling activity and changes in cellular phenotype is not understood.

B. Parathyroid The parathyroid glands develop from the endoderm of the cranial portions

of the third and fourth pharyngeal pouches. These fuse with the developing thyroid, and the parathyroid glands come to lie either adjacent to the thyroid or embedded in its substance. Ret is expressed in the pharyngeal pouch endoderm from which the parathyroids are derived, but the role of yet activity in parathyroid development is still unknown.

C. Adrenal Medulla An analysis of the antigenic and biochemical phenotypes of cells in situ in developing embryos, as well as in vitro manipulation of precursor cells, indicates that the chromaffin cells of the adrenal medulla (from which pheochromocytomas arise) and the sympathetic neurons are both derived from a common sympathoadrenal progenitor cell. Multipotent cells from the trunk neural crest migrate to the region of the aorta, where they coalesce to form the anlagen of the sympathetic ganglia, following which differentiation into sympathetic neurons or chromaffin cells is dependent upon local environmental signals (Anderson, 1993). In yet homozygous knockout mice, the adrenal medulla, like the thyroid “C” cells, appears grossly to be normal, again suggesting that yet activity is not essential for the development or maintenance of this tissue at least up to the time of birth.

210

Bruce A. I. Ponder a n d Darrin Smith

During embryogenesis in the mouse and rat, ret is expressed in the migrating neural crest cells as they coalesce alongside the aorta to form the sympathetic ganglia. Although expression persists in the sympathetic ganglia after their formation is complete at day 14.5 in the mouse, no expression is seen in the adrenal chromaffin cell lineage (Pachnis et al., 1993). In infant and adult rats, ret expression is detected in a few cells in the adrenal medulla (Tsuzuki et al., 1995). As in the case of the thyroid “C” cells, studies on human tissue show that ret mRNA is expressed (by the criterion of Northern blotting) in most pheochromocytomas, but only faint signals are obtained from normal adrenal tissue (Miya et al., 1992; Santoro et al., 1990).

D. Enteric Nervous System The majority of the cells that form the ganglia of the enteric nervous system are derived from the vagal neural crest, originating from the region of the hindbrain (Pachnis et al., 1993). These cells first migrate ventrally through the posterior branchial arches to enter the foregut mesenchyme, and then migrate caudally inside the gut wall, where they eventually coalesce and form the enteric ganglia (Gershon et al., 1993). Ret expression is seen in cells that lie along the migrating pathway of the presumptive enteric neuroblasts. Strong expression is seen in the mesenchyma1 cells of the posterior branchial arches in the 9.0- to 9.5-day-old mouse embryo, and subsequently in the posterior arch mesenchyme migrating toward the foregut, and then at increasingly caudal levels of the gut itself and in the myenteric plexus. Studies of cultured cells isolated from 14.5-day-old embryonic rat gut (Lo and Anderson, 1995) suggest that the ret-expressing postmigratory cells form a population of precursor cells that can differentiate along either neuronal or glial pathways. In the homozygous vet knockout mouse, the enteric ganglion plexus is absent, indicating an essential function for ret in the development of these cells; however, the precise nature of this function is still to be defined.

E. Other Sites of ret Expression Ret expression is also seen in several regions of the central nervous system, in the developing cranial nerve ganglia and a subset of cells within dorsal root ganglia, in motor neurons in the spinal cord and hindbrain, in neuroretina, and in the ureteric bud epithelium and growing tips of the renal collecting ducts in the developing kidney. In adult animals, ret mRNA has been detected in a number of tissues, including salivary gland, thymus,

The MEN II Syndromes and ret

21 1

spleen, and lymph node (Tsuzuki et al., 1995). In a few cases, phenotypic abnormalities can be linked to these sites of expression: homozygous yet knockout mice and some human patients with Hirschsprung disease have severe developmental abnormalities of the kidney (Schuchardt et al., 1994); neonatal lethality of the yet knockout in mice may be due to defective respiratory control due to abnormalities of the superior cervical ganglion (Burton et al., 1995); and it has been speculated that the distribution of the skin abnormality in cutaneous lichen amyloid in MEN IIA (see Section II.D.4) may be due to scratching resulting from itching in a dermatomal distribution, which is due to an abnormality of cells in the corresponding dorsal root ganglia (Nunziata et al., 1989).

V. SPECULATIONS ON HOW DIFFERENT ret MUTATIONS RESULT IN THE ASSOCIATED PHENOTYPES AND IN TUMOR FORMATION At least two sets of observations must be accounted for in any scheme of inherited and sporadic tumorigenesis in C cells. These are (1) the comparative rarity of cysteine codon mutations, compared with the MEN IIB codon 9 18 mutation, in sporadic tumors; and (2) the apparently contradictory reports that differentiation of C cells and related neuroectodermal cells is induced by yet overexpression (Santoro et al., 1990; the transfection experiments described in Section III.E.l) or is accompanied by silencing of yet expression (Carson et al., 1995).

A. The Extracellular Cysteine Mutations Carson et al. (1995) showed that induction of raf-1 signaling in TT cells (an MTC-derived C cell line) caused both differentiation and silencing of vet expression. They suggest that, during development, signaling in the ras-raf pathway causes a switch of C cells from a “preterminal” to a terminally differentiated state in which yet is no longer expressed and the potential for further proliferation is lost. In this scheme, cells that harbor a gain of function yet mutation would be able to override the differentiation signals and retain the ability to proliferate, resulting in the hyperplasias seen in MEN 11. Elaborating on this scheme, one could suppose that the different effects of mutations in different cysteine codons reflected the degree of activation of yet that resulted, and that the Hirschsprung phenotype occasionally seen with MEN IIA mutations might be the result of either cell cycle arrest of the early

212

Bruce A. j, Fonder and Damn Smith

enteric neuroblasts as a result of direct activation of a MAP kinase pathway or of premature neuronal differentiation, or possibly of apoptotic death of enteric neuroblasts, in response to inappropriate ret activation (V. Pachnis, personal communication). The absence of developmental abnormalities in MEN IIA compared with MEN IIB might be because, although the timing of yet activation is inappropriate, the downstream signaling pathways are normal. Although this scheme does not appear to be consistent with the observations of induction of differentiation by activated vet in cells in culture, the inconsistency can possibly be argued away by invoking different responses to specific signals in different cellular contexts (Carson et al., 1995). The prepondecance of codon 9 18 MEN IIB mutations in sporadic tumors suggests that these mutations, but not the cysteine mutations of MEN IIA, are able to cause transformation of mature C cells. In fact, not all sporadic MTC appear to have MEN IIB-type mutations, and data from microdissection of primary MTC tumors (Eng et al., 1996a) suggest that these mutations are distributed in a heterogeneous manner in subpopulations of cells within the tumor. If this is correct, it seems that some other, as yet unidentified, event is likely to be responsible for the initiation of sporadic MTC, and that the codon 918 MEN IIB mutation (but not the cysteine mutations of MEN IIA) can contribute to tumor progression. Whether sporadic MTC can arise from any C cell, even those postulated in the scheme of Carson et al. to be terminally differentiated, or arises only from a subset of cells that retain their proliferative potential, is unknown.

B. The M918T Mutation of MEN IIB This mutation alters the substrate specificity of the ret tyrosine kinase, and this is the most plausible explanation for the spectrum of developmental abnormalities that is seen. It may also account for the different frequency of MEN IIB- and MEN IIA-type mutations in sporadic tumors (see earlier). However, there are some experimental results (described in Section III.E.2) that, although far from conclusive, suggest that the mutation may also confer a degree of ligand-independent activation and, possibly more relevant, an increased catalytic activity of the tyrosine kinase domain. Either of these could also be significant in the effects of both the germline and somatic Met 918 T h r mutations. The absence of parathyroid involvement in MEN IIB is a further puzzle. One explanation could be lack of the ret ligand in parathyroid tissue (so that MEN IIA mutations would be effective but MEN IIB not, assuming they are ligand dependent). Alternatively, if altered signaling pathways are important for MEN IIB tumorigenesis, these pathways may be absent in parathyroid.

