Advances in Cancer Research Volume 65

ADVANCES IN CANCER RESEARCH VOLUME 65

Foundations in Cancer Research

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ADVANCES IN CANCERRESEARCH Edited by

GEORGE F. VANDE WOUDE ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center Frederick, Maryland

GEORGE KLEIN Microbiology and Tumor Biology Center Karolinska lnstitutet Stockholm, Sweden

Volume 65

Foundations in Cancer Research

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, inlcuding photocopy, recording, o r any information storage and retrieval system, without permission in writing from the publisher.

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International Standard Serial Number: 0065-230X International Standard Book Number: 0-12-006665-3 PRINTED IN T H E UNITED STATES OF AMERICA 94 95 96 97 98 99 BB 9 8 7 6 5 4 3 2

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CONTENTS

CONTRIBUTORS TO VOLUME65 .......................................

ix

PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Genetics in the Thirties JAMES

1. 11. 111.

IV.

F. CROW

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics in the 1930s ............................................. Viewpoints and Paradigms in the 1930s ............................. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 10 14

15

Historical Origins of Current Concepts of Carcinogenesis P. D. LAWLEY I. 11. 111.

I v. V. VI. VII.

VIII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. Exogenous Sources of Cancer Mutation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Carcinogenesis: Early History ............................. Mode of Action of Carcinogenic Polycyclic Aromatic HydrocarbonsEarly Historical Development ............................. The Scope of Chemical Carcinogenesis BroadensDNA as an in Viuo Receptor . . . . . . . . . . . . . . The Advent of Mutational Spectra: Attempts to Correlate Carcinogen-DNA Reactions and Carcinogenic Genetic ................ Changes at the Molecular Level . . . . . . . . Summary and Conclusions .............................. ..... References . . . . . . . . . . . . .... ........................

18

23 28 39

44 49

75 92 97

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CONTENTS

WALLACEH . CLARK, JR. An Introduction to an Essay on the Nature of Cancer T h e Only Research Plan. T h e Present: Studies of 'rum in Maine, at the Beth Israel Hospital, and at Harvard . . . . . . . . . . . I l l . T h e Melanocyte. T h e Years at Tulane University . . . . IV. T h e Biologic Forms of Primary Cutaneous Melanoma and Levels of Invasion. The Melanoma Studies at the Massachusetts General I.

11.

V.

V1.

Lesion Clinic ............. Tumor Progression, the Dysplastic Nevus, and the Precursor State of Neoplasia. T h e Years in Philadelphia: Temple University and the Pigmented Lesion Group of the University of Pennsylvania . . . Maine, the Beth Israel Hospital, Harvard Medical School, and Pathology Services, Inc. Back to the Future ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121

123 131

158

The Origins of the Small DNA Tumor Viruses

ARNOLD J. LEVINE I. 11. 111.

IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e Viruses of Monkey Kidney Cells ............................ T h e Origins of the DNA Tumor Viruses ....................... T h e Molecular Biology of the DNA Tumor Viruses Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . ..........

147

163 164

Retroviruses and Wild Mice: An Historical and Personal Perspective

MURRAYB. GARDNER .........

169

..........

173

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

175 183

of the FwZ* MuLV-Resistance Allele from Inbred Mice . . . . . . . . . . . . . . .

188

I. 11.

in the Laboratory ..................................

Ill. Retrovirus (MuLV) Infection in Aging Wild Mice I v. Pathogenesis of Lymphomas . . . . . . . . . . . . . . . . . . . V. Pathogenesis of MuLV Neurologic Disease . . . VI. Nongenetic Control of Type C Virus and Asso VII.

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CONTENTS

VIII . Genetic Control of Type C Virus in Wild Mice by Natural Segregation of the Fv-4 Ecotropic MuLV-Resistance Allele ....................... IX . Type B Mammary Tumor Viruses in Wild Mice ..................... X . Lessons Learned ................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

188 191 192 197

Sol Spiegelman GUNTHER S. STENT Text . .

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

203

Growth Dysregulation in Cancer Cells ARTHUR B . PARDEE ....................... I . Introduction . . . . . . . . I1 . College and Graduate ..................... 111. Cancer Research in 195 I v. Regulatory Processes ....................... V. Regulations in Higher Organisms . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . V I . Cancer and the Cell Surface . . . . . . . . . . . . . . . . VII . The Restriction Point Hypothesis .................................. ...................... VIII . A Short-Lived Regulatory Protein IX . Kinase (TK) Gene . . . ....................... X . Yi Complexes ................................. XI . Cyclin E Is a Candidate R-Point Protein ............................ XI1 . Cyclin Changes in Breast Cells . . . . . . . . . . . . . . . . . . XI11 . Breast Cancer in Vim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV. Future Problems ......................

213 2 14 215 215 217 218 218 219 220 221 222 224 226 226 227

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INDEX .............................

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CONTRIBUTORS

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

WALLACE H. CLARK, JR., Department of Pathology, Harvard Medical School, and The Beth Israel Hospital, Boston, Massachusetts 021 15, and Consultant Pathology Services, Pathology Services, Inc., Cambridge, Massachusetts, and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 16802 ( 1 13) JAMESF. CROW,Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706 (1) MURRAY B. GARDNER, Department of Pathology, School of Medicine, University of California, Davis, Davis, California 9561 6 ( 169) P. D. LAWLEY, Section of Molecular Carcinogenesis, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey S M 2 5NG, United Kingdom

(17) ARNOLD J. LEVINE, Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544 (14 1) ARTHUR B. PARDEE, Department of Biologzcal Chemistry and Molecular Pharmacology, Division of Cell Growth Regulation, Dana-Faber Cancer Institute, Haward Medical School, Boston, Massachusetts 021 15 (2 13) GUNTHER S. STENT,Department of Cell and Molecular Biology, University of California, Berkeley, Berkeley, California 94720 (203)

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PREFACE

T h e chapters in this volume are all part of the Foundations in Cancer Research series we inaugurated with Volume 59. Our purpose has been to capture the views of those who participated in the several disciplines of cancer research that helped to form the present field. These contributions have come in the form of reviews, o r personal views, that are both biographical and autobiographical. The contributions to this volume include reviews of genetics, carcinogenesis, DNA tumor viruses, retroviruses, tumor biology, and the cell cycle. Fifteen years ago these were considered separate research disciplines. It is one of humankind’s most significant achievements that they are now, materially and intellectually, mutually interdependent. George F. Vande Woude George Klein

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GENETICS IN THE THIRTIES James F. Crow Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706

I. Introduction 11. Genetics in the 1930s A. Then and Now B. The 1932 International Congress of Genetics C. Population Genetics D. Human Genetics E. The Nature of the Gene 111. Viewpoints and Paradigms in the 1930s A. A Search for and Belief in Generality B. A Preference for the Mendelian Paradigm C. The Importance of Techniques IV. Conclusion References

1. Introduction

H. J. Muller once complained of having to study genetics in the guise of cancer research. Muller, a leading figure in the first half-century of genetics (perhaps the leading figure), had difficulty getting the support he needed unless it carried the cancer label. Although Muller was confident that genetics would one day play an important role in our understanding of cancer, he was not sure that his research was of immediate relevance. To us now, who are reminded daily of the central role that genetics and its techniques play in cancer research, it is hard to realize that the subject once seemed peripheral. Despite the early writings of Boveri, the recognition of chromosome aberrations in cancer cells, and sundry genetic theories of cancer, cancer research, and genetics marched to different drummers. In the 1930s there was doubt as to whether cancer began as a single cell. In this series of historical articles, my assignment is early genetics. I took my first genetics course in 1936. I was especially fascinated by linkage mapping and told friends that it would be interesting to work on gene mapping in humans. The naivete of this quickly became apparent. In fact the subject waited 50 years for a sufficient number of suitable

1 ADVANCES IN CANCER RESEARCH, VOL. 65

Copyright 8 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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markers; then they came in a grand rush. In no place is the contrast between then and now greater than in the state of the human chromosome map. 1936 will serve as a good reference point. T h e 1930s represented a high point in transmission genetics. T h e rules of inheritance were largely solved. Linkage mapping was an established art; extensive maps were available for Drosophila and maize, and those in other plants were well under way. Cytogenetics and chromosome mechanics were highly sophisticated. Yet the central question, the nature of the gene and how it goes about its business, was as mysterious as ever. T h e answer had to wait for a totally new battery of techniques after World War 11. T h e high resolving power of experiments with microorganisms and new ways to deal with very large molecules led to the Watson-Crick model of DNA, and genetics took off in a different direction. In this article, I shall discuss what was known in the 1930s. In some areas knowledge was about as advanced as it now is; in other areas, especially those that depend on molecular advances, the subject has changed so as to be barely recognizable. I shall also mention what seem to me to have been the prevailing attitudes and assumptions that existed in my student days, viewed through a somewhat cloudy “retrospectroscope.” I have reused some material from two earlier papers (Crow, 1992a,b).

II. Genetics in the 1930s A. THEN A N D Now T h e decade of the 1930s was the golden era of transmission genetics. Mendel had provided the basic rules of inheritance in 1866, but these had to await rediscovery in 1900. With this rediscovery came the almost instant realization, probably by many, that Mendel’s factors were carried by the chromosomes. Sex determination and sex-linked inheritance were worked out during the next decade. In the 1910s, T. H. Morgan and his students, following A. H. Sturtevant’s lead, mapped the location of genes in linear order along the chromosomes. T h e discovery of giant chromosomes in the salivary glands in 1936 permitted the precise physical location of genes, with the pleasing result that the physical gene order agreed with the linkage order determined by breeding tests. T h e concept of mutation was introduced by Hugo DeVries in the early part of the century, even though his “mutations” turned out to be segregants from complex chromosome rearrangements. In 1926 Muller showed that the mutation rate was temperature dependent and could be greatly

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3

enhanced by radiation. By the 1930s cytogenetics was very sophisticated (although not in man); translocations, inversions, and other rearrangements were well understood, as was polyploidy. Cytogenetics had a very pleasing aspect; you could draw pictures based on simple rules of synapsis and segregation and almost always the data agreed. T h e champion at this game was C. D. Darlington, who was simultaneously admired for his cleverness and castigated for his excessive theorizing. T h e dominant organisms were Drosophila and maize. Curt Stern, using Drosophila, and Harriet Creighton and Barbara McClintock, using maize, demonstrated the reality of physical exchange of chromatids in the process of crossing over. The two groups used essentially the same idea, employing heteromorphic chromosomes by which they could correlate a visible exchange of chromosome parts with a correspondingly altered pattern of inheritance. The four-strand nature of crossing over during meiosis and the relationship of exchange to chiasmata were also well understood. Earlier, Eleanor Carothers had taken advantage of heteromorphic chromosomes in the grasshopper to demonstrate the independent segregation of nonhomologous chromosomes. T h e XY basis of sex determination had been worked out early in the century. C. B. Bridges had used nondisjunction as a final proof of the chromosomal basis of inheritance-no longer needed by 1916. Nondisjunction had also provided the opportunity to study genotypes other than XX and XU, with the conclusion that the Y chromosome played no role in sex determination; rather it was the balance between X chromosomes and autosomes. American geneticists assumed that this was generally true, despite evidence from Japan regarding the importance of the Y chromosome in the silkworm. I t later turned out that the silkworm was the better model for mammals. Aneuploidy and polyploidy were studied in many species. Radiation allowed chromosome rearrangements to be produced on a large scale, and colchicine allowed polyploids to be produced almost at will. A. F. Blakeslee had recognized characteristic phenotypes associated with trisomy for chromosomes and chromosome parts in the Jimson weed, Datura, foreshadowing trisomy phenotypes in humans. Although Lindegren had taken advantage of spore order in the ascus of Neurospora to verify and amplify the less direct analyses of meiosis in Drosophila, the biochemical experiments of Beadle and Tatum were yet to come. T h e genetics of yeast, bacteria, and viruses did not exist. Bacteria were thought to be sexless, and even the physical nature of viruses was in doubt. Although Muller had suggested that bacteriophages might provide the way for a chemical attack on the gene, the necessary techniques were not at hand.

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B. THE1932 INTERNATIONAL CONGRESS OF GENETICS

1932 was an exciting year for geneticists, the year of the Sixth International Congress of Genetics at Cornell University. Despite the worldwide depression, more than 500 people from 35 countries attended. (The registration fee was $10 for full members and $6 for students; those short of funds could pay in installments. Rooms were $1.75.) Looking through the proceedings of this Congress (Jones, 1932) gives one a clear representation of the state of genetics in the 1930s. There were two kinds of papers at the Congress. One was an extension of what had been learned, mostly about transmission genetics, to a great many species. Mutant genes, cytogenetics, breeding experiments, and studies of natural populations were reported for an astonishing number of species. T h e Congress featured hundreds of demonstrations, including 15 groups of invertebrates, many of them with living specimens, 15 vertebrate species, and 35 genera of wild plants, along with an equal number of cultivated varieties. The success of linkage mapping was dramatized by a “living chromosome map” of maize, in which representative mutant phenotypes had been planted in appropriate positions along 10 rows corresponding to the haploid chromosome number. Plant and animal breeding received considerable attention. One paper, prescient at least in name, was entitled “Genetic Engineering”; it was a plea for combining genetic and breeding knowledge to improve livestock. Another paper reported locating quantitative traits on specific chromosomes in the tomato. Also, homozygous strains were produced from doubled haploids, and polyploids were utilized for breeding. I wonder how many people who today read excitedly of mapping quantitative trait loci know of these impressive beginnings sixty years ago. A project that was outstanding both for its vision and for its sheer immensity was described by the Soviet geneticist N. I. Vavilov. With a large team of workers, Vavilov traveled the world collecting wild relatives of domestic plants to be used as a reservoir of germ plasm for improving cultivated plants. By 1940, more than 250,000 plants had been collected. These were not just herbarium specimens, but actual seeds and growing plants. Alas, Vavilov fell victim to Lysenko and Stalin. He was unable to attend the Seventh International Congress in 1939 (Punnett, 1941), was arrested soon after, and died in prison in 1943. The second kind of paper is exemplified by most of the plenary addresses. These addresses accepted the information on transmission genetics and attempted to probe further into remaining mysteries. Sturtevant pointed out that early embryonic mutations created mosaics

GENETICS IN THE THIRTIES

5

which could be used to study cell lineage. McClintock used doubly heteromorphic chromosomes to demonstrate four-strand crossing over, which was important at the time because a currently popular hypothesis (due to J. Belling) involving copy switching between newly synthesized strands would predict only two-strand doubles. L. J. Stadler presented his thoughtful arguments for X-ray mutations being deletions, while N. Timofeeff-Ressovsky reported reverse mutations that seemed to imply that at least some radiation-induced mutations were true gene changes. Muller showed how deficiencies and duplications could be used to identify the absolute effect of a mutation. Since ordinary breeding experiments could only substitute genes, not add or subtract them, this introduced a new level of understanding. In this way he showed that most mutations were loss of function, complete or partial. The concept was extended and quantified by Sewall Wright. Several workers reported the creative use of mutant genes to study other processes. J. W. Gowen reported an asynaptic mutation that marked the beginning of the dissection of meiosis, a process that is still actively going on. Others used interacting mutations to determine the sequence of developmental steps, another technique still actively employed. Looking back, I am still impressed by the sheer analytical power of breeding tests, especially in Drosophila. Simply counting phenotypes from carefully planned matings, using specially contrived stocks, revealed deep details about such processes as meiosis and segregation in rearranged chromosomes. Specially constructed stocks, such as Muller’s C B , provided a technique for studying mutation quantitatively, thereby opening u p an active area of research. Analysis of mutation for decades after depended on this stock and its frequently improved successors. An astonishing amount of information was acquired by the absurdly simple operation of breeding flies and counting the different offspring phenotypes-a triumph of careful reasoning and experimental design. I n short, the rules of transmission genetics were established, and were being applied in one species after another. The more thoughtful geneticists were trying to probe deeper and, given the limitations of their techniques, were making some progress. Yet, little happened from the 1930s until after World War I1 to change any fundamental views. Detail after detail was added, but the central mystery-the nature of the gene-remained elusive. Of course the war placed a damper on genetic research, especially in Europe and Japan. The real breakthrough had to await the post-war development of microbial genetics and DNA chemistry.

