Advances in Cancer Research Volume 70

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Advances in

CANCER RESEARCH Volume 70

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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.

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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.

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

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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

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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

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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

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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)

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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.

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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).

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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-

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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

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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

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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

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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.

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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

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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

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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

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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,

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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

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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-

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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,

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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

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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

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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.

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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.

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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.

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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

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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

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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.

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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.

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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

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(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.

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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

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-\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).

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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;

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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-

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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

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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

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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

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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.

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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.

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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,

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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

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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

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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?”

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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

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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

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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.

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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.

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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.

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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.

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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.

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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

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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-

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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

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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

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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

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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-

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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

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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

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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

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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,

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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

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(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

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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-

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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.

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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.

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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

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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).

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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.

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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.

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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

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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.

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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

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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