The MEN 11 Syndromes and ret

213

VI. OTHER EVENTS IN TUMOR PROGRESSION There is still little information about the molecular events in tumor progression in MEN 11-related tumors. Studies using X chromosome markers show that the established tumors are clonal (Baylin et al., 1976). However, it is not known whether the “multifocal hyperplasia” that precedes tumor formation is the direct consequence of the inherited mutation (in which case it should be polyclonal), or whether a somatic event is necessary (in which case it should be clonal). Abnormalities of N-ras, Ha-ras, and K-ras are not seen in MEN 11-related tumors (Moley et al., 1991), nor were there frequent abnormalities in the structure or expression of N-myc, c-myc, L-myc, c-mos, p-nerve growth factor, or the low-affinity nerve growth factor LNGFR (Moley et al., 1992b). There is, however, one report of overexpression of N-myc in 6 of 21 MTCs analyzed by in situ hybridization (Boultwood et al., 1988). Loss of heterozygosity (LOH) studies indicate a low level of chromosomal instability in MTC and pheochromocytoma. Mulligan et al. (1993a) found losses at a frequency of 10% or greater at only six chromosomal locations in a systematic search: lp, 3p, 3q, l l p , 13, and 22. Losses on other chromosomes were absent or infrequent, with the exception of 17p (3of 36; 8%). In particular, losses are rarely seen at the ret locus on chromosome 1Oq. Chromosome l p is consistently the most frequently involved chromosome in all reports of LOH in MTC and pheochromocytomas; in almost all cases, the entire chromosome arm is lost. Allele dosage studies indicate that this is not associated with isochromosome l q formation. A localized region of loss around the c-myc locus at lp32 has been suggested (Moley et al., 1992a), but this appears not to be a general finding in different studies. No mutations have been reported so far in familial or sporadic MTC in candidate genes that lie within the regions of allele loss-for example, the tyrosine phosphatase LAR on chromosome lp, the Von Hippel-Lindau gene on 3p, the retinoblastoma gene on 13q, the neurofibromatosis type 1 gene on 17q, or the neurofibromatosis type 2 gene on 22. None of the somatic changes observed in sporadic MTC (LOH, codon 918 mutations) have been reported in more than a subset of tumors. This suggests that all of these may be progressional events, and that an initiating event, as yet unidentified, may also be required.

MI. ANIMAL MODELS OF MEN I1 Thyroid C cell tumors and pheochromocytomas have been reported in certain Wistar-derived and other strains of rat (Boorman et al., 1972; Gill-

214

Bruce A. 1. Fonder a n d Damn Smith

man et al., 1953; Hoffman, 1987). F344 rats develop a variety of tumors with age, including components of both MEN I1 and MEN I syndromes (pituitary adenoma, adrenocortical ademona; Sass et al., 1975). It is not known whether ret mutations are involved in these tumors. Tumors resembling MTC are common in bulls (Krook et al., 1969). A dog has been reported with evidence of concomitant pheochromocytoma and hyperparathyroidism (Wright et al., 1995). No mouse strain has been described that has a high spontaneous incidence of these tumors, but a strain of transgenic mice, expressing the c-mos oncogene linked to a Moloney mouse sarcoma virus long terminal repeat, have developed a syndrome resembling MEN 11. Interestingly, the pattern of tumors in these mice differed between lines of the same inbred strain derived from different founders, and there was some evidence of an effect on penetrance when the transgene was crossed into different genetic backgrounds. Ret mutant transgenic mice are under construction and are likely to be available shortly.

VI11. CLINICAL IMPLICATIONS OF THE IDENTIFICATION OF ret MUTATIONS IN MEN I1 Because MEN IIA-FMTC families show considerable variation in spectrum of tumors and age at onset, prediction of phenotype would be of considerable help to guide screening and management. However, the genotype-phenotype correlations that have so far been described are not, in the authors’ opinion, yet sufficiently consistent or well established to be used for this purpose. In known MEN I1 families, the main value of DNA testing for the ret mutation is to determine in early childhood which family members require surveillance and which do not. For those who are found to have the ret mutation, there are two possible courses: either to have regular careful biochemical screening or to go immediately to prophylactic thyroid surgery. This is a controversial topic, and one where there is room for individual choice, but current opinion is moving toward prophylactic thyroid surgery in childhood rather than repeated screening. This is because of the perceived disadvantages of repeated biochemical screening, which include the discomfort of the stimulation tests (see Section KC), the anxiety associated with borderline or fluctuating results-which are quite common-and the possibility of default from screening. (By contrast, because of the much greater disadvantages of adrenalectomy, as well as the lower frequency of pheochromocytoma and the rarity with which small adrenal tumors are malignant, surgery for adrenal tumor is not generally recommended unless there is clear

The MEN II Syndromes and ret

215

evidence of abnormality.) DNA testing also reduces the possibility of thyroidectomy in an unaffected individual based on false-positive biochemical screening results, which has been a problem in the past (Lips et al., 1987, 1994). A potentially larger clinical role for yet mutation testing is in the management of the family of a patient who presents as an isolated case of MTC. Most of these cases will indeed be nonhereditary, but a few will have heritable disease (see Section II.D.l), with implications for the family. Even though the probability of heritable disease may be quite low, the consequences of missing the opportunity for early diagnosis and surgical cure are such that most families have been entered into a program of regular biochemical screening. Given the high proportion of MEN 11-FMTC families in which mutations have been found, and the limited spectrum of mutations to test, DNA testing of exons 10, 11, 13, an 14 now allows the rapid exclusion of heritable disease with a false-negative rate that is probably less than 2%, in individuals with a negative family history and no evidence of C cell hyperplasia. DNA testing should allow a large amount of biochemical screening and possible “medicalization” of family members, especially children, to be avoided.

IX. FUTURE PROSPECTS The story so far has been exciting, but it has raised more questions that it has answered. The identity of the y e t ligand;* the details of the downstream signaling pathway and the differences between wild-type and MEN IIB mutant yet; the role of y e t in normal development and how this is perturbed by the MEN I1 mutations; and the structure of the extracellular domain, the effects of the cysteine mutations, and the explanation for the differences in phenotype between them are some of the most obvious. Further ahead, the mechanisms of genetic (and perhaps environmental) interactions and their role in determining phenotype, already exemplified by the families with both MEN I1 and Hirschsprung disease, will be a challenge. Almost nothing is known about C cell development, but the developmental biology of the sympathoadrenal and enteric neuronal lineages is beginning to emerge. While the impact of studies of MEN I1 and of yet will perhaps be greatest in developmental and tumor biology, the clinical benefits should also not be overlooked. Recognition of gene carriers for screening and prophylactic surgery is simple and effective, and ret mutation testing is already among the first DNA tests for cancer susceptibility to enter routine medical practice. “See Note added in proof.

216

Bruce A. 1. Ponder and Damn Smith

ACKNOWLEDGMENTS The authors thank the members of the CRC MEN 2 Group, and clinicians and families and the many colleagues and collaborators who have contributed to their work. B. Nelkin, M. Takahashi, G. C. Vecchio, and the International RET Mutation Consortium kindly made available data from their laboratories in advance of publication. B. A. J. Ponder is a Gibb Fellow of the Cancer Research Campaign (CRC).