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C. POPULATION GENETICS

T h e three great leaders who developed the field of population genetics, R. A. Fisher and J. B. S. Haldane in Britain and Sewall Wright in the United States, all published their greatest work in the 1930s. Each in his own way added to the mathematical theory and collectively they created a new science. T h e subject has become much more sophisticated and rigorous since that time, largely because of trained mathematicians entering the field. One who added mathematical sophistication and rigor to the field was G. Malecot. Although he did tnuch of his work in the 1930s, he published in obscure French journals and remained largely unknown in English-speaking countries until the late 1940s. T h e range of problems that can be studied has since been greatly increased by computers. Yet, I think it is fair to say that the three pioneers took the cream. The 1930s saw the beginnings of the genetics of natural populations. T h e greatest effort went to Drosophila, thanks to the enthusiasm of Th. Dobzhansky (1937), who popularized the theories of the three masters along with his own experimental studies. Dobzhansky’s work had great influence on me. At the same time many plant species were studied. Genetic analysis of closely related species and their hybrids showed beyond any doubt that any differences in species were due to the same kind of mutational and cytogenetic changes that occurred within a species. This conclusion was important in resolving doubts of the time. T h e genetic bases of hybrid sterility and other isolating mechanisms were worked out in great detail in one species after another. Molecular methods have refined these techniques, but the ideas have remained essentially the same. Studies at that time had an important limitation. They were necessarily confined to species that could be hybridized. One of Vavilov’s tricks was to use cytological similarity as a lead in identifying plant species that could be crossed. Molecular biology has removed this limitation, and it is now commonplace to study comparative genetics of distantly related species, even plants versus animals. In addition, molecular and computer methods have given new life to the moribund subject of phylogenetic reconstruction. D. HUMAN GENETICS

Almost immediately after the rediscovery of Mendel’s rules, examples of human traits showing Mendelian inheritance were found. One of the first was brachydactyly, a dominant finger-shortening condition, discov-

GENETICS IN THE THIRTIES

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ered by W. C. Farabee, a Harvard graduate student. More important in its future influence was A. E. Garrod’s discovery of alkaptonuria, the first of the “inborn errors of metabolism.” By the mid- 1930s a large number of traits had been described with varying degrees of certainty as Mendelian. One that attracted much attention was phenylketonuria, reported in 1934, which convincingly demonstrated genetically caused mental retardation. Treatment by withholding phenlyalanine came several years later. Although pedigree analysis was useful for rare conditions, especially dominants and X-linked recessives, studies of common traits such as blood groups were often inconclusive. Decisive answers came from gene frequency analysis, by which population frequencies and the assumption of random mating could be used to produce testable predictions. It now seems hard to believe that it was not until 1924 that F. Bernstein used gene frequency analysis to demonstrate the correctness of the multiple allele hypothesis, although the ABO blood groups had been known to be inherited since the turn of the century. T h e mutation rates of a few human genes were known, most notably the gene for hemophilia. Haldane not only measured the mutation rate, quite accurately as it turns out, but in an analytical tour de force also deduced that the male rate is about 10 times as high as the female rate. T h e latter conclusion was substantiated by the finding of a six-year increase in average age among the normal fathers of children affected by dominant diseases, or, in the case of X-linked recessives, in the maternal grandfathers. This pattern is presumably caused by the much greater number of cell divisions ancestral to a sperm than to an egg, but the relationship to cell division of different molecular kinds of mutations is only now beginning to be worked out. Human cytogenetics in the 1930s was primitive. Even the accepted number of chromosomes was wrong. It was thought to be 48 and the correct number, 46, had to wait for the improved techniques of the 1950s. One of the more interesting plenary talks in the 1932 Congress, the only one on human genetics, was delivered by C. B. Davenport. Astutely suspecting that Down syndrome might be caused by a chromosome irregularity, he sent material to the cytologist T. S. Painter, who (surprising to us now) found nothing abnormal in the chromosomes. Painter, incidentally, is also the person responsible for reporting the wrong chromosome number. He is to be forgiven, however, for two reasons: first, although he was using the best techniques of the time, they were still miserable; second, he discovered the giant salivary chromosomes, thereby revolutionizing Drosophila cytogenetics. Davenport is a controversial figure in genetics history. He made some solid contributions

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to human genetics, but his eugenics views were simplistic at best. His Eugenics Record Office was an early attempt to collect pedigree records for future study. Although it has not turned out to be of much value, it did foretell the CEPH families now used for linkage studies. Charles Cotterman long ago suggested that blood samples from families likely to be informative, such as those for three generations with a large number of children, be stored. These samples could then be used to study the inheritance of other blood traits as these were discovered without having to make additional blood collections. The suggestion was too far ahead of its time to attract any support. Human chromosome mapping hardly existed. A few linkage values were known, but there was no coherent map. Haldane’s analysis of linkage of hemophilia with X-linked night blindness involved solving a 22nd degree equation, a heroic accomplishment in those days before computers. Nowhere is the contrast between quantitative knowledge than and now more striking than the human chromosome map. A happy feature of human genetics, now almost forgotten, was the cleverness of techniques for getting information that would be trivially easy in experimental organisms. Wilhelm Weinberg, of Hardy- Weinberg fame, was a pioneer in devising such tricks. For example, he studied Mendelian ratios of recessive genes in small sibships, where the heterozygosity of the parents was ascertained only by their having affected children. He also worked out the way to make appropriate age corrections for twin studies and was the first to point out (as early as 1912!) that one might expect an increased age in the normal fathers of children with dominant mutations. Further advances, especially for analyzing common traits such as blood groups that d o not lend themselves to pedigree analysis, were made by Haldane and Fisher. There were also sophisticated methods for linkage analysis, but the methods were far ahead of the data; it was like using a main-frame computer to do a simple addition. It is interesting that human genetics was more highly developed in England than in the United States. I think there were two main reasons. One is that the two leading British theoreticians, Haldane and Fisher, were interested in humans while their American counterpart, Wright, was not. T h e other reason is the great influence of Morgan and Muller. Muller, especially, who was deeply interested in human genetics and later in his life would devote much of his energy to the subject, was convinced in the 1930s that substantive advances would have to wait for deeper knowledge of fundamental genetics. So he worked with Drosophila.

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E. THENATURE OF THE GENE T h e central mystery of genetics was, of course, the nature of the gene. Considering the importance of the problem, there was surprisingly little speculation, at least in print. This was perhaps due to the great influence of Morgan, whose disdain for ideas unsupported by data was widely known. Yet, there was inevitably speculation when geneticists got together to talk. For example, at the conclusion of the 1932 Congress, the editor of the Journal of Heredity (Cook, 1932) wrote, “Oceans of words were spilled in formal and informal gatherings to discuss the vital question: ‘What is the gene?’ but that important entity is still elusive.” Some thought that further study of mutation would uncover something of the gene’s nature. The patterns of forward and reverse mutation and of multiple allelism were important, but they did not reveal the desired answer. Nor were the high temperature coefficient and the effectiveness of high-energy radiation on mutation, although arguing for a high activation energy. Numerous attempts were made to estimate the size of the gene (assuming it to be spherical), using the radiationsensitive volume. This was troublesome because the different mutation rates in different tissues seemed to imply an unwelcome difference of gene size. Other calculations were based on the chromosome volume divided by the presumed number of genes, or more convincingly, at least as an upper limit, on the amount of chromosome material removed by the smallest deletion. An important source of controversy in the 1930s was the contrasting view of X-ray mutations by Muller and Stadler (Carlson, 1966). Muller thought that radiation induced the same kinds of mutations that occur spontaneously, although not necessarily in the same proportions. Stadler believed that radiation-induced “mutations” were mainly, if not entirely, small deletions. T h e question is still not completely settled. T h e strange mosaic patterns in corn kernels observed by R. A. Emerson and similar phenomena in Drosophila vim‘lis by M. Demerec suggested that the gene could be subdivided into separately mutating units. But I don’t think this entered into the thinking of most geneticists, who preferred to regard these occurrences as aberrations rather than providing any general insight. This wasn’t clarified until McClintock’s realization that moving elements were involved. We know now that mainy of the classical mutations in Drosophila were also caused by transposons. In 1936 it was universally agreed that the gene was protein; nucleic acid was thought of as scaffolding. Furthermore, animals had “thymus nucleic acid” and plants “yeast nucleic acid,” now DNA and RNA,

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respectively. T h e prevailing idea of nucleic acid structure was that of a monotonous repeating tetranucleotide, which was unsuited to the carrying of genetic information. In addition, only protein was thought to be complex enough to d o the wondrous things that a gene had to do. Most speculations regarded the gene as essentially two dimensional. On the one hand, a one-dimensional gene could not carry sufficient information (it was thought) but on the other hand, it was hard to imagine how a three-dimensional gene could come apart after the self-copying of individual internal components. So, a two-dimensional lattice in which each unit attracted its counterpart seemed most reasonable. In the 1920s Muller had stated clearly what the gene had to do: it had to carry enormous amounts of information; it had to copy itself with great accuracy; it had to mutate, that is replicate errors with the same fidelity as it copied the original message; and it had to be translated into control of development and metabolism. T h e astonishing thing is that none of the speculations came anywhere near the correct picture. ‘The simple Watson-Crick idea of a duplex structure, separating into halves with each copying the other, was not part of the thinking of the time. Ill. Viewpoints and Paradigms in the 1930s

In looking back, it seems to me that the genetic Zeitgeist was different from what it is now. There was an emphasis on generalities and an attempt to explain new phenomena in terms of these generalities, with a consequently diminished interest in exceptions. This contrasts with current research where discovery of new phenomena (introns, trinucleotide instabilities, imprinting) creates great excitement. There was also a curious preference for randomness. Finally, the subject then was less technique driven than it now is. A. A

S E A R C H FOR AND BELIEF IN GENERALITY

There was, it seems to me, great faith in the generality of the rules of genetics. T h e widespread occurrence of Mendelian inheritance, the general similarity of mitosis and meiosis in the various species studied, the linear order of the genes, and the way in which the behavior of rearranged chromosomes could be predicted from simple rules encouraged a belief in the generality of what had been discovered in a few favorite species. An example is the widely held belief, especially among Drosophilists of course, that the X-autosome balance theory of sex determination was the rule. Although it was known that the Y chromosome was the sex-

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determiner in Japanese silkworms, American geneticists persisted in expecting the human Y chromosome not to be functional. At least one human pedigree was explained by attached X chromosomes, following the Drosophila pattern. The postwar discovery that an XO mouse is female and an XXY mouse is male came as a great surprise, although perhaps not to Japanese geneticists. Despite the Bateson-Bridges admonition to “treasure your exceptions,” many exceptions were dismissed either as experimental errors or as being special cases and therefore not of general interest. The mutable genes in Drosophila uirilis and the variegated pericarp of maize were not regarded as of much general significance. Imprinting in mice might have been found early, but it wasn’t. It was assumed, because it was true in Drosophila, that the phenotype did not depend on the source of chromosomes; a diploid fly with entirely maternal chromosomes was fully normal and complementary aneuploid gametes produced a normal zygote (two wrongs make a right, as Muller said). It was not discovered that mice were different until much later. Anticipation-the finding that an inherited disease has an earlier onset in later generations-was explained as ascertainment bias. Cases are more likely to be detected early if severe, and simple regression explains the lesser severity in their parents. At least in Huntington’s disease, the phenomenon has turned out to be real, not statistical, and caused by unstable trinucleotide repeats. Among my associates in graduate school there was the general assumption that Pneumococcus transformation was some tricky form of selection rather than a change in the genetic material. I also remember a few years later not believing Lindegren’s evidence of gene conversion in yeast, because I could account for his results by assuming polyploidy. When we discussed this, he said that he knew of this possibility, but liked the conversion idea better; and he turned out to be right. There were some reports, and undoubtedly many other instances that were not reported, that would now be interpreted as crossing over within the gene. They were explained away as technical errors or mutations, since the gene was assumed to be atomistic, and not separable into functional components. Early attempts to induce mutations with chemicals were not regarded as convincing. The general belief was that the gene was “canned”; evolution had wisely arranged to sequester it from transitory chemical influences. This view led the genetics community to demand what now seems to be an excessively high standard of proof for chemical mutagenesis, a standard resembling the extremely rigorous standards demanded by those skeptical of genetic influences on human behavioral traits. Charlotte Auerbach’s discovery of the mutagenicity of

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nitrogen mustard-made during World War I1 but not revealed until later because of military secrecy-was immediately convincing because the effect was so enormous (and because she used the best Drosophila techniques). The “beads on a string” analogy prevailed. Mutation happened in the bead and function resided in the bead, but exchange was caused by breakage of the string between beads. Seymour Benzer’s dramatization of these three concepts in the words muton, cistron, and recon awaited the finer resolution possible in bacteriophage. FOR B. A PREFERENCE

THE

MENDELIAN PARADIGM

In a casual conversation in the 1950s, George Beadle mentioned what he regarded as a curious preference in the 1930s for randomness. Mendel’s laws are a perfect example. Deviations from Mendelian ratios were almost always attributedundoubtedly correctly most of the time-to viability differences or technical errors. But interesting exceptions such as preferential segregation in maize and transmission ratio distortion in mice were discovered only because some people “treasured exceptions.” Slight distortions probably were found and dismissed many times, and promising leads may have been missed. Mendel’s laws and their meiotic basis appealed to geneticists’ love of symmetry. Regular ratios based on Mendelian symmetry, with random deviations caused by finite samples and calculable by well-known methods, had an inviting simplicity. The cytoplasm, for example, had little attention. This preference showed up in the general acceptance of no chromatid interference in meiosis; that is, no relationship between the particular pair of chromatids involved in one exchange and those in another. T h e evidence for this in the 1930s, which was dependent largely on a complicated, indirect analysis of attached X chromosomes in Drosophila, was far from rigorous. My own memory agrees with Beadle in that the geneticists of the time preferred symmetry and randomness, which might have prevented or delayed the discovery of new phenomena. Another place where Mendelism and randomness played a large role was in population genetics. The Hardy- Weinberg principle was extremely useful for analyzing common human inherited traits, as I mentioned earlier. Randomness played a large part in Sewall Wright’s shifting balance theory of evolution and he regularly simplified by ignoring linkage disequilibrium. Random changes are easy to deal with statis-

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tically, and since so much of early genetics depended on ratios, it is natural that statistical theories became an important part. Was this preference for symmetry and randomness good or bad? 1 think both. Clearly, the simple assumption aided in working out the general principles. Population genetics flourished by making simple assumptions. Part of the wisdom of the three pioneers lay in their ability to simplify while retaining the essence. Yet, I also suspect that some phenomena that have been discovered since World War I1 might have been found earlier (imprinting, for example), had geneticists been more willing to follow up on exceptions. Kuhn may be right; the Mendelian paradigm held out until the deeper insights based on DNA chemistry took over. There is an interesting contrast between American and German genetics in the 1920s and early 1930s. Harwood (1993) has called attention to the greater emphasis on development in German genetics during the time that the dominant influence in the United States was on the rules of transmission. Sewall Wright, one of the minority who was continuously interested in development, tells of his pleasure in conversing with GoldSchmidt during World War I. Yet Goldschmidt bet on too many wrong horses to have any status, at least in the Drosophila community. Whether a greater emphasis on development might have paid off will have to remain uncertain, for molecular advances came from a different direction. C. THEIMPORTANCEOF

TECHNIQUES

Present-day genetics is strongly driven by technical advances. In contrast the genetics of the 1930s had three basic techniques: breeding, microscopy, and examination of (usually superficial) phenotypes. The advances were made by clever experimentation, not by new techniques. Although electron microscopy existed at the time, it contributed relatively little to genetic research until later. Many experiments, especially in Drosophila and maize, were masterpieces of inventive use of limited techniques. Mutant genes were used to explore developmental problems, and the genetic control over some chemical processes was also studied. In general, however, genetic research was limited to breeding experiments, with occasional help from the microscope. T h e discovery of new ways of analyzing DNA and RNA, ever more rapid sequencing methods, ways of getting out cDNAs, and site-specific mutation mean that the questions asked are largely determined by the availability of a new powerful technique. A Rip van Winkle from 1936 would be astonished not only to find that the gene is DNA, not protein,

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but that such things as PCR and sequencing mean that, at least in principle, a gene can be isolated and treated as a chemical. He would probably remember Muller’s hope that one day we would be able to study genes in the test tube; but he would also be utterly astounded by how rapidly this had happened. Research in the 1930s was less technique driven and more idea driven than now; it had to be, techniques were so limited. One who cut his genetic teeth in the 1930s, a time when designing an experiment that could give an unequivocal answer to an important question required great ingenuity, can only stand amazed at the precision and clean outcomes of current molecular experimentation. Yet the thought intrudes that too often an observation is made, a sequence determination perhaps, simply because “it’s there” and a beautiful experimental technique is waiting to be used. IV. Conclusion

By 1936 the major problems of transmission genetics had been solved. The rules of inheritance, the chromosomal basis of heredity, the linear order of genes on the chromosome, and the phenomenology of mutation were well understood. Cytogenetics was a highly developed art, especially in Drosophila thanks to salivary chromosomes, and also in maize and other plants. Likewise, population genetics was highly developed, thanks to the pioneering work of Fisher, Haldane, and Wright. One can admire the ingenuity of experiments that produced remarkably deep insights with the very primitive techniques available. Knowledge of human genetics was weak in the 1930s. Although there were highly inventive methods for obtaining information that would be trivially easy in experimental organisms, the subject lagged far behind Drosophila and maize. A human chromosome map was nonexistent. T h e gene was completely mysterious and was thought to be protein. The general hope was that the study of allelism and mutation would provide insights, but this did not happen. There were conjectural gene models, but none came close to what the structure ultimately turned o u t to be. It seems to me that research in this period was characterized by a search for, and a belief in, generality with a consequent tendency to ignore exceptions. There was also a preference for symmetry and randomness-the Mendelian paradigm-with an attempt to fit all observations into this framework. In summary, although the 1930s represented a high point in trans-

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mission genetics, a deeper understanding of the gene had to wait for microbial genetics and molecular techniques. REFERENCES

I have not given specific references to the work mentioned, but instead have listed general references in which original sources can be found and which reflect the knowledge and spirit of the time. Carlson, E. A. (1966). “The Gene: A Critical History.” Saunders, Philadelphia. Cook, R. C. (1932). J . Hered. 23, 355-369. Crow, J. F. (1992a). FASEB 6,2867-2869. Crow, J. F. (1992b). Gtnttics 131, 761-768. Dobzhansky, T h . (1937). “Genetics and the Origin of Species.” Columbia Univ. Press, New York. Harwood, J . (1993). “Styles of Scientific Thought: T h e German Genetics Community, 1900-1933.” Univ. of Chicago Press, Chicago. Jones, D. F., ed. (1932). “Proceedings of the Sixth International Congress of Genetics. Vol. 1. Transactions and General Addresses. Vol. 2. Condensed Articles and Descriptions of Exhibits.” Brooklyn Botanic Garden, Brooklyn, New York. Punnett, R. C., ed. (194 1). “Proceedings of the Seventh International Genetical Congress.” Cambridge Univ. Press, Cambridge. Stern, C. (1949). “Principles of Human Genetics.” Freeman, San Francisco. Sturtevant, A. H. (1965). “A History of Genetics.” Harper & Row, New York. Sturtevant, A. H., and Beadle, G . W. (1939). “An Introduction to Genetics.” Sdunders, Philadelphia.

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HISTORICAL ORIGINS OF CURRENT CONCEPTS OF CARCINOGENESIS P. D. Lawley Section of Molecular Carcinogenesis, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG, United Kingdom

I. Introduction A. General Considerations: Proliferation and Mutation B. Carcinogenesis from Humoral to Cellular 11. Exogenous Sources of Cancer A. Radiation and Soot B. Other Industrial Carcinogens of the 19th Century-Shale Oil and Aromatic Amines C. Tobacco Carcinogenesis 111. Mutation and Cancer A. How Many Mutations Are Involved in the Carcinogenic Process? B. Viral Carcinogenesis, Oncogenes, and Immunosurveillance IV. Chemical Carcinogenesis: Early History A. General Introduction B. The First Pure Chemical Carcinogens Detected through Their Fluorescence Spectra C. Benzo[a]pyrene-An Archetypal Environmental Carcinogen V. Mode of Action of Carcinogenic Polycyclic Aromatic HydrocarbonsEarly Historical Development A. Their Suggested Relationship to the Sterols B. Carcinogenic Hydrocarbons and Two-Stage Carcinogenesis VI. The Scope of Chemical Carcinogenesis Broadens-DNA as an in Viuo Receptor A. Carcinogens as Growth Inhibitors and Radiomimetic Compounds B. Chemical Methylation of DNA and Its Significance for Radiomimetic Action of Alkylating Agents C. Reaction of Mustards with DNA: In Viuo Alkylation and Its Repair D. Polycyclic Aromatic Hydrocarbons: DNA as in Viuo Target E. I n Viuo Chemical Modifications of DNA: Aromatic Amines and Nitrosamines F. “Ultimate” Carcinogens: Metabolic Generation of Electrophiles G. Specific Chemical Modifications of DNA Are Required to Initiate Cancer H. “Ultimate” Carcinogens: Their Structural Requirements Interpreted through Physical Organic Chemistry I. Metabolic Activation of Carcinogens and Pharmacogenetics VII. The Advent of Mutational Spectra: Attempts to Correlate CarcinogenDNA Reactions and Carcinogenic Genetic Changes at the Molecular Level A. Alkylating Agents B. Polycyclic Aromatic Hydrocarbons 17 ADVANCES IN CANCER RESEARCH, VOL. 65

Copyright 6 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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C. Aflatoxin B , D. Overall Evaluation of Mutational Spectra VIII. Summary and Conclusions References

I. Introduction

A. GENERAL CONSIDERATIONS: PROLIFERATION AND MUTATION Two main themes have run through the development of our ideas of what causes cancer, considered at a fundamental level. The most immediately obvious is that o f growth that has ceased to respond to normal controls; in general terms this suggests an analogy between carcinogenesis and the most profound and enduring problem of biologyevolution. One of the earliest 19th-century writers on the etiology of cancer, preceding Darwin, referred to cancer as “playing the part of a parasite of an organic species, whose object is to substitute itself for others. . . . species (are) destined to maintain a perpetual struggle, (and) have no other object but to destroy each other, each in its turn being destroyed by incessant substitutions” (Velpeau, 1854). This remarkable statement clearly echoes one of the most influential concerns of its period (once again returning to haunt us), Malthusianism, that uncontrolled increase in population must outstrip the means of its sustenance. Malthus also pointed out that, under normal conditions, many families died out from chance infertility. We are not surprised to find a citation to Malthus’ “Essays on the Principle of Population” of 1798 in a relatively recent text on the growth kinetics of tumors (Steel, 1977). The cellular progeny o f a dividing stem cell may be two proliferating daughters, no proliferating daughters, or one of each; according to the relative proportions of these, the cellular family may die out o r avoid extinction (as Malthus found for human families). The central problem in carcinogenesis remains how the balance between cell proliferation and cell death becomes disrupted in favor of proliferation. For recent reviews on the role of cell death, including apoptosis, in tumorigenesis, see Thompson et al., 1992; Wyllie, 1993. It is a truism of the history of biology that Darwin was profoundly influenced by Malthus, and furthermore that his interpretation (Darwin, 1859) of the origin of species, after a brief period of contentiousness, became widely accepted among the scientific community. But toward the close of the 19th century Darwinism tended to languish under the realization that no plausible mechanism of inheritance was available through which evolution could be envisaged to have occurred.

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T h e received wisdom is now, of course, that Darwinism was “saved by its fusion with Mendelism” (Jones, 1993). The famous report by Mendel (1866) on plant hybridization was apparently ignored for over 30 years [see, e.g., Correns (1900)l and it was then realized that had Darwin read this publication, he would have been able to provide a firmer foundation for his ‘‘Origin of Species.” T h e acceptance of Mendelian inheritance was, of course, important because it provided a mechanism for the survival through subsequent generations of a mutated gene, whereas the concepts available to Darwin, of inheritance through particles passing from organs through the blood to sperm and ova, would predict rapid loss of a mutated characteristic through dilution. No immediate solution was available to the question of the physicochemical nature of the gene. This had to wait for another half-century, until the advent of the Watson-Crick model for DNA replication (Watson and Crick, 1953a,b). Remarkably, two years after this was announced, a fascinating and otherwise thorough review of the “significance of mutation in relation to the origin of tumors” by Burdette (1955) did not include the names of Watson and Crick in the 32 1 references cited, and the gene was “tentatively accepted as corpuscular.” Burdette’s review is important because it reflected accurately the uncertainties at that time surrounding the need of oncologists for “a common etiology for neoplastic diseases.” He inclined to skepticism with regard to the somatic mutation hypothesis and noted that, “with surprising frequency, two investigators cannot reach agreement because they do not define mutation in the same way.” T h e problem of how to define mutations that are significant in biologic processes has continued to excite interest in both the broad fields of evolution and carcinogenesis. It is no mere coincidence that the currently predominant technique for investigating the molecular structure of genes, the celebrated polymerase chain reaction (Saiki et al., 1985), is now being applied to “archival” DNA in both these respective contexts, as represented by 7500-year-old skeletal material (mitochondrial, Paabo et al., 1988; genomic, Lawlor et al., 1991), or, perhaps more mundanely at least as regards age of sample, to genomic DNA from par1988; reviewed by Eeles affin blocks of tumor material (Shibata et d., et al., 1993). T h e principal difference that has emerged between these two areas of study is that now we are much more certain about the nature of mutations involved in carcinogenesis than of those mutations in the evolutionary process. Lewontin (1974) commented that “we know virtually nothing about the genetic changes that occur in species formation.” This

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can be ascribed presumably to their rarity, whereas in the supposed parallelism between oncogeny and phylogeny, in so far as, in Huxley’s words, autonomous growths are “equivalent to a new biological species” (Huxley, 1956; Hauschka, 1961), the mutations involved turn out to be in many instances of the types commonly encountered in both spontaneous and experimentally induced mutagenesis. Broadly, mutations have been classified as “alterations in DNA sequence and alterations in DNA topology” (Rainey and Moxon, 1993) and their relative importance in evolution is a matter of continuing debate (e.g., Lenski and Mittler, 1993). The earliest protagonists of what became termed the “somatic mutation” theory of carcinogenesis were confined to observations of chromosomal abnormalities (i.e., the second of these categories), which they established in principle as involved in the etiology of cancer before the concept of mutation itself was in fact recognized. Historians of carcinogenesis have often cited Theodor Boveri as the originator of the somatic mutation hypothesis, by virtue of his publication “Zur Frage der Entstehung malagne Tumoren” (Boveri, 1914). One relatively recent (and critical) reviewer of evidence relevant to the somatic mutation concept (Nery, 1986) concluded that “available data on chromosome anomalies in human neoplasia give little o r no support to the basic tenet of Boveri’s hypothesis that such anomalies initiate the tumorigenic process,” although the reviewer was prepared to concede that “certain selected anomalies may play some role in one or more of the late stages of development of some tumours.” (For a recent review, see Cheng and Loeb, 1993.) Nery (1986) also pointed out that Boveri himself had never examined tumors cytogenetically, having studied mitosis mainly in sea urchin eggs and in Ascaris. The evidence relating to cancer cells had been documented by previous investigators, notably Arnold (1879a,b) and Von Hansemann (1890). The latter became well known as the proponent of the importance of “asymmetrical karyokinesis,” with unequal distribution of chromosomes in daughter cells, as the cause of carcinomas, but as Nery (1986) notes, he later retracted his theory in the light of evidence that the phenomenon was also found in noncancerous regenerating tissues. Boveri (19 14) modified Von Hansemann’s views by attributing cancer to an “abnormal chromatin-complex, no matter how it arises. Every process which brings about this chromatin condition would lead to a malignant tumour” (see Burdette, 1955). This statement appears at first to presage modern knowledge of carcinogens as mutagens acting

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through their various mechanisms. However, somewhat paradoxically, Burdette (1955) goes on to stress that “no mention is made of point mutation in Boveri’s monograph,” thus apparently eliminating him as an early exponent of the now well-accepted role of many highly potent chemical carcinogens as inducers of “point” mutations through base substitutions, following the type of mechanism specified by Watson and Crick ( 1953b). It may be interpolated here, again jumping ahead to contemporary preoccupations, that aneuploidy has been implicated as the essential cause of the first stage of cellular transformation (often termed in vitro carcinogenesis) (Tsutsui et al., 1983). This constitutes a premutational stage in carcinogenesis [although there is uncertainty whether it is obligatory in experimental induction of cancer in the whole animal, as for example in the classical “mouse skin system” (Balmain and Brown, 1988)l. Although aneuploidogens are regarded as genotoxic, their effectiveness as inducers of immortalization correlates positively with their cytotoxic potency, rather than with their mutagenic efficiency (i.e., in contrast to their effectiveness as carcinogens in classical test systems). In a sense, therefore, the concept of immortalization re-echoes the views of Von Hansemann by invoking the significance of induction of aneuploidy at possibly the earliest stage in the carcinogenic process (Oshimura and Barrett, 1986; for recent reviews, see Boukamp et al., 1993; Shay et al., 1993). FROM HUMORAL TO CELLULAR B. CARCINOGENESIS

T h e view that cancer originates in single cells and develops through proliferation of their progeny now seems generally accepted, but the essentially humoralist concept that it is a disease of the whole person, continues to survive in parallel to the cellular concept. T h e historical precedence of humoralism is a well-documented theme in oncology, having been traced back to Hippocrates and Galen (see Shimkin, 1977). Smithers (1962) preferred the term “biocybernetics,” to include cancer as a “disease of organisation.” In this “attack on cytologism,” his supporting evidence included the “observed multicentric origin of neoplasia” and spontaneous regressions of cancers unexplained by conventional theory (see also Boyd, 1977; Challis and Stam, 1990). In the secondcentury AD, Galen expressed his conviction that “black bile” was associated with melancholic characteristics and also with a predisposition in women to breast cancer. Psychosomatic factors are, of course, still invoked as significant in the etiology of cancer and have been subject to

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sporadic review. For example, Le Shan (1959) traced in detail its historical survival and presented evidence that “severe emotional trauma contributed markedly to the onset and development of cancer,” as supported by epidemiologic data. Occasional experimental studies have also linked stress with enhanced carcinogenesis, as for mammary tumors of C3H mice (Riley, 1975). Subsequently, Riley and others (1979) reviewed considerable evidence showing associations between cancer and stress in both experimental and epidemiologic studies. The male hormonally associated counterpart, prostate cancer, has also been linked to psychosocia1 factors. For example, the review of prostate cancer etiology by Fergusson (1967), quotes Trunnell (1956) to the effect that “almost without exception those who get the disease are men with a constricted outlook on life, who are tractable in the extreme and whose wives are somewhat domineering.” He went on to note that “an accurate assessment of these traits is probably beyond the capacity of the cancerologist and in any case, prophylaxis is unlikely to be easy.” Nevertheless, hormonal factors (which presumably would be implicated among others in humoralist theory) continue to be recognized as important, if not predominant, causative factors in carcinogenesis; but they continue to defy detailed biochemical analysis in most instances. Expressing the frustrations of breast cancer etiologists, Fentiman ( 1990) found that “after study of the descriptive epidemiological literature, it is hard to escape the conclusion that much of this has been reinvention of the wheel, namely, confirming that the human breast is an endocrinesensitive organ.” He estimated that three-quarters of breast cancer cases have no identifiable cause; this might well be considered an optimistic estimate. Although estrogens have been invoked as promoters of breast cancer (Thomas, 1984), they are assessed as less effective in this respect than for endometrial cancer. Their role is still under discussion (Pike et al., 1993). “Modern” cancer research largely abandoned the humoralistic approach except that it has been revived by recognition of hormones as carcinogens, and by the concept of immunosurveillance, and the main emphasis shifted to cellular aspects. The acknowledged doyen of the cellular pathology of cancer is Johannes Muller, the centenary of whose publication Ueber den feinern Bau und die Formen der Krankhaften Geschwiilste was celebrated in an informative review by Haggard and Smith ( 1938). Muller’s students included Theodor Schwann, the first microscopist to discover the cellularity of animal tissues, as previously found for plants, and Rudolph Virchow, a dominant pioneer of classical cellular pathology, who was one of the first to stress the need for a comprehensive theory of carcinogenesis. [For a full account of the 19th-century

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foundations of cancer research, see Triolo (1964, 1965)l. Virchow (1858) regarded the dominant factor as “chronic irritation,” and this theme has been re-echoed occasionally in experimental studies, as for example by Deelman (1924, 1927), who essentially showed that in mouse skin, wounding was what later became termed a promotional factor (for a recent discussion, see Fiirstenberger et al., 1993). Experimental carcinogenesis, however, took on a different and highly significant direction from the work of Virchow’s student Katsusaburo Yamagiwa, who returned to Japan to conduct his celebrated work on the experimental induction of skin cancer (by continuous painting of benzene solutions of tar onto the ears of rabbits) (Yamagiwa and Ichikawa, 1915). This, of course, provided the basis of what has now become almost universally accepted as the basis of the comprehensive theory of carcinogenesis that the 19th century founders of cellular pathology sought, and which is essentially the subject of this brief historical review.

I t . Exogenous Sources of Cancer A. RADIATION A N D SOOT T h e humoralists regarded cancer as essentially originating from certain characteristics associated with susceptibility of the individual who contracted the disease. Early 19th century cancer theorists [for example, Velpeau (1854) on breast cancer already cited] recognized heredity as a possible predisposing factor (this author was not, however, impressed with the currently popular theory of “blows to the breast,” perhaps a variant of Virchow’s subsequent emphasis on physical trauma, as causative). Along with the emergent predominance of the cellular theory, a variety of exogenous causative factors became apparent, associated mainly with the use of coal as industrial fuel and of oils as a lubricants, together with the development of the chemical industry in general. Some occupational causes of cancer had been documented before the period we term the industrial revolution, the first generally acknowledged to be among miners for silver and other metals (including uranium) in the Black Forest, as described by Paracelsus in 1531 as “mala metallorum.” This was later interpreted as radiation-induced lung cancer, as a result of epidemiologic studies of American uranium miners (see, e.g., Altshuler et al., 1964; Wagoner et al., 1965). T h e introduction of X-rays by Roentgen in 1895 was followed quite rapidly by striking demonstrations of the carcinogenic potency of ionizing radiation both in man (squamous cell carcinoma of the hand; Frieben, 1902) and experimentally (sarcoma in the rat; Clunet, 1910).As

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pointed out by Mustacchi and Shimkin (1956), the latter was “the first malignant neoplasm to have ever been deliberately induced in an experimental animal.” As w e shall see later, if radiation-induced carcinogenesis had been established from then on as an academic study, it might well have pre-empted the dominance of chemical carcinogenesis as the foundation for a comprehensive theory of carcinogenesis. This was because X-rays became in turn the first established exogenous cause of mutation, with H. J . Muller’s induction of recessive lethal mutations in Drosophilu melunoguster (Muller, 1927, 1928). However, nearly a quarter of a century elapsed before he turned his attention to a consideration of the mechanisms of the carcinogenic process (Muller, 1951). In the meantime, the emphasis had shifted from radiation to chemicals as the most informative agents in experimental carcinogenesis. Chemical sources causing cancer in the environment became largely obvious as a result of the industrial revolution, initially in Britain and then in Germany. T h e widely acknowledged father of modern epidemiology is Percivall Pott (1775) who attributed skin cancers (mainly of the scrotum) to prolonged exposure to soot in chimney sweeps. A comprehensive appreciation of this outstanding historical figure was given by Potter (1963). He notes that Pott’s publications soon led other observers to attribute cancer of various sites to soot, and as with Velpeau’s (later) opinions on the etiology of breast cancer, Pott’s grandson Henry Earle suggested that “a constitutional predisposition is required which renders the individual susceptible to the action of the soot” (Earle, 1823); he may thus be the first to propose the “genetic-environmental paradigm” of carcinogenesis. He further noted that exposure to soot could take over 20 years to manifest as cancer. In addition, Curling (1856) noted a case in which after early exposure, 20 years of lack of contact with soot had occurred; the existence of latent periods in chemical carcinogenesis was thus acknowledged. T h e present-day epidemiologist will not perhaps be surprised at the lack of effective impact of Pott’s work on British public health practice during the succeeding century. Butlin (1892) in his celebrated “three lectures on cancer of the scrotum in chimney-sweeps and others” reported that the prevalence in Britain showed no sign of declining, whereas the disease was almost unknown in other countries which had by then become industrialized with dependence on coal, such as Germany and the United States. He attributed this to the low standards of hygiene in Britain and to the practice of exposing young “climbing boys” to soot. His descriptions of the developmental stages of the soot-induced cancers, from precancerous warts to metastasis, parallel those subsequently observed in experimental skin carcinogenesis. His comparative data con-

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trasting Britain and other countries showed clearly that the spontaneous incidence of scrota1 cancer was comparatively low. He speculated on the chemical basis for the carcinogenicity of soot, and although noting that, in a tar factory, “liquid anthracene, the last product of the distillation of gas-coal tar” appeared to be the most active fraction, he was unable to specify further what classes of compound were most likely to be involved. B. OTHERINDUSTRIAL CARCINOGENS OF THE 1 9 ~ CENTURY-SHALE OIL A N D AROMATICAMINES

~

T h e majority of the 19th century theorists of carcinogenesis were more concerned with cellular and biologic aspects, and did not appear to realize the significance of various industrial causes of cancer. Among these, “paraffin cancer” of workers in the shale-oil industry and “mulespinners’ cancer” among users of these oils as lubricants, were skin cancers showing characteristics similar to those of chimney-sweeps’ cancer, sometimes involving skin of the arms and face as well as the scrotum. Two geographic locations in Germany and Scotland were involved as sources of shale oil, which was independently identified as carcinogenic by Volkman (1875) (referring to oil distilled from brown coal at Halle) and by Bell (1876) (referring to shale oil distillation at Broxburn), respectively. The latter report became of particular importance with regard to subsequent development of theories of carcinogenesis, through the later, more detailed report by Scott (1922). This encouraged Archibald Leitch (1922) to pursue experimental studies as Director of the Institute of Cancer Research, London; Leitch was succeeded by Ernest Kennaway, who, in turn, was succeeded by Alexander Haddow, himself a native of Broxburn. These successive directorships ensured a period of highly significant contributions to studies of carcinogenesis at this Institute (see Brunning and Dukes, 1965), as discussed in more detail later. Another important theme was initiated by a report on incidence of bladder cancer in the German dyestuff industry by Rehn (1895), a Frankfurt surgeon, who found four cases in a group of 45 men involved for 20 years in the manufacture of the dye rosaniline. At first, he referred to “aniline cancer,” because this aromatic amine was a frequent intermediate in dye chemistry, but Leichtenstern (1898) suggested naphthylamines as likelier causative agents. Subsequently, 2-naphthylamine, benzidine, and 4-aminodiphenyl were definitely implicated as human bladder carcinogens (see Case et al., 1954; Parkes and Evans, 1984), thus presaging extensive studies on the mode of action of aromatic amines.