Note A d d e d in Proof The ret ligand has recently been identified as dial cell line derived neurotrophic factor (GDNF) (Durbec, P., Marcos-Gutierrez, C. V., Kilkenny, C., Grigoriou, M., Wartiowaara, K., Suvanto, P., Smith, D., Ponder, B., Costantini, F., Saarma, M., Sariola, H., and Pachnis, V. (1996).Nature (London) 381,789-793; Treanor, J. J. S., Goodman, L., de Sauvage, F., Stone, D. M., Poulsen, K. T., Beck, C. D., Gray, C., Armanini, M. P., Pollock, R. A., Hefti, F., Phillips, H. S., Goddard, A., Moore, M. W., Buj-Bello, A., Davies, M. A. M., Asai, N., Takahashi, M., Vandlen, R., Henderson, C. E., and Rosenthal, A. (1996). Nature (London) 382, 80-83; Trupp, M., Arenas, E., Fainzilber, M., Nilsson, A.-S., Sariola, H., Saarma, M., and Ibanez, C. F. (1996). Nature (London) 381, 785-789).

REFERENCES Anderson, D. J. (1993).J. Neurobiol. 24, 185-198. Angrist, M., Bolk, S., Thiel, B., Puffenberger, E. G., Hofstra, R. M., Buys, C. H. C. M., Cass, D. T., and Chakravarti, A. (1995).Hum. Mol. Genet. 4, 821-830. Asai, N., Iwashita, T., Matsumama, M., and Takahashi, M. (1995).Mol. Cell. Biol. 15,16131619. Attie, T., Pelet, A., Edery, P., Eng, C., Mulligan, L. M., Amiel, J., Boutrand, L., Beldjord, C., Nihoul-Fekete,C., Munnich, A., Ponder, B. A. J., and Lyonnet, S. (1995).Hum. Mol. Genet. 4, 1381-1386. Badner, J. A., Sieber, W. K., Garver, K. L., and Chakravarti, A. (1990).Am.]. Hum. Genet. 46, 568-580. Baylin, S . B., Gann, D. S., and Hsu, S. H. (1976). Science 193,321-323. Beldjord, C., Desclaux-Arramond, F., Raffin-Sanson,M., Corvol, J.-C., De Keyzer, Y., Luton, J.-P., Plouin, P.-F., and Bertagna, X. (1995). J. Clin. Endocrinol. Metub. 80, 2063-2068. Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B. (1994).J. Biol. Cbem. 269,32031-32034. Block, M. A., Jackson, C. E., Greenawald, K. A., Yott,J. B., and Tashjian, A. H. (1980). Arch. Surg. 115, 142-148. Bolino, A., Schuffenecker, I., Luo, Y., Seri, M., Silengo, M., Tocco, T., Chabrier, G., Houdent, C., Murat, A., Schlumberger, M., Tourniaire, J., Lenoir, G. M., and Romeo, G. (1995). Oncogene 10,2415-2419. Bongarzone, I., Monzini, N., Borrello, M. G., Carcano, C., Ferraresi, G., Arighi, E., Mondellini, P., Della Porta, G., and Pierotti, M. A. (1993). Mol. Cell. Biol. 13, 358-366. Boorman, G. A., Van Noord, M. J., and Hollander, C. F. (1972). Arch. Putbol. 94, 35-41. Bork, P., and Margolis, B. (1995).Cell 80, 693-694.

The MEN II Syndromes and ret

217

Borrello, M. G., Pelicci, G., Arighi, E., De Filippis, L., Greco, A., Bongarzone, I., Rizzetti, M. G., Pelicci, P. G., and Pierotti, M. A. (1994).Oncogene 9, 1661-1668. Borrello, M. G., Smith, D. P., Pasini, B., Bongarzone, I., Greco, A., Lorenzo, M. J., Arighi, E., Miranda, C., Eng, C., Alberti, L., Bocciardi, R., Mondellini, P., Scopsi, L., Romeo, G., Ponder, B. A. J., and Pierotti, M. A. (1995). Oncogene 11,2419-2427. Borst, M. J., van Camp, J. M., Peacock, M. L., and Decker, R. A. (1995).Surgery 117, 386389. Boultwood, J., Wyllie, F. S., Williams, G. D., and Wynford Thomas, D. (1988).Cancer Res. 48, 4073-4077. Burton, M. D., Kianane, B., and Kizemi, H. (1995).FASEB J. 9, A666. Caillou, B. (1991).In “Medullary Thyroid Carcinoma” (Colloques INSERM, Vol. 211, John Libbey Eurotext, Montrogue, France) (C. Calmettes and J. Guliana, Eds.), pp. 53-57. Carlson, K. M., Bracamontes, J., Jackson, C. E., Clark, R., Lacroix, A., Wells, S. A. Jr., and Goodfellow, P. J. (1994a).Am. J. Hum. Genet. 5 5 , 1076-1082. Carlson, K. M., Dou, S., Chi, D., Scavarda, N., Toshima, K., Jackson, C. E., Wells, S. A., Goodfellow, P. J., and Donis-Keller, H. (1994b). Proc. Natl. Acad. Sci. U.S.A. 91, 15791583. Carney, J. A., Go, V. L. W., Sizemore, G. W., and Hayles, A. B. (1976).New Engl. J. Med. 295, 1287-1291. Carney, J. A,, Sizemore, G. W., and Hayles, A. D. (1979).Cancer 44, 2173-2183. Carney, J. A., Roth, S. I., Heath, H. 111, and Sizemore, G. W. (1980). Am. J. Pathol. 99,387398. Carson, E. B., McMahon, M., Baylin, S. B., and Nelkin, B. D. (1995). Cancer Res. 55,20482052. Ceccherini, I., Hofstra, R. M. W., Luo, Y., Stulp, R. P.,Barone, V., Stelwagen, T., Bocciardi, R., Nijveen, H., Bolino, A., Seri, M., Ronchetto, P., Pasini, B., Bozzano, M., Buys, C. H. C. M., and Romeo, G. (1994). Oncogene 9,3025-3029. Chabre, O., Labat, F., Berthod, F., Jarel, V., and Bachelot, Y. (1992).Henry Ford Hosp. Med. J. 40,245-248. Chong, G. C., Bearhs, 0. H., Sizemore, G. W., and Woolmer, L. H. (1975).Cancer 35,695704. Cunliffe, W. J., Hudgson, P., Fulthorpe, J. J., Black, M. M., Hall, R., Johnston, I. D. A., and Schuster, S. (1970).Am. /. Med. 48, 120-126. Dodd, G. D. (1985).Semin. Roentgenol. 20, 64-90. Donis-Keller, H., Dou, S., Chi, D., Carlson, K. M., Toshima, K., Lairmore, T. C., Howe, J. R., Moley, J. F., Goodfellow, P., and Wells, S. A. J . (1993).Hum. Mol. Genet, 2, 851-856. Dou, S., Chi, D., Carlson, K. M., Moley, J. A., Wells, S. A. Jr., and Donis-Keller, H. (1994). Abstracts of the Fifth International Workshop on Multiple Endocrine Neoplasia 73. Karolinska Institute, Stockholm, Sweden, Abstract 617. Dralle, H., Schurmeyer, T. H., Kotzerke, T. H., Kemnitz, J., Crosse, H., and von zur Muhlen, A. (1989).Horm. Metab. Res. Suppl. Series 21, 34-38. Dyck, P. J., Carney, A., and Sizemore, G. W. (1979).Ann. Neurol. 6, 302-314. Easton, D. F., Ponder, M. A., Cummings, T., Gagel, R. F., Hansen, H. H., Reichlin, S., Tashjian, A. H. Jr., Telenius-Berg, M., and Ponder, B. A. J. (1989).Am. J. Hum. Genet. 44,208-215. Easton, D. F., Ponder, M. A., Huson, S. M., and Ponder, B. A. J. (1993).Am.J. Hum. Genet. 53, 305-3 13. Edery, P., Lyonnet, S., Mulligan, L. M., Pete, A., Dow, E., Abel, L., Holder, S., Nihoul-Fekete, C., Ponder, B. A. J., and Munnich, A. (1994). Nature (London) 367, 378-380. Emmertsen, K., Erno, H., Henriques, U., and Schroder, H. D. (1983). Dan. Med. Bull. 30, 353-356. Eng, C., Smith, D. P., Mulligan, L. M., Nagai, M. A., Healey, C. S., Ponder, M. A., Gardner, E.,