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The importance of this class of chemical carcinogen lies in its systemic action as opposed to the locally acting soot and oils. C. TOBACCO CARCINOGENESIS

Historians delight in quoting early monarchial anathema to the bCte noire of most cancer researchers, expressed in the “Counterblast to ‘Ib-

bacco” of James I in 1604. Although the king instigated the public exhibition of snioke-blackened viscera, as indicative of a general health hazard, at a meeting in Oxford (see Nery, 1986), the “first clinical report” linking tobacco and cancer is generally regarded as that by Hill (1761) referring to snuff (see Redmond, 1970). Six cases of “snuff users’ polypus,” one of which developed into an “open cancer” were reported. Soemmering (1795) was the first to implicate pipe smoking as carcinogenic, finding that “the lower lip is particularly affected by cancer when it is compressed between the tobacco pipe and the teeth” (see Shimkin, 1977). Nineteenth-century etiologists of cancer were well aware of carcinogenesis as a concomitant of injurious effects of tobacco, as evidenced by an Editorial in the British Medical Journal of 1890 (Anonymous, 1890). Thus, Thiersch (1865) implicated tobacco smoke and juices as labial carcinogens comparable with soot as a scrota1 carcinogen. Tillmanns (1880) commented on the similarity of smoke tar distillates and industrial products such as creosote, mentioning in particular the bicyclic aromatic hydrocarbon, naphthalene. The first report on experimental tobacco carcinogenesis is that of induction of skin cancer in the rabbit by painting tobacco-derived tar (Roffo, 1931). This was confirmed by Graham et al. (1957), who considered tobacco tar to be a “weak carcinogen.” T h e modern phase of tobacco research could well have started with the unequivocal demonstration of the association between cigarette smoking and lung cancer reported by F. H. Miiller (1939). The Second World War intervened, and, no doubt, as in the First World War, the cigarette was regarded as essential for maintaining morale; the majority of males in the western world became cigarette devotees in the interwar years as did many women. Eventually it became obvious that this caused the almost explosive increase in lung cancer revealed by several subsequent epidemiologic studies, most notably by Doll and Hill (1950), and Wynder and Graham (1950) [see IARC (1986)l. Although 19th century theorists included smoking as probably irritant in action following the views of Virchow (1858), interest later switched to the nature of the carcinogenic chemicals involved, with important consequences, as discussed in more detail later. Epidemiologic

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analyses of lung cancer induced by cigarette smoking have led to fundamental reappraisals of the conceptual basis of theories of carcinogenesis. Broadly speaking, these are reminiscent of what followed from the earlier work of Pott and Earle: disappointment that a major cause of cancer shows no sign of its being eliminated, and a renewed interest in the question of individual susceptibility to the action of carcinogens. R. Pet0 (1984) pointed out that the discovery of what is now known to be the most prominent cause of death from cancer (“about one-third”) “did not depend on any serious understanding of the mechanism(s) of carcinogenesis, nor on reliable identification of the importantly causative component(s) of tobacco smoke” (although considerable progress has been made on the latter topic since he wrote). He therefore commended the strategy of “black-box” epidemiology in order to detect other major carcinogens, noting that, for example, cancer of the large intestine is the most common type of fatal cancer next to lung cancer among U.S. blacks, but “only about one-tenth as common among blacks in West Africa from where most of the U.S. blacks originated.” Therefore, appropriate epidemiologic studies were expected to reveal the carcinogen(s) responsible, which were indicated to be most likely dietary (Doll and Peto, 1981). Unfortunately, so far, this hope has not been realized (for more recent discussions, see Vandenbroucke, 1988; Vineis and Brandt-Rauf, 1993). Humans as a species appear to be (perhaps uniquely) susceptible to the carcinogenic action of cigarette smoke. Although Roffo (1931) and others later showed tobacco tar to be a relatively weak skin carcinogen, induction of lung cancer in animals by tobacco smoke proved even more difficult to demonstrate. Dontenwill et al., (1973) could not induce lung cancer in hamsters, and, although Dalbey et al., (1980) obtained positive results in F344 rats, the amounts of smoke involved were much higher on a comparative basis than those ingested by even heavy human smokers. Another outstanding causative agent of human lung cancer, mustard gas (Wada et al., 1968), had also been difficult to demonstrate as a carcinogen in animal tests (Heston, 1950). This evident lack of carcinogenic potency of cigarette smoke in conventional tests, coupled with the occurrence of spontaneous bronchial carcinoma, has prompted interest in whether individuals succumb to smoking-induced cancer because of inherited defects in their ability to metabolize the carcinogens involved. This question remains controversial, especially since it implies the, largely unspoken, possibility that genetic tests could identify those not at risk from smoking, which one imagines would be not unwelcome to the tobacco industry. It has also led to some divergence of opinion between the “black-box” epidemiologists and pharmacogeneticists. Idle ( 199 l), a

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pioneer of the pharmacogenetic approach, quoted Darwin ( 1859) that “no-one supposes that all the individuals of the same species are cast in the same actual mould. These individual differences are of the highest importance to us for they are often inherited,” and thought it axiomatic that genetic constitution would influence susceptibility to carcinogens (for a recent review, see Shields, 1993). Epidemiologic studies generally found it difficult to detect such individual susceptibilities (Peto, 1980; Easton and Peto, 1990). But, in a comprehensive review of epidemiologic data, relating cancer incidence and age, Stein (1991) deduced the concept of a susceptibility factor (p) for “the risk that a member of the population will fall victim to the cancer in question during her or his lifetime,” a value of 100% being interpreted to mean that there is no subsection of the population that is especially at risk. For cancer of the bronchus, Stein’s factor p was 13% for males and 1.2% for females, but he did not comment on whether these limited proportions of the population at risk could be attributed to pharmacogenetic factors. With respect to cigarette smoking, Stein (1991) used data from the classical British doctors study (Doll and Peto, 1978) to deduce that the dependence of cancer risk on the number of cigarettes smoked per day was of the quadratic form, i.e., proportional to the square of the dose of carcinogen, and the most likely interpretation was that this reflected the increase in rate of two somatic mutations involved in lung carcinogenesis. This agrees with the analysis of these data by Moolgavkar et ul. (1989) that there is little or no effect of cigarette smoke on what the experimentalist would term tumor promotion, i.e., increasing the rate of growth of transformed (initiated) clones, but contrasts with the earlier analysis of the data by Moolgavkar and Knudson (198 1). Ill. Mutation and Cancer

A. How MANYMUTATIONSAREINVOLVED IN THE CARCINOGENIC PROCESS? As previously noted, the authors credited with originating the somatic mutation theory of carcinogenesis had to rely on microscopic observations of chromosomal abnormalities as their experimental evidence, and, despite the biologic definition of the term “mutation” in 1900 following the rediscovery of Mendel’s work, no molecular mechanisms were available. T h e period that followed was characterized by considerable progress in the identification of specific chemical carcinogens, which, paradoxically, failed to offer support to the somatic mutation hypothesis.

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T h e simple fact was that no chemical mutagens were regarded as established until the Second World War, and because the first chemical mutagen to be acknowledged was an agent of chemical warfare, mustard gas, even this was subject to delay in publication (see Auerbach and Robson, 1947a,b; for fuller accounts of their work first published as Auerbach and Robson, 1946). One of the reasons for investigating mustard gas was its vesicant action, which resembled that of X-rays (Auerbach and Robson, 1947a), the latter being the only known mutagen for the conventional test species used at that time, Drosophila melanogaster (Muller, 1927). In view of the intensive interest and progress in isolation and characterization of chemical carcinogens that began around the 1920s, radiation became somewhat neglected as an experimental object of study. Nevertheless, reports subsequently appeared showing that the mouse ovary was susceptible to carcinogenesis by X-rays (Furth, 1949; Lorenz et al., 1947) at what Muller (195 1) regarded as impressively low doses (down to 25r). This prompted Muller (1951) to write two paragraphs which can still stand today as expressing the fundamental characterization of the carcinogenic process. There are, however, reasons for inferring that many or most cancerous growths would require a series of mutations in order for the cells to depart sufficiently from the normal. This, if true, would cause the frequency of such growths to depend upon some exponent of the dose higher than one, rather than upon the dose itself in the simple proportionate way characteristic of individual gene mutations. In that case, too, the time element would constitute an influential factor unlike what is found to be the case in ordinary mutation production; for cells in which one step had occurred might because of it have proliferated sufficiently, by the time of a later treatment, to give better opportunity for another step to occur on top of the first. Of course, this mechanism would not at all exclude the influence of predisposing conditions outside the genes, which allowed the mutated cells to respond in the given way. Thus the mutant cells might have to wait many years after the exposure to radiation before the effects of the induced mutations became visibly expressed. In this connection we may recall the fact that cancers induced by overexposure of a part of the body to radiation often fail to show up until some 10 to 20 years after the cessation of the irradiation.

Although this embodies the salient characteristics of the time course of carcinogenesis, it remains remarkable, and perhaps disappointing, that Muller apparently never published a mathematical expression of his views. However, shortly afterwards, such an expression, relating cancer incidence (i.e., the number of cancer deaths appearing per year) (I,)and age at death ( t ) was put forward by Nordling (1952, 1953). He introduced the now well-known method of plotting log I , versus log t, and obtained approximately straight lines that have become referred to as “log-log’’ plots.

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Nordling’s interpretation was that the slope of these lines (m)indicated that (m + 1 ) mutations were required to cause cancer (from the overall data then available he found that m = 6). In remarkable prescience of later modifications of this type of theory, Nordling (1953) noted that the slope of his log-log curve was much reduced for the incidence of “cancer in the specifically female organs” above the age of 45 and therefore that “altered hormonal conditions in connection with the menopause might play a part in determining the actual cancer frequency among women.” He also quoted Bauer (1949) as the originator of the somatic mutation concept of carcinogenesis (evidently discounting the claims of such prior contenders as Boveri), arid noted that Bauer had stressed that, by then, the well-established causations of cancer by exogenous agents had shown the existence of latent periods of “9 years for x-rays, 12 years for paraffin, 18 years for aniline, and 40 years €or sunlight.” Nordling therefore specified the main features of the relationship between cancer incidence and age, but did not deal satisfactorily with the question of cellular proliferation, although he showed his awareness of this factor. Platt (1955) in a short letter to “The Lancet” entitled “Clonal ageing and cancer” interpreted the latent period as necessary for clonal expansion of initiated cells in order to provide sufficient numbers for subsequent mutation(s) to become likely. Others began to challenge the requirement for as many as six or seven successive mutations on the grounds that the observed frequency of mutations in mammalian cells occurring in the large, but finite, number of cell divisions occurring during a lifetime would not in fact suffice to produce enough cancer mutations. For example, Stein (1991) quotes consensus values of rates of mutation per gene per cell generation of the order of 10-5 to lo-‘;, and number of cell generations per human lifetime of 1016. From these values, even a 5-hit model would predict a negligible cancer incidence of less than per lifetime. Some authors have been prepared to accept much higher mutation frequencies in order to accommodate the multihit requirement; Burch ( 1 976) referred to mutation frequencies of the order of low3per cell per generation at the H-2 locus in mice, and noted that this would be adequate to account for cancer on the multihit model; Strauss (1992) reviewed literature reporting data consistent with mutation frequencies claimed to be highe r than those conventionally accepted; Morris ( 1989), while accepting the relatively low mutation frequencies per cell division of round 1 0 - 7 for the classical interpretation of incidence of retinoblastoma (see discussion later), derived a value of‘ 10-6 for a four-mutation model of acute lymphoblastic leukemia.

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R. Pet0 (1984) offered a somewhat different interpretation of the agedependence of cancer. He stressed the requirement for mechanisms of protection against mutations which render human epithelial cells “a million or a billion times more cancer-proof than are the epithelial cells of rodents.” Without these, he states that “any individuals who could not keep Darwinian evolution in check among the cells of their own body would die of cancer long before they had children.” Whereas the “late stages” of carcinogenesis according to theories of the type promulgated by Nordling would be further mutations, Peto differentiated them from the “early stages,” quoting Cairns (1975, 1981) to the effect that the early stages of cancer are “just trivial molecular biology,” and that the “late” stages were quantitatively more important as determinants of cancer incidence. These various interpretations have been synthesized into what now appears, judging by literature citations, to be a well-accepted expression of data on cancer incidence versus age. Following Platt (1955) it was realized that if allowance was made for a requirement that clonal expansion of an intermediate cell type resulting from mutation of‘ a normal stem cell should take place to enable sufficient mutant cells to give a reasonable chance that a second mutation could occur, even at the low rates of mutation generally accepted, then the log-log type of relationship could result. This concept thus postulates only two, rather than the six or seven, successive mutations envisaged by Nordling (see Armitage and Doll, 1957; Moolgavkar and Venzon, 1979; Moolgavkar and Knudson, 1981). As a “minimalist scheme,” this concept also fits the consensus of a large body of evidence from experimental carcinogenesis. Nordling (1953) did not take such evidence into consideration, although the foundations of “two-stage carcinogenesis” had already been laid (see discussion later) through the work of Berenblum (1941a,b). In addition, Friedewald and Rous (1944) had introduced the terms “initiation” and “promotion” to denote two distinct processes shown to be essential for “complete” carcinogenesis. However, as will be discussed in more detail later, the connection between these major advances in chemical carcinogenesis and the somatic mutation theory was far from clear at that time; as already noted, Burdette (1955) in fact reviewed available evidence as contraindicative of such a positive connection. In his major classical review of neoplastic development, Foulds (1969) was unenthusiastic, but not dismissive, of the role of mutagenesis. In his overall scheme, “precancerous lesions, group B” are a critical feature, and can be formally equated with the intermediate cells of Moolgavkar and Knudson (1981). In experimental carcinogenesis they are typified by skin papillomas or hormonally dependent breast tumors in mice, or

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precancerous nodules in rat liver. Progression of these cells into nialignancy will occur in “only a small proportion” in Foulds’ scheme, and most persist without qualitative change, or regress and disappear. In the Moolgavkar and Knudson scheme, progression depends on a second mutagenic process, and rates of cell division or cell death for intermediate cells are expressed as “a”and “B,” respectively. T h e expression derived for age-specific cancer incidence at age t in these terms is

I(t) =

PlP-2

X ( S ) exp [(a- P)(t - s)lds,

where pl and p2 represent rates of first and second mutations expressed per cell per year; X(s) the number of stem cells in a tissue at times; a and p denote the “birth” and “death” rates respectively of intermediate cells per year; with (a+) positive a clone of intermediate cells grows, and plots of I(t) versus t resemble the log-log plots of Nordling; with (a+) negative, peak incidence occurs at a comparatively early age, as in childhood tumors. The quantitative importance of the later stage, corresponding to Foulds’ progression, follows from the overall dependence on the exponential term exp (a+).In their paper, Moolgavkar and Knudson ( 1 98 1) show plots of cancer incidence versus age according to variation of positive (a+),with mutational and other factors held constant on a linear scale. Transference to log-log plots shows that approximate straight lines are obtained, as in Nordling’s earlier scheme, and that the slope is greater as (a+)increases. Therefore, the slopes of Nordling’s lines could reflect this factor, rather than the number of successive mutations. This interpretation (Moolgavkar et al., 1979) indeed seems more likely to explain the change in slope of the log-log plots for incidence of breast cancer versus age around the menopausal age for women, which Nordling himself noted, whereas for breast cancer in males a simple log-log plot is found (Moolgavkar et al., 1978). In summary, the Moolgavkar and Knudson scheme with appropriate adjustments of the cellular growth and death-rate factors, can account for all the observed shapes of cancer incidence versus age curves (see Fig. 1). Nevertheless, its comprehensive applicability remains uncertain, for reasons already mentioned. Malignancy attributable to two successive mutations only is regarded by some supporters of the basic scheme in terms such as the following (this being from a recent review of “cancerprotective factors in fruits and vegetables, discussing the biochemical and biological background,” by Dragsted et al., 1993): A biologically based two-stage model for carcinogenesis is sufficient to explain all known incidence curves for human cancers, and this model is able to accommodate

0.09

per 100,OOO

10

30

60 100

age (t). year

FIG. 1. “Log-log” plots of incidence of cancer per year (I,)versus age (1) in years. These were calculated according to the equation of Moolgavkar and Knudson (1981)(see the text); the assumed values of the parameters are as for Fig. 2 of their paper, but plotted according to the “log-log’’ format as introduced by Nordling (l953),in order to show how different conclusions, regarding the number of successive mutations in a precursor cell and its progeny that are required to produce a malignant cell, can arise. The exemplary values assume 107 target stem cells in a renewable adult tissue, of which 5% were present at birth; two successive mutations are envisaged to cause cancer, at rates p, and p2 per cell per year, with the product p1p2= 3 x 10-14; the effect is shown of varying the promotional factor (a+),representing the difference between the relative birth and death rates per cell per year of the intermediate cells produced by the first mutation; these are not malignant, but malignancy is conferred by the second mutation. The values denoted as “n” are derived from the slopes of the log-log plots (m);n = m + 1; such plots of epidemiologic data were interpreted by Nordling (1953)as showing that n successive mutations are required to convert a normal into a malignant cell. These approximately staright-line “loglog” plots are fortuitous, since they represent the same data as the curves of Fig. 2 of Moolgavkar and Knudson (1981);see also Stein (1991).The replotting here is to show how a two-mutation theory embodying a requirement for proliferation of intermediate cells can account for the same data as a multimutation theory. The range of values of I, covers observed incidences of the more common cancers, such as those of lung, colon, or stomach, with Nordling’s n values of around 6 to 7; incidence of breast cancer in women shows n around 6 up to age about 50,then a lower value around n = 3.

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not only the actions of initiating, promoting, or converting carcinogens, but also synergistic or antagonistic effects on these actions (Moolgavkar and Knudson, 1981). This is not to say that any cancer follows exactly these steps, but only that in mathematical terms, the model is sufficient. ( I t should be noted that by “converting carciriogens,” these authors are referring to the second mutation, converting an intermediate cell into a malignant cell.)

As already noted, the two-mutation model can also account for important aspects of experimental carcinogenesis. These include, as major examples, the classical two-stage system of carcinogenesis in mouse skin (Berenblum, 194 la,b; Mottram, 1945) as discussed in more detail later, and the dose- and time-dependence of tumorigenesis in the second most widely studied experimental organ-liver of the rat (Druckrey, 1967; Moolgavkar and Luebeck, 1991). But, as with human cancer, the stages of carcinogenesis in these systems are being subject to increasingly detailed analysis (e.g., for mouse skin, Brown et al., 1993; Furstenberger f t d.,1993). Nevertheless, the basic relevance of the model remains. This relevance can be attributed to the element of synthesis between experimental and human carcinogenesis that it embodies. T h e latter factor was particularly important and resulted from the analysis of what had previously been classified as an inherited disease through a single dominant gene (i.e., retinoblastoma), affecting about 1 in 20,000 children. Knudson (1971) showed that the inherited disease exhibited a time-dependence of incidence consistent with “single-hit” kinetics, whereas sporadic cases required two “hits”; incidence was maximal before the age of two, and decreased rapidly with age, reflecting the cessation of cell division in the lineage involved and absence of positive promotional stimulus. Numerical values for the appropriate parameters in the mathematical expression of the two-mutation hypothesis could be deduced in order to account for the observed incidences of the disease. The required rate of mutation calculated, assuming that 2 x 10“ cells per retina are at risk, was about 2 x 10-7 per cell per year; this is of the order of values generally found for mutagenesis in man (Vogel and Motulsky, 1982). At around the same time that the two-mutation model was promulgated on this basis, evidence became available that the genes causing cancer could be identified and isolated. This was achieved through transfection of tumor DNA (Hill and Hillova, 1971; Graham and Van der Eb, 1973) and indicated that the genes involved were dominantly acting (Shih et al., 1979; Reddy et al., 1982), but it was recognized that such action could account for only one stage of the carcinogenic process (Land e f al., 1983; Sinn et al., 1987). Previous evidence for such dominant genes had been obtained for cultured tumor cells through the

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development of the technique of cell fusion by Barski and Cornefert (1962); this technique was used later to show the existence of genes capable of suppressing malignancy (Harris et al., 1969). It thus emerged that the alternative mechanisms of activation of an oncogene, and of inactivation of a tumor suppressor gene, could cause disruption of the control of cell division. T h e Knudson model for genesis of retinoblastoma then became further established with the identification of the predisposing (RB) gene (Friend et al., 1987; Lee et al., 1987), and demonstrations that both copies of the gene were inactivated to cause malignancy. This gene became the archetype of what were denoted “antioncogenes” by Knudson (1985), but subsequently the term “tumor suppressors” supervened. In more recent developments, identifications of gene alterations associated with carcinogenesis have become a major field of investigation. These show involvement of both oncogenes and tumor suppressor genes, the former activated generally by base-substitutions, the latter, inactivated by a variety of mechanisms, with often one copy mutated and the other deleted. An often cited example, perhaps equaling retinoblastoma as a classical archetype, is colon cancer, for which a requirement for more than two mutations has been deduced (Ashley, 1969; Fearon and Vogelstein, 1990). Here, the intermediate cell types are represented by early adenomas, then two stages of intermediate and late adenomas precede the emergence of carcinomas. At least four genes are implicated, including one activated oncogene and three tumor suppressor genes; in addition there is evidence that an epigenetic change involving biomethylation of cytosine in DNA may be involved. In analogy with retinoblastoma, some patients inherit the first mutation with the condition denoted familial adenomatous polyposis. The order in which the various changes occur is not necessarily the same for all tumors. T h e progression of tumors evidently cannot completely be accounted for in all cases by the minimalist form of the two-mutation concept, and there is currently much interest in the need to discern whether the early stages of the carcinogenic process (i.e., the initiating mutation and subsequent proliferation of resultant intermediate cells), can enhance the rates of further mutations causing progression. Evidence favoring this concept emerged from about 100 years of cytogenetic studies (see, e.g., Nowell, 1986). Recent evidence on the genesis of colorectal cancer has suggested that, in addition to the involvement of oncogenes and tumor suppressor genes, mutations within a third type of gene could promote “genomic instability at numerous loci” and “nonspecifically alter the regulation of a wide spectrum of genes, thereby promoting tumour formation” (Thibodeau et al., 1993).

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T h e most frequently inactivated tumor suppressor gene in human carcinogenesis has been found to be that often denoted p53 (strictly, TP53). Lane (1992), a codiscoverer of the p53 protein, proposed as an answer to the question “why is p53 a tumor-suppressor gene?” that it acts as a “molecular policeman monitoring the integrity of the genome.” Therefore, “tumor cells without workingp53 are more susceptible to the killing effect of DNA-damaging agents because they replicate through the damage; at lower doses, they are more susceptible to the mutagenic effects of these agents.” T h e relevant function of p53 was first reported by Maltzman and Czyzyk (1984), when it was shown to switch off DNA replication to allow extra time in the replication cycle for repair of DNA damage due to ultraviolet radiation. In summary, the demonstration that ionizing radiation could cause both mutations and cancer, followed by interpretations of the agedependence of cancer in terms of a requirement in carcinogenesis for multiple serial mutations, of which those subsequent to the first depended on proliferation of mutant cells, gave rise to a comprehensive quantitative theory of the probability of cancer incidence. A minimalist theory embodying a requirement for two mutations was conceptually attractive, but it became evident from studies of the development of specific types of cancer that often more than two genetic changes are involved. B. VIRALCARCINOGENESIS, ONCOGENES, A N D IMMUNOSURVEILLANCE As already noted, the identification and isolation of genes involved in carcinogenesis came from studies in which the causative agents were tumor viruses. Historically, the discovery of the first known tumor viruses preceded reports of experimental carcinogenesis by either radiation or chemicals. Ellerman and Bang (1909) transmitted chicken erythromyeloblastic leukemia by cell free extracts containing “ultravisible virus.” Rous (1910) was successful in parallel experiments using extracts of chicken sarcoma. Somewhat ironically, these demonstrations became regarded as supporting an unorthodox view of cancer etiology that external infective agents could cause the disease, attributed to Borre1 ( 1907), rather than the more widely accepted somatic mutation theory promulgated by Boveri at around the same time. It appears that Rous remained unimpressed by the mutation theory according to a review he published much later (Rous, 1959); after making notable contributions towards defining the two-stage mechanism of chemical carcinogenesis in the mouse skin system and introducing the

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terms “initiation” and “promotion” (Friedewald and ROUS, 1944), he reiterated the evidence that the carcinogens involved had not been convincingly shown to act as mutagens (Burdette, 1955). He also expressed the view that the somatic mutation hypothesis was “fatalistic” and would lead to discouragement of efforts to “cure cancer” through chemotherapy. Subsequently, the first convincing evidence that an oncogene existed was in fact found from studies using the Rous sarcoma virus (Duesberg and Vogt, 1970; Martin, 1970). Previously, Bader (1965) had shown that its replication depended on DNA synthesis, implying the existence of RNA-dependent DNA polymerase, as later discovered (Temin and Mizutani, 1970; Baltimore, 1970). This part of the viral genome required for cellular transforming activity (denoted src) was then found to derive from host-cell DNA (Stehelin et al., 1976), and the transforming genes could be introduced into “normal” cells causing malignancy through “transfection,” a technique first applied by Hill and Hillova (1971) using tumor DNA. This phenomenon was not unique to oncogenes of viral origin, since Shih et al. (1979) found that DNA from cells transformed in uitro by a chemical carcinogen could in turn cause transformation through an analogous procedure of transfection. Previously, Huebner and Todaro ( 1 969) had introduced the term “oncogene” to accord with their concept that the induction of thymic lymphoma in mice by x-rays (Kaplan and Brown, 1952), indicated to involve a murine leukemia virus (Gross, 1961; Kaplan, 1967), depended on some form of “activation of an oncogene.” It emerged that oncogenes could be activated by mutation of cellular proto-oncogenes; in the case quoted, the proto-oncogene is murine c-rusK in which codon 12, sequence GGT, codes for glycine; the viral v-rmK oncogene has AGT (serine) (Tsuchida et al., 1982) and an x-ray induced thymoma oncogene has GAT (aspartic acid) (Guerrero et al., 1984). This codon also turned out to be frequently mutated by chemical carcinogens, and chemically induced base substitutions at codon 61 of ras could also be effective in activation of this family of oncogenes (reviewed by Barbacid, 1987). T h e first such mutation traced in a human oncogene was reported for a bladder tumor cell line by Reddy et al., (1982), who also noted that the base substitution involved (G + T transversion) was of a type commonly induced experimentally by aromatic amines, a class of chemical known to cause bladder cancer. This presaged a period of intense interest in the possibility that the type of mutation associated with a given cancer could be correlated with

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the carcinogenic agent involved, thus opening up the field of “molecular etiology.” With regard to activation of oncogenes, the scope for this was limited because of the paucity of sites of which such activation could result, essentially limited to 3 out of 188 codons in ras, for example. In view of this small target size, the widespread occurrence of oncogene activation as a critical event in human carcinogenesis (Bos, 1989) was somewhat unexpected, and a requirement for inactivation or deletion of tumor suppressor genes was thought likely to be least as, if not more, frequent. Detection of such deletions (as for the RB gene) through loss of heterozygosity (Cavanee et al., 1983) became an important method for tracing and isolating tumor suppressor genes. But, somewhat paradoxically, mutations, through base substitutions, emerged as important for the inactivation of tumor suppressors, as had been found for activation of oncogenes associated with viral carcinogenesis. For example, Whyte et al. (1988) showed that products from a DNA-containing virus (adenovirus E1A proteins) bound to the RB gene product, thus providing a mechanism for its inactivation (see the review by Goodrich and Lee, 1990). Previously, in parallel experiments, the oncogenic protein product of another DNA tumor virus, SV40, had been shown to bind to a host-cell protein (p53)of molecular weight around 53,000 daltons [Lane and Crawford, 1979; Linzer and Levine, 1979 (the latter authors estimated the molecular weight as 54000)l. After the identification of the corresponding gene as a tumor suppressor, in colon cancers in which one copy was deleted and one mutated (Baker et al., 1989), p53 mutations were found in virtually all types of tumor (Hollstein et al., 1991). Although no tumor type has yet been found that shows 100% incidence, p53 mutations now appear to be the most prevalent in human carcinogenesis (for a recent succinct review, see Stratton, 1993). Apart from its quantitative importance, this gene has become a preferred object of study linking human carcinogenesis with causative agents such as chemicals, virus, or radiation. This follows from the comparatively large size of the effective target it presents to mutagens, i.e. the number of codons within the p53 gene in which mutations can cause its inactivation (the “domains highly conserved during evolution”); 95 such codons (as opposed to 3 in the rus oncogene) were documented by Caron d e Fromental and Soussi (1992). Investigations into p53 mutations relevant to the action of a human dietary chemical carcinogen, aflatoxin B (notably prevalent in South China and parts of Africa) have done much to clarify its co-operative action with the viral hepatocarcinogen, hepatitis B virus (Bressac et ul., 1991; Hsu et al., 1991). Inactivating mutations are induced by the cheniical in the p53 gene at a n aflatoxin-specific “hotspot”; the protein product

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of the remaining copy of the gene may be inactivated by binding of a viral protein product (Hsu et al., 1993). T h e other principal tumor viruses now known to be involved in human carcinogenesis are certain papilloma viruses and Epstein-Barr virus (DNA-containing), human T-cell lymphotropic virus, and immunodeficiency virus (RNA-containing retroviruses) (see Cooper, 1992). T h e possible significance of interactions between environmental carcinogens in general and tumor viruses appears to be attracting increasing attention, particularly with regard to leukemogenesis (see, e.g., Aboud et al., 1992). Another particularly important characteristic of virus-induced tumors is that they are subject to immunosurveillance (Ioachim, 1990; Kinlen, 1992), and can be induced with unusually short latent periods after administering immunosuppressive drugs. This was first noted in the 1970s (Hoover and Fraumeni, 1973), along with the phenomenon of regression of resultant malignant tumors after cessation of immunosuppression (Stribling et al., 1978). T h e apparent requirement for viral involvement contraindicates immunosurveillance as a protective mechanism against induction of cancer in general. This was first proposed by Ehrlich (1909) as “natural immunity not due to the presence of antimicrobial bodies (as previously proposed by Borrel) but determined by cellular factors. These may be weakened in the older age groups in which cancer is more prevalent” (see Alexander, 1972; Himmelweit et al., 1957). Although this concept of immunosurveillance has obvious attractions, and was revived by the eminent theoreticians of medical research, Thomas ( 1 959) and Burnet (1970), evidence from both epidemiologic and experimental studies failed to provide support for its relevance to carcinogenesis by chemicals (see, e.g., reviews by Alexander, 1972; Stutman, 1975). However, subsequent comparative studies of syndromes associated with inherited deficiency in repair of DNA damage induced by ultraviolet radiation have suggested that an additional deficiency in imniunosurveillance is required to confer marked susceptibility to the carcinogenic action of sunlight (Bridges, 1981; Lehmann and Norris, 1989). A case of spontaneous regression of malignant melanoma, possibly due to cell-mediated immunosurveillance, was cited in support of this view (Anstey et al., 1991). IV. Chemical Carcinogenesis: Early History A. GENERAL INTRODUCTION This historical survey of theories of carcinogenesis has so far stressed that studies with ionizing radiation and viruses as the causative agents

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had the most influence on the conceptual development of the subject. Yet, because it is generally acknowledged that quantitatively most cancer cannot be attributed to these agents, chemical carcinogenesis must account for the remainder. For example, Boyland (1969) estimated that 5% of human cancer was due to viruses, 5% to radiations; the residual 90% was attributable to chemicals, with “at least half due to environmental factors.” His estimates appear to have stood the test of time; subsequently around 30% was assigned to tobacco, and the largest remaining proportion (40-6076) to diet (see, e.g., Doll and Peto, 1981; Higginson and Muir, 1979; Wynder and Gori, 1977). However, although “diet” manifestly consists of chemicals, it has proved difficult to define molecular mechanisms in diet-associated carcinogenesis, with some notable exceptions, such as aflatoxin, as already noted. Also, as previously discussed, the general concept of chemical carcinogenesis derived from observations implicating industrial products such as soot, tars, and oils as locally acting skin carcinogens, and dye intermediates as systemic bladder carcinogens. Experimental carcinogenesis, following Yamagiwa and Ichikawa (1915), began by choosing the first type of causative agent Isee Henschen (1968) for a discussion of the historical significance of Yamagiwa’s work]. Extensive investigations were made possible through the demonstration by one of Yamagiwa’s students, Tsutsui (1918), that the mouse could replace the rabbit as the model animal in studies of skin carcinogenesis. In the 1920s, experimentation began directed to the isolation, in the words of Kennaway (1955), in his authoritative memoir, of the “cancerproducing compound in coal-tar.” Thus began over ten years of fascinating work in the history of carcinogenesis. CARCINOGENS B. THEFIRSTPURECHEMICAL DETECTED THROUGH THEIR FLUORESCENCE SPECTRA The work of Leitch (1922) on “experimental paraffin cancer” was the stimulus for this period, in which the dominant theme was carcinogenesis by polycyclic aromatic hydrocarbons, and the outstanding center for research on this topic was the Research Institute of the Cancer Hospital (Free) in London, of which Leitch was the second director; this later became the Institute of Cancer Research of the Royal Cancer Hospital, of which a prominent part was the Chester Beatty Institute (see Haddow, 1961). Kennaway joined Leitch in 1922 and his research programme was determined by previous reports suggesting that the carcinogen(s) he

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sought would be found among the polycyclic aromatic hydrocarbons. As he acknowledges in his memoir (Kennaway, 1955), Berthelot (1866) had produced benzene by pyrolysis of acetylene, and Kennaway (1924, 1925) went on to show that mixtures, resulting from pyrolysis, in an atmosphere of hydrogen, of isoprene, contained aromatic hydrocarbons that were carcinogenic. He was dissatisfied with this preparative procedure mainly because the purpose of his research was to study the carcinogenic effect of tar in order to “throw light upon the far more important cancers which are caused by some internal factors. Chemical reactions which in nitro require the temperature of red heat, must, if they occur within the body, be brought about in some other way” (Kennaway, 1955). Although admirable in principle, this concept proved to be somewhat misleading. Toward the end of the intensely productive period of research by Kennaway’s group, Cook et al. (1937) had later to admit that, even though they could obtain a potent carcinogen (3-methylcholanthrene) from a bile acid, deoxycholic acid, by “simple chemical transformation” it was not possible to “secure evidence that production in the body of methylcholanthrene or a related compound is an etiological factor in human cancer.” T h e immediate effect of the search for milder conditions of production of polycyclic hydrocarbons was that Kennaway, at around that time (in 1924),joined by Hieger, turned to the use of aluminum chloride as a catalyst, following the methods due to Schroeter (1920, 1924) which permitted the use of lower temperatures. They were disappointed that carcinogenic mixtures could not be obtained at body temperature, but the mixture obtained at 60 to 70°C was powerfully carcinogenic ( 1 18 cancers in 496 mice; Kennaway and Hieger, 1930). The search for the putative highly active hydrocarbon(s) in these mixtures was brought to a successful conclusion because of a positive correlation between carcinogenic potency of the fractions and the intensity of characteristic blue fluorescence bands at around 400, 418 and 440 nm, when they were irradiated with ultraviolet light. This was first noted by Mayneord in 1927, and Kennaway’s memoir of 1955 is at pains to emphasize that this rapid test was an essential part of the work, but he was evidently embarrassed at his failure to mention Mayneord in the earliest series of papers on pure chemical carcinogens. The first of these (Kennaway and Hieger, 1930) described how over 20 hydrocarbons were tested for carcinogenicity towards mouse skin by prolonged painting of, generally, 0.3% solutions in benzene twice weekly. Three of the compounds available at that time were reported to be potent carcinogens in an experiment lasting 285 days and using 10 mice per compound. These were

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designated 1,2,5,6-dibenzanthracene(which gave 2 papillomas in 10 mice); 3’-methyl-l,2,5,6-dibenzanthracene (4 papillomas); and 1,2,7,8dibenzanthracene (4 malignant cancers). While these compounds all showed the fluorescence bands associated with carcinogenic fractions of tars, oils, and synthetic mixtures of the “Schroeter” type (Hieger, 1930), it was emphasized that the wavelengths of these bands were not identical with those of the carcinogenic fractions, and the attempts to obtain the highly active constituents of tar were still in progress at that time. The report on the “fluorescence spectra of carcinogenic substances” (Kennaway and Hieger, 1930) thus became accepted as the first clear demonstration that pure chemical compounds were able to induce cancer, and a further account with more data appeared two years later, including the co-authorship of Mayneord (Cook et al., 1932). Later publications cleared u p two further awkward points. Firstly, the carcinogen previously designated 1,2,7&dibenzanthracene turned out because of an ambiguity to be identical with 1,2,5,6-dibenzanthracene, resulting from the method of synthesis (due to Clar, 1929); the authentic 1,2,7&dibenzanthracene was subsequently synthesized by Cook (1932), and showed carcinogenic potency, but weaker than its isomer. [It should be noted here that the nomenclature of‘ polycyclic aromatic hydrocarbons used at that time became obsolete, and the carcinogens involved are now denoted dibenz[a,h]anthracene ( 1,2,5,6-isomer) and dibenz[aj]anthracene(see Dipple et al., 1984).] Secondly, priority in the identification of a carcinogen known to be a constituent of’tar was acknowledged by Kennaway and Hieger (1930) to have been claimed by Twort and Fulton (1930) for chrysene, from twice weekly painting of a 2.6% (w/v) suspension in liquid paraffin. Starting with 800 mice, high mortality and 15 tumors were reported from various mixtures, containing chrysene and other hydrocarbons, and in one group, from an unstated number of mice, one wart (papilloma) and one epithelioma (after 65 weeks) were obtained from “purified chrysene.” Twort and Fulton (1930) noted that the synthetic tars they used contained “large quantities” of chrysene, and that its carcinogenic potency “must be very low indeed.” They considered that, with this proviso, “chrysene (if our specimens have been completely purified) appears definitely to be carcinogenic.” Kennaway and Hieger (1950) were not impressed by this evidence, since they had tested the compounds used by Twort and Fulton and obtained negative results, and they also noted that “the fluorescence spectrum of chrysene is very similar to that of benzanthracene, but its position is displaced considerably towards the region of shorter wave-