218

Bruce A. 1. Ponder and Darrin Smith

Scheumann, G. F. W., Jackson, C. E., Tunnacliffe, A., and Ponder, B. A. J. (1994).Hum. Mol. Genet. 3,237-241. Eng, C., Crossey, P. A., Mulligan, L. M., Healey, C. S., Houghton, C., Prowse, A., Chew, S. L., Dahia, P. L. M., O'Riordan, J. L. H., Toledo, S. P. A., Smith, D. P., Maher, E. R., and Ponder, B. A. J. (1995a).J. Med. Genet. 32, 934-937. Eng, C., Mulligan, L. M., Smith, D. P., Healey, C. S., Frilling, A., Raue, F., Neumann, H. P. H., Pfragner, R., Behmel, A., Lorenzo, M. J., Stonehouse, T. J., Ponder, M. A., and Ponder, B. A. J. (1995b) Genes Chromosomes Cancer 12, 209-212. Eng, C., Smith, D. P., Mulligan, L. M., Healey, C. S., Zvelebil, M. J.,Stonehouse, T. J., Ponder, Oncogene 10,509M. A., Jackson, C. E., Waterfield, M. D., and Ponder, B. A. J. (1995~). 513. Eng, C., Mulligan, L. M., Healey, C. S., Houghton, C., Frilling, A., Raue, F., Thomas, G. A., and Ponder, B. A. J. (1996a). Cancer Res. 56, 2167-2170. Eng, C., Clayton, D., Schuffenecker,I., Lenoir, G., Cote, G., Gagel, R. F., Ploos van Amstel, H. K., Lips, C. J. M., Takai, S-I., Marsh, D. J., Robinson, B. G., Frank-Raue, K., Raue, F., Xue, F., Noll, W. W., Romeo, G., Pacini, F., Fink, M., Niederle, B., Zedenius, J., Nordenskjold, M., Komminoth, P., Handy, G. N., Ghanib, H., Thibodeau, S. N., Lacroix, A., Frilling, A., Ponder, B. A. J., and Mulligan, L. M., (1996b).J. Am. Med. Assoc. (submitted). Fabien, N., Paulin, C., Santoro, M., Berger, B., and Grieco, M. (1994). Int. J. Oncol. 4,623626. Farndon, J. R., Leight, G. S., Dilley, W. G., Baylin, S. B., Smallridge, R. C., Harrison, T. C., and Wells, S. A. Jr., (1986). BY.J. Surg. 73, 278-281. Gagel, R. F., Tashjian, A. H., Cummings, T., Papathanasopoulos, N., Kaplan, M. M., Delellis, R. A., Wolfe, H. J., and Reichlin, S. (1988). New Engl. J. Med. 318, 478-487. Gagel, R. F., Levy, M. L., Donovan, D. T., Alford, B. R., Wheeler, T., and Tschen, J. A. (1989). Ann. Intern. Med. 111, 802-806. Gaisie, G. (1989). Padiatr. Radiol. 20, 136-137. Gardner, E., Mulligan, L. M., Eng, C., Healey, C. S., Kwok, J. A. J., Ponder, M. A., and Ponder, B. A. J. (1994).Hum. Mol. Genet. 3, 1771-1774. Gershon, M. D., Chalezonitas, A., and Rothman, T. P. (1993).J. Neurobiol. 24, 199-214. Gibson, W. G. H., Peng, T. C., and Croker, B. P. (1981). Am. J. Clin. Pathol. 75, 347-350. Gibson, W. G. H., Peng, T. C., and Croker, B. P. (1982). Am. J. Pathol. 106, 388-393. Gillman, J., Gilbert, C., and Spence, I. (1953). Cancer 6, 494-511. Grieco, M., Santoro, M., Berlingieri, M. T., Melillo, R. M., Donghi, R., Bongarzone, I., Pierotti, M. A., Della Porta, G., Fusco, A., and Vecchio, G. (1990). Cell 60,557-563. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). Science 241, 42-52. Hill, C. S., Ibanez, M. L., Samaan, N. A., Ahearn, M. J., and Clark, R. L. (1973).Medicine 52, 141-171. Hoffman, B. B. (1987). Clin. Invest. Med. 10,555-560. Hofstra, R. M. W., Landsvater, R. M., Ceccherini, I., Stulp, R. P., Stelwagen, T., Luo, Y., Pasini, B., Hoppener, J. W. M., Ploos van Amstel, H. K., Romeo, G., Lips, C. J. M., and Buys, C. H. C. M. (1994).Nature (London) 367,375-376. Howe, J. R., Norton, J. A., and Wells, S. A. J . (1993). Surgery 114, 1070-1077. Iwamoto, I., Taniguchi, M., Asai, N., Ohkusu, K., Nakashima, I., and Takahashi, M. (1993). Oncogene 8,1087-1091. Iwashita, T., Asai, N., Murakami, H., Matsuyama, M., and Takahashi, M. (1996). Oncogene 12,481-487. Khairi, M. R. A., Dexter, R. N., Burzynoki, N. J., and Johnson, C. C. Jr. (1975).Medicine 54, 89-112. Khalil, M. K., and Lorenzetti, D. W. C. (1980). BY. J. Ophthalmol. 64, 789-792. Kinoshita, S., Tanaki, F., Ohasi, Y., Ikeda, M., and Takai, S. (1991). Am. J. Ophthalmol. 111, 307-311.