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length (i.e., away from the “‘Schroeter’ bands”). They appeared to remain convinced that the highly carcinogenic hydrocarbon in coal tar responsible for this fluorescence had yet to be isolated. Subsequently, Cook et al. (1937) reported on synthetic chrysene, which gave one tumor in 20 mice after 853 days by skin painting, but was judged to be a more potent carcinogen by subcutaneous injection into rats (two tumors in ten rats). Chrysene from coal-tar, stated to be indistinguishable from the synthetic compound, was found to be more weakly carcinogenic (one papilloma from 50 mice u p to 775 days; Barry and Cook, 1934). Van Duuren et al. (1966), using the later more sensitive technique of twostage carcinogenesis in mouse skin, found chrysene was a powerful initiator using croton resin as promoter. T h e question of which hydrocarbon was the first shown to be carcinogenic may therefore still be regarded as unresolved. However, this proved to be irrelevant to the resolution of the task to which Kennaway and Hieger had applied themselves starting in 1924, that is the isolation and identification of the constituent of tars which shared the attributes of high carcinogenic potency, and the characteristic fluorescence spectrum found to be associated with this.

c. BENZO[a]PYRENE-AN

ARCHETYPAL ENVIRONMENTAL CARCINOGEN

By the beginning of 1932, Hieger had eventually arrived at a powerfully carcinogenic product, from ethanol extracts of a material designated as a “distillate of 2 tons of pitch,” obtained from a London gasworks. This “red to orange sealing wax-like material,” after numerous fractionations and crystallisations, with tests of fluorescence of fractions as a guide to carcinogenic potency, was concentrated into about 7 g of yellow crystals with intense fluorescence spectra of the required type (photographs of which are reproduced in Hieger, 1937; 1961). This product was further purified by classical methods of recrystallisation, and elemental analysis showed it to be an isomer of 1,2,5,6-dibenzanthracene, most probably 1,2-benzpyrene (as then designated; subsequently 3,4-benzpyrene, and now benzo[a]pyrene). This structure was confirmed by synthesis (Cook et al., 1933), which left no doubt that this was “the only potent carcinogenic compound which has been shown to be present in coal tar” (Cook et al., 1936). Contemporary readers may question the prolonged and laborious nature of the experimentation that led to what in Hieger’s words was “something like a revolution in cancer research.” Apparently chromatography, which might well have proved useful, was not used by Hieger

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and his collaborators, although Cook et al. (1937) refer to chromatographic analyses of coal tar, showing that 3,4-benzypyrene was present in “relatively large amounts in the highest-boiling fractions” (Winterstein and Schdn, 1934). By whatever precise nomenclature it is designated, “benzpyrene” became the archetypal carcinogen and has been the subject of comprehensive reviews (see, e.g., Phillips, 1983; Osborne and Crosby, 1987). Acknowledged as ubiquitous in the environment, it became the most intensively studied chemical carcinogen. Tumors in neonatal mice could be obtained using extracts from the air of American cities; the lower carcinogenicity of air from Los Angeles when compared with that of Cincinnati paralleled their contents of benzpyrene (Epstein et al., 1966). But, more recently, emphasis shifted away from air pollution as a significant exogenous source of cancer towards cigarette smoking (Greenberg, 1983). Hieger (1961) considered that “carcinogenesis by benzpyrene is of special interest because it is assumed, without direct proof, that it is the responsible carcinogen present in town smoke and in cigarette smoke, for it is formed during the pyrolysis of any carbonaceous material provided that combustion is not complete.” His remarks raise the question of what would be accepted as “direct proof,” and the evidence perhaps closest to answering this requirement has been obtained only recently. This is based on a chain of events linking benzo[a]pyrene through its metabolic activation to reaction with DNA, thus implicating the carcinogen as a source of mutation in a target tissue. How this came about requires delineation of the historical development of the mode of action of the polycyclic aromatic hydrocarbons. V. Mode of Action of Carcinogenic Polycyclic Aromatic Hydrocarbons-Early Historical Development

A. THEIR SUGGESTED RELATIONSHIPTO

THE

STEROLS

As noted, Kennaway sought endogenous biochemical pathways for generation of carcinogenic hydrocarbons, and his co-workers Hieger and Cook enthusiastically followed what must have seemed at the time this rather obvious idea; obvious because at the time this group of compounds were the only known chemical carcinogens. However, a priori, the idea seemed implausible; it was clear that exogenous carcinogens such as soot, tars, and oils contained carcinogens of this chemical type. T h e only endogenous compounds that appeared to

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be chemically related were the then relatively recently discovered steroids. In the early 1930s a concatenation of discoveries occurred which appeared to lend credence to the significance of this otherwise apparently superficial relationship. Perhaps the most dramatic was the demonstration by Lacassagne ( 1932) that the steroidal ovarian hormone, estrone, then designated “folliculin” by Doisy et al. (1930), who had isolated it from the urine of pregnant women, could induce mammary cancer in male mice. This provided an experimental basis for the long-suspected involvement of hormones (a term introduced in 1906) in carcinogenesis, generally regarded as originating with Ramazzini (1713), who, in an early treatise on occupational health, “Diseases of Workers,” noted a high incidence of breast cancer in nuns. However, Von Siebold (1824) associated positively what he regarded as excessive sexual activity with cancer of the uterus, finding this to be more prevalent in women with frequent pregnancies. These apparently contradictory factors were confirmed by more recent epidemiologic studies; MacMahon et al. (1973) confirmed that early pregnancy and multiple births are associated with decreased risk of breast cancer; Rotkin (1962) found early coitus and multiple sexual partners increased the risk of cancer of the uterine cervix; but this latter is now associated with predominantly a viral, rather than hormonal, etiology (Kiviat and Koutsky, 1993). Beatson (1896), the eminent Scottish surgeon, introduced castration as a treatment for breast cancer in the general belief that “the etiology of cancer lies in an ovarian or testicular stimulus.” Cook et al. (1934) took as their starting point the then “justifiable assumption” (the precise structures of steroids were not at that time completely elucidated) of “a biological relationship between estrogenic hormones and the sterols and bile acids.” Using a method by Allan et al. (1930) for testing estrogenic activity in ovariectomized rats, they found that certain potent carcinogens, including 1,2-benzpyrene (i.e., benzo[a]pyrene), were positive, unlike 1,2-benzanthracene (benz[a]anthracene) and other weakly or noncarcinogenic hydrocarbons. They thought it more likely that the carcinogens themselves had estrogenic activity rather than that they were metabolically converted into estrogens. Further support for the possible association between carcinogens and hormones was adduced from the discovery of a hydrocarbon more potent than benzo[a]pyrene. This was “methylcholanthrene” (now designated 3-methylcholanthrene), synthesized independently by Wieland and Dane (1933) and Cook and Haslewood (1933). The particular interest in this carcinogen was clearly delineated by Cook (1935), who noted

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that the side chain of cholesterol-derived bile acids could possibly cyclize to yield a cholanthrene nucleus. As Cook pointed out, this cyclization had already been achieved by Wieland and Schlichting (1925) in their preparation, starting from the bile acid deoxycholic acid, of dehydronorcholene, further dehydrogenation of which yielded methylcholanthrene. As previously noted, Cook et ul. (1937) became resigned to accepting that these conversions were unlikely to occur in uiuo, although they also noted at that time that, while several investigators found that the carcinogens were eliminated from the body of experimental animals, there was no knowledge of the metabolic processes involved.

B. CARCINOGENIC HYDROCARBONS AND TWO-STAGE CARCINOGENESIS T h e experimental induction of skin cancers in mice was the essential test system enabling the first chemical carcinogens to be identified. Hieger (1961) has given detailed experimental details of the methods used, as for example, “mice were painted interscapularly twice a week with one stroke of a camel-hair brushful of benzpyrene solution (0.75% in benzene:liquid paraffin, 9: l).”The latent period (i.e., time to appearance of first papilloma), ranged from 13 to 54 weeks. These procedures were clearly prolonged and laborious, but they did enable a consensus for grading carcinogenic potency of the various polycyclic aromatic hydrocarbons that became available during the 1930s, in terms of yield of tumors and average latent periods following standard applied doses. These procedures stimulated attempts to devise structure-activity relationships. Progress towards understanding mechanisms accompanied attempts to speed up and simplify the test system. Rous and Kidd (194 1) reverted from mice to rabbits, and found that if skin painting with tar was interrupted, the tumors disappeared, but could then reappear at precisely the same sites when application of tar was continued. They also found that noncarcinogenic stimuli such as application of turpentine, or wound healing by boring holes in the rabbits’ ears, could produce the same effect (this latter stimulus had also been shown before, e.g., by Deelman ( 1924), as previously noted). These experiments suggested that the tarred skin contained “cells which had not yet completely attained to the neoplastic state” and that a reversible process was occurring. Later, Friedewald and Rous (1944) referred to the latent cells as having undergone the process of “initiation”; the formation of papillomas was termed “promotion”; a further step was the conversion of papillomas (which

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might or might not be subject to regression) into malignant cancers. As has already been pointed out, these stages broadly resemble the descriptions of the stages of development of human skin cancers, induced by soot, published about 50 years earlier by Butlin (1892). In the same year that the observations of Rous and Kidd were reported, Berenblum (1941a) introduced what has proved to be the archetypal promoter of carcinogenesis, in the mouse skin system that had by then become widely used. He found that dissolving carcinogenic hydrocarbons in croton oil, before painting a solution diluted in conventional solvents such as acetone, markedly enhanced both the yield of tumors and reduced the latent period. From then on the concept that two distinct biochemic phenomena were involved in carcinogenesis became inescapable. Mottram (1945) further clarified and extended this concept by showing that an initiating single dose of hydrocarbon, that did not of itself suffice to induce tumors, could be promoted by multiple applications of croton oil to produce papillomas, some of which could, on prolonged further promotion, progress to malignancy. T h e important additional finding was that this latter progression could be enhanced by repainting the papillomas with the hydrocarbon. With hindsight, this two-stage carcinogenesis accords in excellent fashion with the two-mutation theory, as previously discussed in Section 111, A. T h e experimental approach to this theory could, in principle, have preceded the epidemiologic approach, provided that initiation of cancer had been equated with mutagenesis. However, the available evidence not only failed to suggest this, but was interpreted as contraindicating it. It took 20 more years until the demonstration that the carcinogenic potency of a spectrum of hydrocarbons correlated positively with their covalent binding to DNA in mouse skin (Brookes and Lawley, 1964) provided biochemic evidence in favor of the ability of these classical carcinogens to act as mutagens. T h e attempts to correlate carcinogenic action with hormonal action, based on structural analogies between hydrocarbons and steroids (Cook, 1935), began to appear irrelevant when chemicals of different structural groups became revealed as carcinogenic. Thus, the review by Cook et al. (1937) was somewhat of a turning point by noting carcinogens not chemically related to benzanthracene, such as a p-aminostyrylquinoline derivative (a potential trypanocide; Browning et al., 1933), and a systematically acting hepatocarcinogen in the rat, o-aminoazotoluene (Sasaki and Yoshida, 1935). T h e two-stage methodology introduced by Berenblum (194 la), Berenblum and Shubik (1947; 1949), and Mottram (1945) permitted a rapid expansion in the number and variety of experimentally verified

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carcinogens. It also revealed initiators which, although shown to be carcinogens when administered by other routes, gave few, if any, skin tumors when tested without promotion (see, e.g., the review by Salaman, 1958). Some hydrocarbons fell into this category, as did the aliphatic carcinogen, ethyl carbamate (urethane) (discovered as a carcinogen for mouse lung by Nettleship and Henshaw, 1943). The question also arose whether promoters should be classified as carcinogens. Various types of chemical were represented, including iodoacetic acid (a thiol reagent) and various detergents. Berenblum ( 1954) recognized their ability to cause a hyperplastic reaction in mouse skin, but also that this of itself was inadequate to explain promoting activity. Hieger ( 1962) found evidence that, in certain strains of mice, croton oil was a “complete” carcinogen (i.e., did not always require an initiator). T h e tumor promoters were also deemed skin “irritants,” but this property was also thought to be not necessarily related to promoting ability, especially since Berenblum ( 1935) had previously shown that mustard gas, a notable “irritant,” was able to act as an inhibitor of hydrocarbon-induced tumorigenesis in mouse skin. As noted previously, mustard gas, by virtue of its x-radiation-like vesicant action, was tested as a mutagen and found to be positive (Auerbach and Robson, 1946). Therefore, the corollary from these combined biologic actions appeared to contraindicate the involvement of somatic mutations in mouse skin carcinogenesis (Berenblum and Shubik, 1949), although it could also be interpreted as showing that mustard gas was an inhibitor of the hyperplasia required for promotion. Subsequently, Heston ( 1950) obtained experimental evidence that mustard gas was carcinogenic. Correlations between the phenomena of initiation and promotion with other biologic actions of the chemicals exhibiting these properties had evidently failed to emerge, according to the reviews of “the significance of mutation in relation to the origin of tumors” by Burdette (1955), or of the general field of carcinogenesis by Hieger (1961). Burdette was not convinced by various reports of mutagenic activity of carcinogenic hydrocarbons, whereas the established chemical mutagen, niustard gas, was an inhibitor of carcinogenesis. Hieger found no theory of the mode of action of promoters to be satisfactory; progress in this area had to await the isolation of the active principles of croton oil, the phorbol esters (Hecker, 1967). Subsequently, a protein receptor of phorbol esters was identified (Castagna et al., 1982) as protein kinase C; the pleiotropic effects of these classical promoters are still being studied (e.g., Brown et al., 1993). It is remarkable that one strain of mouse, much used in carcinogenesis studies, C57BL, is resistant not only to phorbol

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esters, but also to physical promotion through wound healing (DiGiovanni et al., 1993). It gradually emerged, therefore, that initiators and promoters had different receptors, DNA, and protein, respectively. An important requirement was to resolve the apparent paradox that carcinogens could not only induce uncontrolled cell, proliferation but also act as cytotoxic agents. This was embodied in the introduction of the concept of radiomimetic chemicals. VI. The Scope of Chemical Carcinogenesis Broadens-DNA as an in Vivo Receptor

A. CARCINOGENS AS GROWTH INHIBITORS AND RADIOMIMETIC COMPOUNDS Haddow (1935), then working in the Department of Bacteriology at Edinburgh, obtained purified hydrocarbons from Cook, and tested their ability to inhibit the growth of a transplantable sarcoma in rats, using a method of administration introduced by Boyland ( 1932), injection in colloidal suspensions in gelatin. He found positive correlation between growth-inhibitory activity and carcinogenic potency sufficient to warrant a comparison with the corresponding dual action of X radiation. T h e term “radiomimetic” had been introduced in 1925 by A. P. Dustin, (see the review by his son; Dustin, 1947) initially referring to acriflavine, and extended to various mitotic poisons, including colchicine, methylcholanthrene, urethane, and mustards (Dustin, 1947). The site of action of these compounds was unequivocally indicated to be the cell nucleus, and the original drugs under this heading, the acridine dyes, being positively charged, were assumed to bind reversibly to nucleic acids. Boyland (1952a) summarized the outcome of the rapid expansion in both the number and types of carcinogen that was heralded by Cook ~t al. (1937), and, developing the analogies between carcinogen chemicals and radiations, he attempted to devise a unifying concept to account for the mode of action of these apparently diverse carcinogens. His review, under the heading “different types of carcinogens and their possible modes of action” (Boyland, 1952a) represents a further landmark in the history of carcinogenesis. Using the classical hydrocarbons as his starting point, he referred to the interest in structure-activity relationships, pioneered by Schmidt (1939), who applied valence-bond theory to discern the importance of high-electron density, in polycyclic aromatic hydrocarbons regarded as derivatives of phenanthrene, at a

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region denoted “K” for Krebs (cancer) (i.e., the “phenanthrene bridge” bond). This led to a prodigious number of papers along the same lines (e.g., Robinson, 1946; Pullman and Pullman, 1946) but a comprehensive review by Badger (1948) noted that these correlations were “only partly successful.” Although some further evidence emerged that the structural modifications around the K-region which led to enhanced electron density were generally accompanied by increased reactivity of this bond, for example to oxidizing agents, this gave no indication in general regarding how hydrocarbons would be converted by metabolic oxidation. Badger (1948) offered a somewhat tentative conclusion that “carcinogenic substances are-as far as is known-active as such, and are metabolized to inactive derivatives.” Boyland (1952a) revived the possibilities that the carcinogenic hydrocarbons could act as radiomimetic compounds by reversible binding to purines, and to bases in DNA, through van der Waals’ forces, or that they could act through reactive metabolites. With regard to the latter alternative, he noted that anthracene (Boyland and Levi, 1935) and naphthalene (Young, 1947; Booth and Boyland, 1949) gave dihydrodiol and phenolic metabolites in rats and rabbits. He went on to make the important suggestion that these metabolic oxidations could involve reactive intermediates, epoxides (i.e., aralkylating agents), or free radicals, respectively. T h e radiomimetic analogy was thus encapsulated; epoxides fell into the category of alkylating agents such as mustard gas; X-rays were thought to induce oxidation of biologic material through hydroxyl radicals. However, Boy land appeared to prefer the more classical analogy between hydrocarbons and the original radiomimetic agents such as acriflavine; action through the formation of reversible complexes with DNA (Boyland and Green, 1962). Afterjoining the staff of the Royal Cancer Hospital Research Institute in 1936, Haddow succeeded Kennaway as Director of the Chester Beatty Institute, which the main part of this institute became, in 1946. In view of his work showing growth inhibitory action of carcinogens, he reoriented the effort of research in the direction of cancer chemotherapy. An important aspect of this new emphasis was derived from the work of his predecessors, who, as discussed, had attempted to trace a broad correlation between the action of carcinogens and hormones. Their analogy drawn between certain polycyclic hydrocarbons and steroids became amplified by inclusion of another structural group, derivatives of stilbene, which, in turn, stimulated interest in possible correlations between carcinogenicity and growth inhibition with a further spectrum of structurally related carcinogens.

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An important starting point was the introduction of synthetic estrogens by Dodds et al. (1938), the first of which was diethylstilbestrol. Badger et al. (1942) reported comparative data on growth inhibition by more than 200 compounds that they regarded as structurally related to the carcinogenic polycyclic aromatic hydrocarbons, including heterocyclic analogues containing ring-nitrogen atoms, and derivatives of stilbene and azobenzene. This last group was regarded as structurally equivalent to phenanthrene derivatives with the central ring disrupted. It was pointed out that although “estrogens are to be regarded as growthpromoting agents. . . evidence is accumulating to show that estrogens are capable of marked inhibitory effects, in many cases of the same nature as those produced by carcinogenic polycyclic hydrocarbons.” They went on to discuss whether this action was exerted directly, or indirectly through interference with pituitary function as suggested by Zondek (1936). T h e conclusion was that there was evidence for both; that favoring direct action was provided by the observations of White and White (1939) that growth inhibition in rats fed carcinogenic hydrocarbons in the diet was opposed by feeding additional sulphurcontaining amino acids o r -glutathione; this effect was also observed (White, 1940) with the dye 4-dimethylaminoazobenzene, shown by Kinosita (1937) to be hepatocarcinogenic in rats, thus obviating its use as a food additive “butter yellow.” This last compound proved later to be of particular importance, since it was found to bind firmly to proteins in liver of rats fed with the dye, in sufficient amounts to permit detection of the product by light absorption (Miller and Miller, 1947), thus showing beyond doubt that carcinogens could directly combine with amino acid constituents of proteins, presumably through activated metabolites. As these authors noted, a similar conclusion was indicated for carcinogenic hydrocarbons, since Doniach et al. (1943) and Weigert and Mottram (1946) had reported evidence for firm binding of benzpyrene in mouse skin, on the grounds that the bound metabolite was extractable only after alkaline hydrolysis of the tissue proteins. T h e absence of bound dye in proteins of hepatomas induced by 4-dimethylaminoazobenzene was thought to be significant for the interpretation of the carcinogenic process (Miller and Miller, 1952). However, Hieger (196 1) in a comprehensive review of the “protein-deletion hypothesis” and of the numerous studies of binding of carcinogens by proteins that in the 1950s followed the first report by the Millers remained unconvinced that this was critically important mechanistically. He noted that Woodhouse (1954, 1955) found that noncarcinogenic

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hydrocarbons could bind firmly to mouse skin (using fluorometric methods for detection) and that protein binding of various hydrocarbons did not correlate positively with their carcinogenic potency. Meanwhile, Haddow and co-workers continued to introduce new compounds with tumor growth-inhibitory potential on the principles that this property would be associated with ability to induce tumors and might be related to hormonal action (reviewed by Haddow, 1947; 1951). Among these, an amine embodying the stilbene nucleus of the synthetic estrogens, 4-dimethylaminostilbene, was synthesized and proved to be both a potent systemically acting carcinogen and tumor-growth inhibitor in the rat (Haddow et al., 1948a). However, the emphasis had already shifted away from the supposed relationship between growth inhibition and hormonal action after the introduction of what appeared to be a completely different type of cancer chemotherapeutic agent, the archetype of which was mustard gas. As already noted, mustard gas was known to be both a mutagen and an inhibitor of experimental skin carcinogenesis, but its attraction to cancer chemotherapists derived from a much earlier observation that suggested the possible specificity in its systemic cytotoxicity because deaths from poisoning by mustard gas during the First World War were attributed to the bone marrow as the critical site (Krumbhaar and Krumbhaar, 1919). During the Second World War, the leukopenic action of mustard gas and the homologous nitrogen mustards was confirmed, and nitrogen mustards, notably HN2 (di-2-chloroethylmethylamine) [named after the wartime code H for mustard gas, di-(2-chloroethyl) sulphide] was introduced into cancer chemotherapy (Gilman and Phillips, 1946). The mustards were clearly in the category of radiomimetic drugs (Boyland, 1952b). Their mode of action, based on the much greater cytotoxic action of di- and tri-functional nitrogen mustards than that of monofunctional agents, was suggested to depend on their ability to alkylate and cross-link chromosomal macromolecules (Goldacre et d., 1949). Following this concept, Haddow et al. (1948b) synthesized a mustard, 4-N,N-di(2-chloroethyl)aminostilbene,which they presumably hoped might present some pharmacologic advantage over either separately, by embodying the structural features of growth inhibitors that acted in different ways. In fact, using their standard test of a transplantable rat sarcoma, they found encouragement that this aromatic nitrogen mustard, although less toxic than the aliphatic mustard HN2, was an effective inhibitor of tumor growth. This stimulated the synthesis of further mustards derived from aromatic amines, including melphalan (phenylalanine mustard) (Bergel and Stock, 1953) and chlorambucil (Everett

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et al., 1953), which have proved clinically useful up to the present. The

carcinogenic risk inherent in using most cancer chemotherapeutic drugs was first pointed out by Haddow et al. (1948a). This risk continues because of the lack of effective alternatives, but it should be noted that clinical use of another aromatic nitrogen mustard chlornaphazin, embodying the structure of the bladder carcinogen, 2-naphthylamine, was abandoned because it caused high yields of bladder cancer (Videbaek, 1964), which were suggested to result from metabolism to the parent amine (Boyland, 1969). T h e introduction of the mustards as cancer chemotherapeutic agents was followed by a virtually complete reappraisal of the mechanisms involved in chemical carcinogenesis. Reviewing the topic of “our present understanding of the cancer cell,” Haddow (1955) summarized the situation as follows: Chemical carcinogenesis can be regarded as a very special case of the action of drugs on cells and Ehrlich’s adage applies here as elsewhere: corpora non ugunt nisi fixutu. Accordingly, much work is being directed to the nature of the cellular substrates with which the carcinogens combine. Already there is evidence that the combination may lead, either directly or indirectly, to the elimination of certain key proteins essential in the normal regulation of growth-so liberating more primitive synthetic reactions upon which the process of cell division depends, and from the uncontrolled impetus of which it now proceeds more or less continuously.

T h e question remained of how to interpret the term “fixation.” The essential process could be a strong, but reversible, binding, as envisaged for the specific interaction between a hormone and its receptor, for example. On the other hand, as, in fact, Ehrlich (1913) himself had suggested, with respect to the mode of action of his first successful chemotherapeutic agents, including Salvarsan, “these arsenical drugs contain unsatisfied avidities (i.e., valencies) which render them capable of additive reaction with other compounds.” Evidently he thought that drugs capable of covalent reactions with cellular constituents would prove the most biologically effective (for assessments of the historical origins of drug receptor theory see Parascandola and Jasensky, 1974; Travis, 199 1). It was during the 1950s when these considerations became even more obviously significant with respect to the mode of action of chemical carcinogens, that I became involved. Having carried out some studies on the interactions between positively charged dyes and DNA (Lawley, 1956a), in line with the earliest concepts of the mode of action of radiomimetic drugs as represented by acriflavine, as discussed previously, I was assigned to d o analogous work with the carcinogen, trans-4aminostilbene, and homologous aminostyrylquinolines. This showed

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that reversible binding of the drugs to DNA occurred as expected, but that of the noncarcinogen 3-aminostilbene was no less than for its carcinogenic isomer. Binding to chromosomes could be shown for related basic dyes, quaternary salts of aminostyrylquinolines, by fluorescence microscopy, but these were biologically inactive (Haddow et al., 1960). These observations suggested that physicochemical complexing between carcinogens and chromosomal components, including DNA, was largely irrelevant to biologic action, and that covalent reactions of metabolites of carcinogens, as shown for butter yellow and protein (Miller and Miller, 1947), was more likely to be significant. However, despite the crosslinking hypothesis of Goldacre et al. (1949), the involvement of DNA as the cellular receptor of carcinogens had not been invoked, and at that time there was no evidence of its occurrence, in contrast to the numerous reports of reaction between proteins and carcinogens (presumably mediated by metabolic activation). It may be interpolated here that Haddow’s principle, derived from his early observations (Haddow, 1935) on carcinogens as growth inhibitors, has resurfaced in an unexpected instance. Tamoxifen, a chemotherapeutic agent in breast cancer with a chemical structure embodying the stilbene nucleus, and thought to act, at least in part, as an antiestrogen, has been found to be a potent hepatocarcinogen in rats of either sex (Greaves et al., 1993; Williams et ad., 1993). As with aromatic amines, including 4-aminostilbene (Gaugler and Neumann, 1979), in vim covalent reaction with DNA has been detected (Han and Liehr, 1992); this has also been invoked as possibly significant for the carcinogenic action of the archetypal synthetic estrogen, diethylstilbestrol (reviewed, Metzler, 1990). However, a recent comprehensive appraisal of the carcinogenicity of diethylstilbestrol in humans concluded that its mechanism of action “remains obscure” (Marselos and Tomatis, 1992). B. CHEMICAL METHYLATION OF DNA A N D ITS SIGNIFICANCE FOR RADIOMIMETIC ACTION OF ALKYLATING AGENTS T h e mutagenicity of the mustards (Auerbach and Robson, 1946) suggested that an important target of their cytotoxic and carcinogenic action [the latter shown for HN2 by Boyland and Horning (1949)], and for mustard gas by Heston (1950), would be the cellular component of the nucleus embodying the “hereditary material.” This had already been proposed as the target of molecular cross-linkage by mustards (Goldacre et al., 1949). It was remarkable, therefore, that few studies of the reactions of carcinogens or related compounds with DNA had been reported

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by the 1950s, despite the demonstration by Avery et al. (1944) that DNA, rather than protein, was the “transforming factor” capable of transmitting heritable characteristics in bacteria. The molecular model for replication of DNA (Watson and Crick, 1953b) embodied a mechanism for mutagenesis involving miscoding of DNA bases if their tautomeric configurations were different at the time of replication from the predominant forms maintaining the normal base pairing. T h e bases of DNA were implicated in its reaction with nitrogen mustard HN2, by Chanutin and Gjessing (1946), who showed that this altered the ultraviolet absorption spectrum of DNA. The reactions of mustard gas with DNA were reported by Young and Campbell (1947) to involve amino groups of guanine (at N-2) and adenine (at N-6), while Elmore et al. (1948) interpreted evidence, based on electrometric titrations, as indicating reaction at phosphodiester groups in the macromolecular chain. The present author, having reported on the effect of acid on the ultraviolet absorption spectrum of DNA (Lawley, 195613) and noting the essentially similar changes due to alkylation of DNA by HN2, hypothesized that the basic sites in DNA would also be the nucleophilic sites reactive to alkylation and that these would be ring-nitrogen atoms; since adenine and cytosine were more basic than guanine, they might be expected to be the more easily alkylated. Independently, Lawley and Wallick (1957) and Reiner and Zamenhof (1957) turned to dimethyl sulfate as a suitable rapidly reacting agent of simple structure for investigation of DNA alkylation, and both found the N-7 atom of guanine to be the most reactive site. The sites of alkylation in adenine and cytosine, predicted on what turned out to be somewhat simplistic grounds as major sites, but in DNA, found to be quantitatively minor, were detected in the corresponding deoxyribonucleotides (Lawley, 1957). Their identification required isolation and characterization of novel methylated bases, l-methyladenine (Brookes and Lawley, 1960a) and 3-methylcytosine (Brookes and Lawley, 1962) (the latter then numbered as 1 -methylcytosine). Apart from the, at first, unexpected high reactivity of N-7 of guanine, the next most reactive site in DNA turned out to be N-3 of adenine (Lawley and Brookes, 1963), also unexpected in the sense that this atom, together with N-7 of adenine (Lawley and Brookes, 1964), was a minor reactive site in adenine nucleotides. Evidently, the base pairing in DNA through hydrogen bonds involving the N-1 atom of adenine and N-3 atom of cytosine suppressed their reactivity. T h e N-7 atom of guanine was predicted to be the most nucleophilic site in a wave-mechanical study (Pullman and Pullman, 1959). Alkylation of DNA was found to affect markedly its stability to hydrolysis at neutral pH; this was expected since protonation of purines caused

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their release from the macromolecule more easily than for pyrimidines, and the alkylating agents attacked the same basic groups as did acid; at neutral pH, 37”C, 7-methylguanine hydrolyses out from methylated DNA with a half-life of around 100 hours; 3- and 7-methyladenines more rapidly with half-lives of about 30 and 3 hours, respectively (Lawley and Warren, 1976). T h e resultant apurinic sites further facilitate hydrolysis of the macromolecular chain, an alkali-catalyzed, as well as an acid-catalyzed, process. Alkaline treatment of alkylated DNA before loss of the alkylpurines results in ring fission of the destabilized imidazole moiety of the guanine nucleotide alkylated at N-7, to yield a 2,6-diamino-4-oxy-5-alkylforrnarnidopyrimidine moiety (Lawley and Wallick, 1957; Brookes and Lawley, 1961; Lawley and Brookes, 1963). Although, at neutral pH, too slow to be of any great biologic significance, the analogy between the reaction and the analogous imidazole ring fissions of purines due to X irradiation (Hems, 1958) appeared to provide some justification for the rddiomimetic attributes of the alkylating agents, although the depurinations and subsequent chain fission are no doubt more significant in this respect. The ring-fission reactions of both guanine and adenine sprang back into prominence relatively recently with the emphasis on oxidative attack on the imidazole ring of the purines by carcinogens (including X-rays) thought to act through OH. radicals (Kasai et al., 1986; Floyd, 1990), giving 8-hydroxypurines and corresponding formamidopyrimidines. The ubiquity of these products at comparatively high levels (of the order of 105 molecules per diploid cellular DNA content), apparently from whatever source (animal or human), continues to excite attention; for example a recent study reported markedly different ratios of oxidation products between DNAs from normal and cancerous breast tissues (Malins et al., 1993), in particular the ratio of the content of the formamidopyrimidine product from adenine to that of S-hydroxyguanine was consistently higher in the normal tissue. These oxidations are generally believed to derive from metabolically generated OH. radicals (Totter, 1980). A comparatively high level of 7-methylguanine in human DNA was reported by Park and Ames ( 1 988),but the claim was later withdrawn on the grounds that a misidentification had occurred; the compound “Z” in question does not appear to have been specified. Aberrant methylation of DNA [i.e., not at the sole site of biomethylation, C-5 of cytosine (Lawley at al., 1972)] might be expected to result from the endogenous methylating agent, S-adenosylmethionine, which can alkylate DNA in random fashion in uitro [i.e., when not under enzymic control (Rydberg and Lindahl, 1982)]. However determinations of chemical methylation

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of DNA in human tissues, which have been generally immunoassays of 06-methylguanine, a promutagenic base (see Section VI, F) have shown comparatively small extents of reaction (see, e.g., Foiles et al., 1988, for human placenta). OF MUSTARDS WITH DNA: INVIVO C. REACTION ALKYLATION AND ITS REPAIR

At the time of the first studies on methylation of DNA, the emphasis was on mustards as alkylating agents, and dimethyl sulfate (although subsequently classified as a probable human carcinogen, IARC, 1987) was not regarded as a typical “biological alkylating agent.” In a comprehensive review of the pharmacology of anticancer agents, Mandel(l959) pointed out that only one instance of biologic alkylation in animals had been reported at that time. This instance was reported by Roberts and Warwick ( 1958) who showed ethylation of thiol groups in the rat by ethyl methanesulfonate which they were studying in relation to the mode of action of myleran (busulfan, 1,4-dimethanesulfonoxybutane)a difunctional alkylating agent introduced for treatment of myelogenous leukemia by Haddow and Timmis (1953), the structure of which can be regarded as a dimer of ethyl methanesulfonate. In fact, the nitrogen mustard, HN2, had been shown, through the use of the [ ‘4C]-methyl-labeled drug, to give ‘%-labeled purines from nucleic acids after injection into rats (Wheeler and Skipper, 1957), but a specific alkylation reaction had not been demonstrated. Brookes and Lawley (1960b) used 3%-labeled mustard gas to achieve this, the preference for the 3% radioisotope being its availability (from the Radiochemical Centre, Amersham, near to the Pollards Wood Laboratories of the Institute of Cancer Research at Chalfont St Giles, where the work was carried out), at much higher specific radioactivity than the ”Glabel. Furthermore, no incorporation of 3 5 s into nucleic acids could occur, unlike the possibility that ‘4C could incorporate into the nucleic acid bases through metabolism of “one-carbon fragments” derived from labeled drugs. The reactions of mustard gas with nucleic acids in vitro and in vivo (in the mouse) were demonstrated. Two principal products were identified, resulting from monoalkylation of guanine at N-7 (this was also given by the monofunctional mustard 2-chloroethyl2-hydroxyethyl sulfide) and a product from cross-linking two guanine bases at N-7 specific to the difunctional mustard. In this way, it was established that DNA could be a target of a carcinogen in vivo and that the specific alkylations could be the cause of significant biologic effect, in this case that cross-linking of DNA could

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inactivate this essential template for cell division. Supporting evidence was obtained from a number of analogous studies using model systems, such as the inactivation of bacteriophage with double-stranded DNA as genetic material (Lawley et al., 1969). T h e difunctional mustard was much more effective in this respect than the monofunctional, and cross-links between strands and within strands were equally effective inactivating lesions. When the number of cross-links in DNA, as measured by the diguaninyl derivatives, were correlated with the doses of mustard required to inactivate cell division in bacteria, it was found that in a typical strain of E . coli, B/r, at the level of one lethal hit, there were about 72 such cross-links per genome, whereas approximately one of these sufficed to inactivate bacteriophage T7. This apparent paradox was resolved when a bacterial strain lacking ability to repair radiation-induced damage, E . coli B,- was found to be much more sensitive to the mustard, with a few cross-links per genome constituting a lethal hit (Lawley and Brookes, 1965). T h e repairing strain was shown to be capable of rapidly removing the cross-linked guanine product from its DNA, whereas the monoalkylated guanine remained, and evidently did not interfere with DNA replication to a significant extent. It was apparent from the nature of the bacterial strains used that the repair system removing cross-links was that involved in repair of ultraviolet light-induced damage, at that time recently shown to be pyrimidine dimers (Boyce and Howard-Flanders, 1964; Setlow and Carrier, 1964); the mechanism of repair of cross-links was subsequently worked out in detail by Cole (1973). It thus emerged that cytotoxic action of a carcinogen could occur at comparatively low levels of chemical damage to DNA as cellular target, levels at which the extent of alkylation of other cellular macromolecules, which were generally found to be about the same per unit weight of material, would be so small that most potential targets such as specific proteins or R N A molecules would escape chemical modification. Furthermore, not all chemical damage to DNA would necessarily have biologic effect; cross-linking of guanine was potentially lethal but monoalkylation appeared ineffectual.