The MEN I1 Syndromes and ret

219

Komminoth, P., Kunz, E. K., Matias-Guiu, X., Hiort, O., Christensen, G., Colomer, A., Rother, J., and Heitz, P. U. (1995). Cancer 76,479-489. Krook, L., Lutwak, L., and McEntee, K. (1969). Am. J. Clin. Nutr. 22, 115-118. Kwok, J. B. J., Gardner, E., Warner, J. P., Ponder, B. A. J., and Mulligan, L. M. (1993). Oncogene 8,2575-2582. Lacroix, A., Verdy, M., Hamet, P., Myers, S., Anderson, L., Goodfellow, P. J., and Simpson, N. E. (1992). Henry Ford Hosp. J. Med. 40, 315. Lairmore, T. C., Howe, J. R., and Korte, J. A. (1991). Genomics 9, 181-192. Landsvater, R. M., Jansen, R. P. M., Hofstra, R. M. W., Buys, C. H. C. M. Lips, C. J. M., and Ploos van Amstel, H. K. (1996). Hum. Genet. 97, 11-14. Le Douarin, N. (1982). “The Neural Crest.” Cambridge University Press, Cambridge, England. Lindor, N. M., Honchel, R., Khosla, S., and Thibodeau, S. N. (1995). I. Clin. Endocrinol. Metab. 80, 627-629. Lips, C. J. M., Leo, J. R., Berends, M. J. H., Minder, W. H., Roeland Blok, A. P., Geerdink, R. A., Hackeng, W. H. L., Roelofs, J. M. M., Vasen, H. F. A., and Vette, J. K. (1987). Henry Ford Hosp. Med. J. 35, 133-138. Lips, C. J. M., Landsvater, R. M., Hoppener, J. W. M., Geerdink, R. A., Blijham, G., JansenSchillhorn van Veen, J. M., van Gils, A. P. G., de Wit, M. J., Zewald, R. A., Berends, M. J. H., Beemer, F. A., Brouwers-Smalbraak, J., Jansen, R. P. M., Ploos van Amstel, H. K., van Vroonhoven, T. J. M. V., and Vroom, T. M. (1994). New Engl. 1. Med. 331. 828-835. Lo, L., and Anderson, D. J. (1995). Neuron 15,527-539. Lorenzo, M. J., Eng, C., Mulligan, L. M., Stonehouse, T. J., Healey, C. S., Ponder, B. A. J., and Smith, D. (1995). Oncogene 10, 1377-1383. Marsh, D. J., Andrew, S. D., Learoyd, D. L., Poger, R., Eng, C., and Robinson. B. G. (1996a). Hum. Mutat. (in press). Marsh, D. J., Learoyd, D. L., Andrew, S. D., Krishnan, L., Pojer, R., Richardson, A.-L., Delbridge, L., Eng, C., and Robinson, B. G. (1996b). Clin. Endocrinol. 44, 249-257. Miya, A., Yamamoto, M., Morimoto, H., Tanaka, N., Shin, E., Karakama, K., Toyoshima, K., Ishizaka, Y., Mori, T., and Takai, S. (1992). Henry Ford Hosp. Med. J. 40, 215-219. Moley, J. F., Brother, M. B., Wells, S. A., Spengler, B. A., Bredler, J. L., and Brodeur, G. M. (1991). Cancer Res. 51, 1596-1599. Moley, J. F., Brother, M. B., Fong, C. T., White, P. S., Baylin, S. B., Nelkin, B., Wells, S. A., and Brodeur, G. M. (1992a). Cancer Res. 52, 770-774. Moley, J. F., Wallin, G. K., Brother, M. B., Kim, M., Wells, S. A. Jr., and Brodeur, G. M. (1992b). Henry Ford Hosp. Med. J. 40, 284-288. Mori, S., Ronnstrand, L., Yokote, K., Engstrom, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C.-H. (1993). EMBO J. 12,2257-2264. Mulligan, L. M., Gardner, E., Smith, B. A., Mathew, C. G. P., and Ponder, B. A. J. (1993a). Genes Chromosomes Cancer 6, 166-177. Mulligan, L. M., Kwok, J. B. J., Healey, C . S., Elsdon, M., Eng, C., Gardner, E., Love, D. R., Mole, S. E., Moore, J. K., Papi, L., Ponder, M. A., Telenius, H., Tunnacliffe, A., and Ponder, B. A. J. (1993b). Nature (London) 363, 458-460. Mulligan, L. M., Eng, C., Attie, T., Lyonnet, S., Marsh, D. J., Hyland, V. J., Robinson, B. G., Frilling, A., Verellen-Dumoulin, C., Safar, A., Venter, D. J., Munnich, A., and Ponder, B. A. J. (1994a). Hum. Mol. Genet. 3, 2163-2167. Mulligan, L. M., Eng, C., Healey, C. S., Clayton, D., Kwok, J. B. J., Gardner, E., Ponder, M. A., Frilling, A., Jackson, C. E., Lehnert, H., Neumann, H. P. H., Thibodeau, S. N., and Ponder, B. A. J. (1994b). Nature Genet. 6, 70-74. Mulligan, L. M., Eng, C., Healey, C. S., Ponder, M. A., Feldman, C. L., Li, P., Jackson, C. E., and Ponder, B. A. J. (1994~).Hum. Mol. Genet. 3, 1007-1008. Mulligan, L. M., Marsh, D. J., Robinson, B. G., Schuffenecker, L., Zedenius, J., Lips, C. J. M., Gagel, R. F., Takai, S. I., Noll, N. N., Fink, M., Raue, F., Lacroix, A., Thibodeau, S. N.,

220

Bruce A. I. Ponder and Darrin Smith

Frilling, A., Ponder, B. A. J., Eng, C. for the International RET Mutation Consortium. (1995). J. Intern. Med. 238,343-346. Myers, S . M., Eng, C., Ponder, B. A. J., and Mulligan, L. M. (1995). Oncogene 11,2039-2045. Narod, S . A., Sobol, H., Nakamura, Y., Calmettes, C., Baulieu, J.-L., Bigorgne, J.-C., Chabrier, G., Couette, J., de Gennes, J.-L., Duprey, J., Gardet, P., Guillausseau, P.-J., Guilloteau, D., Houdent, C., Le Febvre, J., Modigliani, E., Parmentier, C., Pugeat, M., Saime, C., Tourniaire, J., Vandroux, J.-C., Vinot, J.-M., and Lenior G. M. (1989).Hum. Genet. 83, 353-358. Narod, S. A., Lavone, M. F., Morgan, K., Calmettes, C., Solbol, H., Goodfellow, P. J., and Lenoir, G. M. (1992). Am. J. Hum. Genet 51,469-477. Nelkin, B. D., De Bustros, A. C., Mabrey, M., and Baylin, S. B. (1989).JAMA261,3130-3135. Norum, R. A., Lafreniere, R. C., O’Neal, L. W., Nikolai, T. F., Delaney, J. P., Sisson, J. C., and Sobol, H. (1990). Genomics 8, 313-317. Nunziata, V., Giannattasio, R., Di Giovanni, G., D’Armiento, M. A., and Mancini, M. (1989). Clin. Endocrinol. 30,57-63. O’Dell, K. (1987).J. Pediatr. Surg. 22, 1019-1020. Ooi, J., Yagnik, V., Immanuel, D., Gordon, M., Moskow, J. J., Buchberg, A. M., and Magolis, B. (1995). Oncogene 10, 1621-1630. O’Toole, K., Genoglio-Prieser, C., and Pushparag, N. (1985).Hum. Patbol. 16, 991-1000. Pachnis, V., Maukoo, B., and Constantini, F. (1993). Development 119, 1005-1017. Padberg, B. C., Schroder, S., Jochum, W., Kastendieck, H., Roth, J., Heitz, P. U., and Komminoth, P. (1995).Am. J. Patbol. 147, 1600-1607. Pandey, A., Duan, H., Di Fiore, P. P., and Dixit, V. M. (1995).J. Biol. Cbem. 270, 2146121463. Pasini, B., Bonello, M. G., Greco, A., Bongarzone, I., Luo, Y., Mondellini, P., Albert, L., Miranda, C., Arighi, E., Bocciardi, R., Seri, M., Barone, V., Radice, M. T., Romeo, G., and Pierotti, M. A. (1995). Nature Genet. 10, 35-40. Pawson, T., and Schlessinger, J. (1993). Cum. Biol. 3,434-442. Pazin, M. J., and Williams, L. T. (1992).Trends. Biochem. Sci. 17, 374-378. Ponder, B. A. J., Finer, M., Coffey, R., Harmer, C. L., Maisey, M., Ormerod, M. G., Pembrey, M. E., Ponder, M. A., Rosswick, P., and Shalet, S. (1986). Q. J. Med. 252, 299-308. Ponder, B. A. J., Ponder, M. A., Coffey, R., Pembrey, M. E., Gagel, R. P., Telenius-Berg, M., Semple, P., and Easton, D. F. (1988). Lancet 8582, 397-400. Puffenberger, G. G., Hosoda, K., Washington, S. S., Nakao, K., De Wit, D., Yanagisawa, M., and Chakravarti, A. (1994). Cell 79, 1257-1266. Robertson, D. M., Sizemore, G. W., and Gordon, H. (1973).Trans. Am. Acad. Opbtbalmol. Otolaryngol. 79, 772-787. Robinson, M. F., Furst, G. J., Nunziata, V., Brandi, M. L., Ferrer, J. P., Bugalho, M. J., DiGiovanni, G., Smith, H., Donovon, R. T., Vilardell, E., and Gagel, R. F. (1992). Henry Ford Hosp. Med. J. 40, 249-252. Romeo, G., Ronchestto, P., Luo, Y., Barone, V., Seri, M., Ceccherini, I., Pasini, B., Bocchiardi, R., Lerone, M., Kaariainen, H., and Martucciello, G. (1994). Nature (London) 367, 377378. Rossel, M., Schuffenecker, I., and Schlumberger, M. (1995). Hum. Genet. 95, 403-406. Saad, M. F., Ordonez, N. G., Rashid, R. K., Guido, J. J., Hill, C. S. Jr., Hickey, R. C., and Samaan, N. A. (1984). Medicine 63, 319-342. Santoro, M., Rosati, R., Grieco, M., Berlinger, M., DAmato, G. L. C., de Franciscis, V., and Fusco, A. (1990). Oncogene 5, 1595-1598. Santoro, M., Dathan, N. A., Berlingieri,M. T., Bongarzone, I., Paulin, C., Grieco, M., Pierotti, M. A., Vecchio, G., and Fusco, A. (1994a). Oncogene 9, 509-516. Santoro, M., Wong, W. T., Aroca, P., Santos, E., Matoskova, B., Grieco, M., Fusco, A., and Di Fiore, P. P. (1994b). Mol. Cell. Biol. 14, 663-675.

The MEN I1 Syndromes and ret

22 I

Santoro, M., Carlomagno, F., Romano, A., Bottaro, D. P., Dathan, N. A., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. H., and De Fiore, P. P. (1995).Science 267,381383. Sapienza, C. (1994).Am. J, Hum. Genet. 55, 1073-1075. Sass, B., Rabstein, L. S., Madison, R., Nims, A. M., Peters, R. L., and Kelloff, G. J. (1975).JNCI I. Natl. Cancer lnst. 54, 1449-1456. Schimke, K. N., Hartmann, W. H., Prout, T. E., and Rimoin, D. L. (1968). New Engl. J. Med. 279. 1-7. Schneider, R. (1992). TIBS 17,468-469. Schuchardt, A., D’Agati, V., Larrson-Blomberg, L., Constantini, F., and Pachnis, V. (1994). Nature (London)367,380-383. Schuffenecker, I., Billaud, M., Calender, A., Chambe, B., Ginet, N., Calmettes, C., Modigliani, E., Lenior, G. M., and the GETC. (1994). Hum. Mol. Genet. 3, 1939-1943. Sizemore, G. W., Carney, J. A., and Heath, H. 111. (1977).Surg. Clin. North Am. 57,633-645. Sizemore, G. W., Carney, J. A., Gharib, H., and Capen, C. C. (1992).Henry Ford Hosp. J. Med. 40,236-244. Smith, D. P., Eng, C., and Ponder, B. A. J. (1994).J. Cell Sci. 18, 43-49. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993). Cell 72, 767-778. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., Ratnofsky, S., Feldman, R. A., and Cantley, L. C. (1994). Mol. Cell. Biol. 14, 2777-2785. Songyang, Z., Carraway, K. L. 111, Eck, M. J., Harrison, S. C., Feldman, R. A., Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C., Lorenzo, M. J., Ponder, B. A. J., Mayer, B. J., and Cantley, L. C. (1995a).Nature (London)373, 539. Songyang, Z., Margolis, B., Chaudhuri, M., Shoelson, S. E., and Cantley, L. C. (1995b).J. Biol. Chem. 270, 14863-14866. Sugaya, R., Ishimaru, S., Hosoya, T., Saigo, K., and Emori, Y. (1994).Mech. Dev. 45,139-145. Takahashi, M., and Cooper, G. M. (1987). Mol. Cell. Biol. 7, 1378-1385. Takahashi, M., Asai, N., Iwashita, T., Isomura, T., Miyazaki, K., and Matsuyama, M. (1993). Oncogene, 8,2925-2929. Takami, H., Ozaki, O., Mimura, T., and Ito, K. (1994). Abstracts of the Fifth International Workshop on Multiple Endocrine Neoplasia, Karolinska Institute, Stockholm, Sweden, Abstract 802. Tamir, H., Liu, K.-P., Payette, R. F., Hsuing, S.-C., Adlersberg, M., Nurez, E. A., and Cershon, M. D. (1989).J. Neurosci. 9, 1199-1212. Telenius-Berg, M., Berg, B., and Hamberger, B. (1984).Henry Ford Hosp. Med. J. 32, 225232. Tsuzuki, T., Takahashi, M., Asai, N., Iwashita, T., Matsuyene, M., and Asai, J. (1995). Oncogene 10, 191-198. van der Geer, P., Wiley, S., Lai, V. K.-M., Olivier, J. P., Gish, G. D., Stephens, R., Kaplan, D., Shoelson, S., and Pawson, T. (1995). Curr. Biol. 5, 404-412. van Weering, D. H. J., Medema, J. P.,van Puijenbroek, A., Burgering, B. M. T., Baas, P. D., and Box, J. L. (1995). Oncogene 11,2207-2214. Vasen, H. F. A., Kruseman, A. C. N., Bertel, H., Beukers, E. K. M., Delprat, C. C., van Doorn, R. G., Geerdink, R. A., Haak, H. R., Haekeng, W. H. L., Koppeschaar, H. P. F., Krenning, E. P., Lamberts, S. W. J., Lekkerkerker, F. J. F., Michels, R. P. J., Moers, A. M. J., Pieters, G. F. F. M., Wiersinga, W. M., and Lips, C. J. M. (1987).Am. J. Med. 83, 847-852. Verdy, M., Weber, A. M., Roy, C. C., Morin, C. L., Cadotte, M., and Brochu, P. (1982). J. Pediatr. Gastroenterol. Nutr. 1, 602-607.

222

Bruce A. 1. Ponder and Damn Smith

Wada, M., Asai, N., Tsuzuki, T., Maruyama, S., Ohiwa, M., Imai, T., Funahashi, H., Takagi, H., and Takshashi, M. (1996). Biochem. Biophys. Res. Commun. 218, 606-609. Wolfe, H. J., Melvin, K. E. W., Cervi-Skinner, S. J., Al Saadi, A. A., Juliar, J. F., Jackson, C. E., and Tashjian, H. Jr. (1973).New Engl. J. Med. 289, 437-441. Wolfe, H. J., Kaplan, M., Cummings, T., Ponder, B. A. J., Ponder, M., Gardner, G., Papi, L., and Reichlin, S. (1992). Henry Ford Hosp. Med. J. 40, 312. Wright, K. N., Breitschwerdt, E. B., Feldman, J. M.,Berry, C. R., Menten, D. J., and Spodnick, G. J. (1995).J. Am. Animal Hosp. Assoc. 31, 156-162. Xing, S., Tong, Q., Suzuki, T., and Jhiang, S. M. (1994). Biochem. Biophys. Res. Commun. 205,1526-1532. Zedenius, J., Wallin, G., Hamberger, B., Nordenskjold, M., Weber, G., and Larsson, C. (1994). Hum. Mol. Genet. 3, 1259-1262.

Index

A Abnormalities, developmental, in MEN IIB, 188- 190 Activins homology to TGF-PI, 65 TGF-p receptors responding to, 69 Adaptations, evolutionary, in cancer development, 41-42 Adhesion, cell and substrate, 12 Adrenaline, biochemical testing, 183 Adrenal medulla, ret expression, 209-210 Age at onset, MEN 11, 190 Alternatively spliced forms, ret, 194- 195 Amyloidosis, cutaneous lichen, with MEN IIA, 186-187 Antigens cancer, molecular identification, 169172 melanocyte, 150 melanoma expressed in normal testes, 161-163 MAGE, 147-149 tumor-specific, 166-169 Antioxidants, induction of new phenotype,

30 AP-1 binding sites, interaction with transcription factors, 78 Apoptosis, and c-Myc, 130-134 AP-2 transcription factor, interaction with C-MYC,112-113 Autoregulation domain, c-Myc protein, 101

B Basal cell carcinomas, hereditary and sporadic, 57-58 bcl-2, and c-Myc, in apoptosis, 132 Betaglycan, TGF-P type Ill receptor, 69-70 Binding proteins phosphotyrosine, and yet, 204-207

TATA box, interaction with c-Myc, 115 TGF-P, 68-71 Biology, cellular, cancer, 1-17 Bone morphogenetic proteins, similar to TGF-P, 65 Breast cancer, progression, estrogen role, 83-84

C Calcitonin, thyroid cells secreting, 208 Calcium, in keratinocytes, 80 Cambridge, England, cancer studies, 3-5 Cancer antigens, molecular identification, 169172 epithelial, development from phenotype to genotype, 21-44 immunotherapy, 145-172 phenotype, liver, 36-37 Cancer research cancer development basic to, 22-23 carcinogens, 41-42 Carcinogens cancer research, 41-42 effects on cell proliferation, 29-30 Casein kinase 11, c-Myc as substrate, 113 p-Catenin, mutated product, 168-169 C cells developmental role of ret, 207-209 tumors derived from, 181-188 Cell adhesion molecules, identification, 12 Cell culture food, 9 foundations, 5 Cell lines and cell strains, development, 8-9 c-Myc-induced apoptosis, 130-13 1 NIH 3T3, transfection, 203 tumor-infiltrating lymphocytes, 151-152 Cell loss, accompanying proliferation, 35

224 Cell proliferation carcinogen effect, 29-30 and cell loss, sequence, 41 c-Myc control, 95-134 hepatocyte, 33-36 TGF-P dual effects, 71-73 Chimera, EGFR-ret, 205-207 Chromosomes 9, NBCCS region, 53-56 loss, and malignancy, 16 Clinical implications, ret mutations in MEN 11,214-215 Clinical varieties, MEN If, 184-191 Clinicopathology, NBCCS, 50-52 Clofibrate, induced hepatocyte phenotype, 28 Clonal expansion, hepatocyte, 25, 29, 3235 Clonal origin, cancers, 2 Cloning, advances, 5-9 c-myc cooperation with bcl-2, 132 discovery, 96-98 downregulation, TGF-P-mediated, 7273 mRNA, 126-127 c-Myc protein and apoptosis, 130-134 and cell proliferation, 116-125 in embryonic development, 125-128 structural and functional features, 101108 as transcription factor, 108-1 16 Colon, cancer development, 38-39 Conflict of interest model, apoptosis, 134 Contact inhibition, and density-dependent inhibition, 10-11 Contamination, cell cultures by bacteria, 5 Cornified envelope, keratinocyte, formation, 79-80 Cosmids, chromosome 9-specific, 54-55 Crypt foci, aberrant, 39 Cryptic determinants, self-peptides, 164165 CUG codon, c-myc, 100 Cutaneous lichen amyloidosis, with MEN IIA, 186-187 Cyclin, transcription, regulation, 119-120 Cytotoxic T lymphocytes, melanoma-reactive, 147-149, 153-158, 165

Index

D Derepression, cdk inhibitors, 120 Development embryonic, c-Myc control, 125-128 epithelial cancer, from phenotype to genotype, 21-44 tissues involved in MEN 11, 207-21 1 Differentiation and c-Myc, 128-130 keratinocyte, retinoid role, 79-82 regulation by TGF-P, 73 Dihydrofolate reductase, interaction with C-MYC,122 Dimerization c-Myc protein DNA-binding domain, 104- 107 with Max, requirement in cell fate, 133 DNA naked, 171 replication, and c-Myc, 124-125 viruses containing, 15 DNA-binding domains, c-Myc protein, 104-107 Dulbecco modified Eagle’s medium, 9

E Earle, W.R., cloning studies, 6-8 E-box motif, binding of Myc/Max dimers, 112-113 E2F transcription factor, and c-myc induction, 119 Embryo, development, c-Myc role, 125-128 Endoglin, TGF-Pl binding, 69 Endoplasmic reticulum, hepatitis B protein accumulation, 35-36 Endothelin B receptor, G protein-coupled, 191 Epidermal growth factor, identification, 10 Epidermis, differentiation, 80-8 1 Epithelial cells, developmental patterns of cancers, 23-27 Epitopes, nonmutated, T cell response, 163166 Estrogens, in breast cancer progression, 8384 Ethnic groups, NBCCS occurrence, 57-58 Etiology, cancer, modulatable, 22-23 Evolutionary conservation C-MYC,114 c-myc, 96-100

index

Expression systems, insertion of cancer antigens, 170-172 Extracellular domain, ret, 194 cysteine mutations, 200-202, 211-212

F Fanconi anemia complementation group, gene mapping, 52-55 Features associated with NBCCS, 51 c-Myc protein, 101-108 Feeder layers, development, 6 Fibrin clots, dissolved by plasmin, 85 Focal proliferation hepatocyte phenotypes, 3 1-32 preceding cancer, 25-27 Food, for cells in culture, 9 Fusion, cell, 15-16

225

H HeLa cells, essential requirements, 9 Helix-loop-helix motif, c-Myc protein, 104-107 Hematopoietic cells, proliferation, TGF-P effect, 74-75 Hepatocytes, rare altered, 28-29, 3 1-32 Hirschsprung disease with MEN IIA, 187, 199 summary, 191-192 Homodimers, Max, 106-1 13 Hormones, steroid-thyroid superfamily, 6386 Human lymphocyte antigen-A2, restricted melanoma-reactive CTL, 147-148, 151-157 Hybrids, human-mouse, 16 Hyperplasia focal, 37, 40 multifocal, in MEN 11, 182-184

G G2, c-Myc role, 117 Gap junctions, identification, 11-12 Gene products, mutated, 166-169 Genes candidate, isolation strategies, 54-57 encoding cancer antigens, 145-172 GAGE, 162 ODC, 121-122, 131 target, c-Myc, 121-123 TRP-1, 159-160 Genetics, nevoid basal cell carcinoma syndrome, 49-59 Genomes, viral and cell, integration, 14-15 Genotype correlation with phenotype, in MEN 11, 198 neoplasia, 40-41 phenotype to, conceptual approach, 4244 Gorlin syndrome, 49-50 gP 100 identification as melanoma antigen, 157159 immunization trials, 171 gp7S, tyrosine-related, 159-161 Growth factors, response of cultured cells, 9-10 Guanine exchange factors, interaction with C-MYC,119

I Immune system, regulatory role of TGF-P, 73-75 lmmunogenicity MART-1 peptides, 153-154 melanosomal proteins, 163-165 Immunotherapy, cancer, 145- 172 Inhibins, homology to TGF-P1, 65 Initiation, hepatocyte phenotypes, 27-32 Interleukin-2, administration, 145-146 Interleukins, interaction with TGF-P, 74 Involucrin, in formation of cornified envelope, 79 Iron metabolism, alterations in hepatocyte nodules, 33

J

Junctional communication, in cell cultures, 11-12

K Keratinocytes, differentiation, 79-82 Knockout mice ret, 211 TGF-P, 74-75

226

L Lambda phage, and work on animal viruses, 3-4 Latency-associated protein, interaction with TGF-P, 67 Latent TGF-P-binding protein, in activation of TGF-P, 67 Lens, maturation, and c-Myc levels, 129 Lesions cancer-precursor, 23-24, 37 focal proliferative, 39 Leucine zipper motif, c-Myc protein, 104107 Linkage, and mapping, NBCCS, 52 Liver, phenotypes, initiation and progression, 27-36 Loss of heterozygosity, in MTC, 213

M a2-Macroglobulin, TGF-Pl binding, 70-71 Madl, interaction with Max, 109-111 Major histocompatibility complex, class I products, 147-148 Malformations, in NBCCS, 50, 57-59 Malignancy, and chromosome loss, 16 Mammary gland, TGF-P regulation in, 8284 Mapping, NBCCS comparative, 55-57 and linkage, 52 MART, see Melanoma antigens, recognized by T cells MART-l/Melan-A, expression and immunogenicity, 152-157 Max association with c-Myc protein, 106-1 13 dimerization, requirement in cellular fate, 133 Medium, conditioned, 6 Medullary thyroid carcinoma derived from C cells, 181-182 familial, 187-188 hereditary and nonhereditary, 184-185 yet mutations familial MTC, 195-198 sporadic MTC, 199-200 Medulloblastoma, in NBCCS, 50-51 Melanocytes, antigens, 150 Melanoma antigens, recognized by T cells, 149- 169

Index

MEN 11, see Multiple endocrine neoplasia type 11 Met 918 Thr, mutation in MEN IIB, 202204,212 Models, animal, MEN 11, 213-214 Molecular aspects, NBCCS, 52-54 M9-27 peptide, in MART-1 protein, 153156 mSin3 proteins, interaction with Madl, 111-112 MTC, see Medullary thyroid carcinoma Multiple endocrine neoplasia type I1 animal models, 213-214 clinical varieties, 184-191 M918T mutations, 212 ref mutations, 195-204 tumors, 181-184 Multiplicity, in phenotypic components, 43 Mutation analysis, for NBCCS, 57-59 Mutations gene products, 166-169 in MEN IIA, frequency, 185 in MEN IIB, 190-191 null c-myc, 127-128 yet, in MEN I1 syndromes, 180-181, 183-191, 195-207

N NBCCS, see Nevoid basal cell carcinoma syndrome Nervous system, enteric, y e t expression, 210 Neuromas, in MEN IIB, 188-189 Nevoid basal cell carcinoma syndrome, genetics, 49-59 Nevus sebaceous of Jadassohn, clinical profile, 58-59 Nodules, hepatocyte, 25-27, 32-36 Nuclear localization, c-Myc protein, 103

0 Oligonucleotides, antisense, c-myc, 128129 Open reading frames C - ~ Y C , 98, 100 ORF1, gp75,160-161 Origin, clonal, cancers, 2 Ornithine decarboxylase, transcription, c-Myc role, 121

Index

P pl5, gene product, 169 p53, interaction with c-Myc, 120-121 in apoptosis, 131-132 plOSKb,interaction with c-Myc, 123-124 p107, interaction with c-Myc, 114-115, 123- 124 p27, interaction with Myc, 120 Pancreas, precursor lesions, 37 Papilloma, regression, 38 Parathyroid disease, association with C634R mutation, 198 hormone, biochemical testing, 183 ret expression, 209 Patterns epithelial cancers, 23-27 MEN IIA disease in families, 185-186 ret expression, 207-21 1 Peptides HLA-A2-binding, 162- 163 MART-1, 153-154 mutated, 166-169 Peripheral blood lymphocytes, sensitization, 146-147 Persistence, hepatocyte nodules, 34-36 Phenotype associated with MEN IIA syndrome, 186-187 ret mutations, 211-212 to genotype, conceptual approach, 42-44 hepatocyte, 27-37 liver cancer, 36-37 Pheochromocytoma in inherited syndromes, 182 in MEN IIA families, 197-198 ret mutations, 199-200 Phosphotyrosine-binding proteins, and ret, 204-207 Plasmin, mediation of TGF-P activation, 67 Plasminogen, activation by steroids, regulation, 84-86 Platelet-derived growth factor and src kinases, 118 TGF-P binding, 70 Plating efficiency, variance, 6-8 Precursor cells, cancer, modulation, 22-23 Promoters c-myc transcription from, 98-100 DHFR, 122 Promotion, hepatocyte phenotypes, 3 1-36

227 Protein complexes, c-Myc association, 108 Proteins melanosomal, immunogenicity, 163-165 Ret, tyrosine kinase activity, 200-202 Proteus syndrome, clinical profile, 58-59 a-Prothymosin, interaction with c-Myc, 122 Proto-oncogenes, ret, role in MEN I1 syndrome, 179-215 PTC, NBCCS candidate gene, 54-55 PTC-ret constructs, 192, 203

R Ras, and c-Myc function, 119 Receptor tyrosine kinase, pYs on, 204-207 Redifferentiation, nodule hepatocytes, 34 Replication, DNA, and c-Myc, 124-125 Repressor, transcriptional, c-Myc as, 115116 Research, cancer basics, 22-23 carcinogens, 41-42 Resistance, to inhibiting effects of carcinogens, 29-32 Response elements, hormone and glucocorticoid, 75-77 ret expression patterns, 207-21 1 identification and structure, 192-195 mutations in MEN I1 syndromes, 195-207, 214215 and tumor formation, 21 1-212 Retinoblastoma protein, phosphorylation, inhibition by TGF-P, 71-72 Retinoids, in keratinocyte differentiation, 78-81 Retinoid X receptor, characteristics, 75-76 Reverse transcriptase, isolation, 15 Reversion, hepatocyte phenotypes, 31 RNA, messenger, c-myc, 126-127

S Screening familial MTC, 197 tumors in MEN 11, 182-184 Sensitization, peripheral blood lymphocytes, 146-147 Sequence homology, c-Myc protein, 97 SH2 domains, optimal binding sites, 205

Index Signaling, downstream from ret, 204-207 Signal transduction pathways c-myc, 117-119 steroid and thyroid hormones, 77 Skin, cancer development, 38 Steroid hormones adrenal, sex, and vitamin D,, 75-76 nongenomic actions, 77-78 regulation of TGF-P isoform expression, 79-84 Steroid receptors, characteristics, 76-78 Strangeways, Thomas, tissue culture studies, 4-5, 14 Structure, ret, 192-195 Syndromes MEN 11,179-215 nevoid basal cell carcinoma, genetics, 4959

T Target genes, c-Myc, 121-123 TATA box-binding protein, interaction with C-MYC,115 T cell receptors, recognizing MART-1, 156157 T cells human melanoma antigens recognized by, 149- 169 response to nonmutated epitopes, 163166 Testes, normal, melanoma antigen expression, 161-163 TGF-P, see Transforming growth factor-p Therapy, cancer, from cancer antigens, 169172 Tissue culture, foundations, 4-5 Tissues adult, c-myc expression, 126 development in MEN 11, 207-211 Transactivation, c-Myc protein, 109-1 15 Transactivation domain, c-Myc protein, 103-104, 113-115 Transcription c-myc, from distinct promoters, 98-100 cyclin, regulation, 119-120 Transcription factor AP-2, interaction with c-Myc, 112-1 13 c-Myc protein as, 108-116 E2F, and c-myc induction, 119

Transformation, cultured cells, 13-14 Transforming domain, c-Myc protein, 101 Transforming growth factor-p dual effects on cell proliferation, 71-73 isoform expression, regulation, 79-84 regulation of cell differentiation, 73 immune system, 73-75 structure and activation, 65-68 treatment of tumor cells, 86 Transforming growth factor-p receptors, characterization, 68-71 Tumor-infiltrating lymphocytes cell lines, 151-152 growing, 146 melanoma-reactive, 152-153, 158-159, 165 potency, 171-172 Tumors in MEN 11, types and development, 181184 MEN 11-related, progression, 213 parathyroid, ret mutations, 199-200 thyroid and adrenal, 190 Tumor suppressor proteins, interaction with C-MYC,123-124 Tumor viruses, transformation of cultured cells, 13-14 Tyrosinase, melanoma antigen, 150-152 Tyrosine related protein-1 (gp75), 159-161 residues, in Ret protein, 206 Tyrosine kinase domain familial MTC, 204 ret, 201-202

U Urinary tract, lower, epithelial cancer development, 39-40

V v-abl, transformation, c-Myc effect, 118 Vav, and c-Myc function, 119 Viruses, tumor, transformation of cultured cells, 13-14

229

index

Vitamin D,, in keratinocyte differentiation, 79-82 Vitamin D, receptor, DNA binding, 75-76

y YY-I, interaction with c-Myc protein, 116

Z X Xenopus, c-myc expression, 126-127 Xeroderma pigmentosum complementation group, gene mapping, 52-55

ZNFl69, NBCCS candidate gene, 54

This Page Intentionally Left Blank