D. POLYCYCLIC AROMATIC HYDROCARBONS: DNA I N VIVOTARGET

AS

Alkylation of DNA by mustards appeared to account for their cytotoxic action in terms of their specific chemical effects, but there still seemed to be little relevance to carcinogenesis. T h e mustards were late-

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comers to the scene and had been investigated because of their mutagenicity and found to be comparatively weak carcinogens experimentally [although, as noted, mustard gas subsequently became recognized as a well-established carcinogen for the human respiratory tract (Wada et al., 1968; Easton et al., 198S)l. Brookes and Lawley (1964) therefore applied methods similar to those used with mustard gas to investigate the possibility that DNA was the significant target of carcinogenic polycyclic aromatic hydrocarbons. In this case, "-labeled carcinogens of what was thought to be sufficient specific radioactivity were available from Amersham. The tissue of choice was that which was used in the previous half-century or so of the classical period of carcinogenesis studies-skin of the mouse. A spectrum of hydrocarbons was available, and their relative carcinogenic potencies had been studied extensively and expressed numerically by Iball (1939), in the form of the ratio of proportion of mice bearing tumors in a standard test involving continuous skin painting of the hydrocarbons, divided by the latent period for appearance of tumors. T h e extent of binding of the hydrocarbons to cellular constituents DNA, RNA, and protein were determined at various times (in some cases up to several days) after single applications of hydrocarbons to skin; from the preparative methods used this firm binding was judged to be irreversible, and to involve covalent reactions of activity metabolites; it was maximal after about 24 hours. T h e ratios of binding of hydrocarbons to DNA to those of RNA or protein were found to increase consistently as the carcinogenic potency increased, and maximal binding to DNA was also positively associated with carcinogenic potency. These data were considered to support the view that DNA was the significant receptor of hydrocarbons in the initiation of cancer, and that the effective mediation of this action was, as with the alkylating agents, covalent reactions between metabolites of hydrocarbons and DNA. The late Dr. Izrael Hieger, then working at Pollards Wood, and up until that time not a convinced supporter of the somatic mutation theory, expressed appreciation of our work, but pointed out that this was not the first report of binding to DNA in mouse skin of a carcinogenic hydrocarbon, referring to the publication of Heidelberger and Davenport ( 196 1 ) on 'Clabeled dibenz[a,h]anthracene. However, DNA binding was subsequently assessed as artefactual by Heidelberger's group (Giovanella et al.,1964), who regarded repressor proteins as more likely to be the significant receptors in carcinogenesis (Pitot and Heidelberger, 1963); but later the DNA reactions were substantially confirmed (Goshman and Heidelberger, 1967). In this way the mode of action of the classical chemical carcinogens was reconciled with the somatic mutation theory, in so far as initiation of

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cancer in mouse skin could plausibly be associated with induction of mutations. Furthermore, considering the demonstration by Mottram (1945) that hydrocarbons, now accepted to be mutagens, could enhance the conversion of papillomas into carcinomas, the two-mutation theory for generating malignant cancer was also supported. T h e earlier methods of cancer induction, involving continuous treatment with hydrocarbons, before the introduction of two-stage methodology, in which the hydrocarbons must also act as promoters, implied that either they had significant protein receptors like phorbol esters, or that their promotional acton derived from their cytotoxic capability. The consequences that followed from this broadening of the spectrum of mutagenic carcinogens were manifold. With regard to the hydrocarbons themselves, there was clearly a need to determine their mode of metabolic activation that enabled reaction with DNA to occur. As previously noted (Section VI, A), Boyland (1950) had suggested epoxidation as a likely possibility, in view of observations that dihydrodiols were among the metabolites of representative polycyclic aromatic hydrocarbons, not necessarily those with carcinogenic activity. Progress towards this end over the next ten years was reviewed by Lawley (1974), and, as often happens, the essential solution to this question was published shortly after his conclusion that it was yet to be found. This came from the study of the relevant aspects of the metabolism of benzo[a]pyrene by Sims et al. (1974), which showed that, somewhat unexpectedly, two successive epoxidations were necessary to enable reactivity towards DNA in viva In the interim period, no doubt reflecting this comparative complexity, attention became directed towards other types of chemical carcinogen which also required metabolic activation in order to attack DNA in vivo.

E. I N VIVOCHEMICAL MODIFICATIONS OF DNA: AND NITROSAMINES AROMATIC AMJNES Among environmental chemical carcinogens, the aromatic amines occupy a unique historical role because “they are the first human carcinogens of established chemical identity and produce cancer in the same organs (i.e., the urinary tract, principally the bladder) in experimental animals” (Hueper, 1969). The first report implicating synthetic cheniicals used in dye manufacture (Rehn, 1895) was at first largely ignored, paralleling the lack of attention to skin cancer earlier found to be caused by the, then undefined, chemical carcinogens in soot, tars, and oils. What Hueper (1969) had characterized as this “negativistic and unenlightened

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attitude” was exemplified by the report by Bachfeld (1898) that 63 cases by poisoning of aniline and other dye precursors exhibited no bladder cancers, and this group of chemicals was regarded as “bladder irritants” (Nassauer, 1920). T h e recognition of aniline dye manufacture as a carcinogenic industrial hazard eventually came from an epidemiologic study in Switzerland (Muller, 1931). Later investigators apparently still found it difficult to obtain information on the incidence of bladder cancer in the dye industry, and it was not until the reports by Case and co-workers (Case et al., 1954) that reliable epidemiologic data, specifically implicating 2-naphthylamine as a very potent carcinogen, were obtained. Experimental evidence that this aromatic amine, which was long suspected to be the most active carcinogen involved in dye manufacture [and later in the rubber industry (Case and Hosker, 1954)], could cause bladder cancer in animals proved difficult to obtain. The studies in Hueper and Wolfe (1937), using daily dosage to dogs by injection o r orally for periods of u p to 32 months, are generally acknowledged to have given the first indisputably positive results. Since, by and large, the aromatic amines and homologous amides were active systemically, the involvement of some form of metabolic activation in their carcinogenic action was more obvious than for the hydrocarbons. They also presented a more heterogeneous spectrum of chemical structures, and were found to undergo a variety of metabolic conversions. T h e key to detecting which of these pathways was significant for carcinogenesis was sought by testing metabolites to ascertain whether they could be more potent than the parent compound [i.e., whether they could be classified as “proximate carcinogens” (Miller et al., 1961)l. This approach succeeded first (Cramer et al., 1960) with 2-acetylaminofluorene. This carcinogen was first investigated because of its proposed use as an insecticide (Wilson et al., 1941); malignant tumors at various sites, mainly in liver and bladder, resulted from prolonged feeding to rats with latent periods of over 100 days. T h e site of metabolism critical for conversion of this biochemically inert carcinogenic aromatic amide into a potentially in vivo-reactive proximate carcinogen was found to be N-oxidation (Cramer et al., 1960; Miller et al., 1960); this proved to be true for carcinogenic aromatic amines and amides in general (Miller and Miller, 1969). Miller et al. (1960) pointed out that the N-hydroxylated aromatic amines could act as arylating agents, but, at neutral physiologic pH, esters such as N-acetoxyacetylaminofluorene proved to be much more reactive (Lotlikar et al., 1966; Miller and Miller, 1969). Therefore, these proximate carcinogens could be envisaged to give, as “ultimate carcinogens,” nitrenium ions,

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ArNH+, which could react either at the nitrogen atom, or as arylating agents. Evidently, they paralleled the alkylating agents in their capability to react with a variety of cellular constituents including proteins and nucleic acids. ‘They also paralleled the polycyclic aromatic hydrocarbons in that not all N-hydroxylated aromatic amines were carcinogenic, and the structural basis of carcinogenicity for both classes of carcinogen became an intriguing field of study. T h e relationship between the aromatic amines and the alkylating agents was evidenced by the demonstrations that metabolic activation of representatives of both types of chemical carcinogen can result in reactions with nucleic acids in vivo (in liver of rats). These were, for aromatic amines or amides, binding to RNA, by Farber et al. (1962) and Marroquin and Farber (1962) for 2-acetylaminofluorene, and Marroquin and Farber (1963) for dimethylaminoazobenzene. Subsequently, binding to DNA of rat liver in vivo was shown for 2-acetylaminofluorene by Sporn and Dingman ( 1966) and for dimethylaminoazobenzene and 2-naphthylamine by Roberts and Warwick ( 1966). T h e alkylating carcinogen was at that time a relatively new addition to the cumulation of types of chemical known to cause cancer, dimethylnitrosamine, (otherwise denoted as N-nitrosodimethylamine) notable for being an aliphatic compound of simple structural characteristics. While there was ample evidence that the group of aromatic amines contained carcinogens which could induce bladder cancer in both humans and animals, the evidence implicating dimethylnitrosamine as an evironmental carcinogen started from sporadic observations of its hepatotoxic action, which has been well established. Unequivocal demonstration of its involvement in the etiology of cancer in specific instances remains elusive, despite its undoubted ubiquity in the environment (see e.g., Wu et al., 1993). Its importance for developing our conceptions of carcinogenesis stems from its mode of action through the simplest of alkylation processes, methylation. As noted, the use of methylation was an obvious first choice to establish the fundamentals of the chemistry through which alkylating agents exert their biologic effects, but the agent chemically most suitable, dimethyl sulfate, was not at that time regarded as biologically significant. Application of the chemical findings and the methods developed from these to demonstrate in vivo alkylation of nucleic acids by Brookes and Lawley (1960a) to investigate the mode of action of the newly discovered carcinogen, dimethylnitrosamine, (Magee and Farber, 1962) brought in vivo chemical methylations, and subsequently the whole range of alkylations through ethylation and so on, into the forefront of mutagenesis and carcinogenesis research. T h e starting point was a visit by Drs. P. N.

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Magee and P. F. Swann to the Pollards Wood Laboratories where effective methods for isolation of DNA from rat liver and other animal tissues had been developed by Kirby (1957), and used by Brookes and Lawley (1960a), who were also able to suggest a methodology for analyzing the suspected alkylated DNA to show the presence of 7-[14C]methylguanine following administration to rats of di[ **C]methylnitrosamine. T h e comparatively high levels of the modified base induced were a consequence of the dependence of in vivo methylation on metabolic activation of the carcinogen, which occurred mainly in the liver. T h e stimulus to these historically important experiments was, initially, the suspected involvement of dimethylnitrosamine as a human hepatotoxin, which in England came from a report by Dr. H. Swaffield that two men who had worked with this “new solvent” in an industrial pilot plant had contracted cirrhosis of the liver. He approached the then Director of the Medical Research Council’s Toxicology Research Unit at Carshalton, Dr. J. M. Barnes (see Barnes, 1974) and this apparently biologically inert water-soluble organic solvent was confirmed as a hepatotoxic agent experimentally by feeding it to rats in their drinking water (Barnes and Magee, 1954). A more unexpected outcome was that rats, after prolonged dosage, sustained hepatocellular carcinoma (Magee and Barnes, 1956). It became evident that metabolic conversion to highly reactive intermediates, occurring mainly in the liver, was involved (Magee, 1956). Rose (1958) suggested that the most likely mechanism was through a-oxidation of the nitrosamine, giving diazomethane as a methylating species, and formaldehyde as a potential hydroxymethylating agent. These discoveries and their interpretation in chemical terms proved of much wider importance than merely leading to a considerable expansion in the numbers of chemical carcinogens [by the inclusion of over 300 N-nitroso compounds (Preussmann and Stewart, 1984)]. This importance derives from the clear association between initiation of cancer and chemical methylation of DNA in target tissues by diniethylnitrosamine, and of the later introduced methylating carcinogen, N-methyl-N-nitrosourea, which reacts through the same mechanism without the requirement for metabolic activation. This association does not imply that chemical modification of‘ DNA will necessarily lead to cancer. Firstly, a limited number of organs of given species have proved to be susceptible to the carcinogenic action of the very wide spectrum of various N-nitroso compounds available (“organotropism,” Druckrey et al., 1967). T h e broad reason why extensive DNA alkylation fails to elicit tumors can be expressed, in terms of the two-stage theory of carcinogenesis, as lack of adequate “promotion” (see, e.g., the review by Lawley, 1990), but we still lack knowledge of what constitutes “endogenous

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promotion.” Secondly, studies comparing the carcinogenic potency of two chemical types of methylating agents were important in showing that methylation of DNA per se was insufficient to initiate cancer (Swann and Magee, (1968). Therefore, specific modes of chemical methylation were indicated to be associated with carcinogenesis. Thirdly, the mode of metabolic activation of the nitrosamines stimulated a re-examination of the corresponding processes in the other, historically precedent, types of chemical carcinogen, in order to ascertain the underlying chemical basis of structure-activity relationships in general.

F. “ULTIMATE” CARCINOGENS: METABOLIC GENERATION OF ELECTROPHILES Much of the previous discussion on the development of theories of carcinogenesis, in so far as it involved considerations of chemicals as carcinogens, has been devoted to searching for the significant in vivo targets of the carcinogens, following Ehrlich’s concept of drug action. As noted, the early emphasis was an analogy between carcinogens and hormones, assumed to have specific protein receptors. This received experimental support from demonstrations that carcinogens did in fact undergo covalent reactions with proteins in their target tissues, notable examples being 4-dimethylaminobenzene in rat liver (Miller and Miller, 1947) and benzo[a]pyrene in mouse skin (Miller, 1951). The McArdle Memorial Laboratory at the Medical School of the University of Wisconsin thus became a major center of fundamental studies on the mode of action of chemical carcinogens, and particularly for developing the hypothesis that carcinogen-binding by proteins was associated with carcinogenesis. It was realized that these reactions were mediated by metabolism; Wiest and Heidelberger ( 1953) distinguished between the “bound compound, provisionally believed to be the ‘true’ carcinogen” and the chemically inert carcinogen as applied to the skin. The concepts of “proximate” and “ultimate” carcinogens, introduced by the Millers, and supported by their studies on N-hydroxylation of aromatic amines and amides, specified further stages in the overall process of metabolic activation of carcinogens, which became of general applicability. Thus the N-hydroxyl derivative (a proximate carcinogen) could ionize in acid solution to yield a more reactive nitrenium cation (an ultimate carcinogen); metabolically derived esters of N-hydroxylated amines would generate this ion under physiologic conditions of pH. The conversion of fat-soluble, water-insoluble carcinogens through metabolic oxidation and subsequent conjugation could be understood as detoxification through conversion to more water-soluble derivatives.

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T h e reactivity of certain metabolites towards cellular constituents was a potentially toxic consequence of a process necessary for this mode of detoxification. T h e generally positively charged nature of the ultimate carcinogens, expressed as “electrophilicity,” was very much in line with the previous description of chemical carcinogens as “radiomimetic,” and suggestive of an ion-pairing association between these reactive cations and the negatively charged nucleic acid macromolecules as a likely immediate precursor of the ultimate establishment of the covalent bond that is envisaged to mark the starting point of tumor initiation. The emphasis on electrophiles as mediators of chemical carcinogenesis thus emerged from studies with aromatic amines (Miller, 1970). This represented a slight shift from the chemical mechanism involved in the earliest demonstrations of in vivo DNA reactions using mustards. Although these agents are broadly electrophilic in the sense that they react at the same sites as the archetypal electrophile, the proton, and conversion into cationic immonium ions precedes the alkylations, they fall into the category of Ingold’s S,2 agents (Ingold, 1969; Swain et al., 1953; Swain and Scott, 1953). T h e lack of enthusiasm for alkylating agents as typical carcinogens, in contrast to the general acceptance of the polycyclic aromatic hydrocarbons and aromatic amines, which characterized the period of early development of comprehensive theories of chemical carcinogenesis, based on the somatic mutation theory, could now be somewhat justified at the fundamental level of chemical reaction mechanisms applicable to the carcinogen-induced chemical modifications of DNA envisaged to initiate cancer.

G. SPECIFIC CHEMICAL MODIFICATIONS OF DNA ARE REQUIRED TO INITIATE CANCER T h e first example of the discounting of the traditional nucleophilic centres of DNA as receptors of cancer-initiating reactions came from the comparative study of methylating carcinogens (see Secton VI, E; Swann and Magee, 1968). They found that dimethyl sulfate and methyl methanesulfonate were able to methylate DNA systemically in rats, but only a few brain tumors were obtained. In contrast, dimethylnitrosamine, through metabolic activation, methylated DNA mainly in liver and kidney, gave hepatomas on prolonged administration, and kidney tumors from single doses. This launched a search for the putative specific in vivo reaction(s) that conferred carcinogenic potency on the nitrosamine. This was revealed by Loveless (1969), who showed that deoxyguanosine, in neutral aqueous

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solution, could be methylated by N-methyl-N-nitrosourea (a directly acting analogue of dimethylnitrosamine), at the extranuclear 0 - 6 atom, in addition to the conventional nucleophilic sites at ring-nitrogen atoms. T h e significance of this finding was that the resultant methylated base, 06-methylguanine, effectively “fixed” an anomalous tautomer of the base that could form an anomalous Watson-Crick base-pair with thymine. T h e first suggestion to account for the action of alkylating mutagens as inducers of base substitutions (GC + AT transitions in the nomenclature introduced by Freese, 1959) according to this model was put forward by Lawley and Brookes (1961) on the basis that, at neutral pH, the 7-alkylguanine moiety in alkylated deoxyguanosine would be extensively ionized with loss of the proton attached to the N-1 atom, thus potentiating the anomalous base pairing with thymine. This mechanism appeared to receive support from the earliest observations that alkylating agents caused GC + AT transitions (reversions) in bacteriophage T4rII (Krieg, 1963). However, whereas all alkylating agents investigated up until that time gave 7-alkylguanines as predominant products in DNA, there were obvious differences in their potency as mutagens for bacteriophage. Thus, Loveless (1959) had already stressed that, while ethyl methanesulfonate was highly effective in this respect, methyl methanesulfonate, although toxic, was not at all mutagenic. This “uniqueness of ethylation” had t o be abandoned when Loveless and Hampton (1969) found that both N-methyl- and N-ethyl-N-nitrosoureas were powerfully mutagenic towards bacteriophage T2. But these observations could readily be explained in terms of the mispairing between 06-alkylguanines produced in DNA by the nitrosoureas, but not by methyl methanesulfonate. It thus emerged that alkylation of the DNA template could have two biologic effects: (1) inactivation, causing reproductive death; or (2) mutagenesis through replication of alkylated DNA. Evidently the various products already known to be produced in DNA by the different types of alkylating agent were specifically associated with either of these actions. Furthermore, these specificities would be expected to influence carcinogenic action; in particular, only those reactions potentiating niutation would be capable of initiating tumors. Two fields of study were thus begun: (1) the mechanism of the r-eactions of carcinogens, or their activated metabolites, with DNA invited study, with the objective of discerning the reasons why carcinogens differed in their abilities to induce the promutagenic reactions; and (2) the carcinogen-derived products in target DNA required isolation, identification, and quantitative determination, in order to permit meaningful correlation between DNA modifications and yields of tumors.

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With regard to reaction mechanisms, Ehrenberg and co-workers had already made pioneering studies with alkylating mutagens, with the objective of correlating their potency as mutagens with the established concepts of their differential reactivities developed from physical organic chemistry (Walles and Ehrenberg, 1969; Veleminsky et al., 1970). They found a negative correlation between mutagenicity and the substrate constant (s) of Swain and Scott (1953), which, in essence, gives numeric expression to the selectivity of alkylating agents. As examples, iodoacetamide (s = 1.3) reacts with macromolecules in vivo almost exclusively with the most nucleophilic site, the thiol group of proteins, and not with DNA. Therefore, it is toxic but not mutagenic. However, it can be shown to inactivate DNA repair enzymes through protein alkylation (Lawley and Brookes, 1968). Methyl methanesulfonate and dimethyl sulfate are in an intermediate category (s = 0.8). They react with DNA in viuo to about the same extent as with protein, but, as previously noted, in selective fashion; about 80% at N-7 of guanine, but only about 0.4% at 0 - 6 of guanine (Lawley and Shah, 1972). Since 7-methylguanine proved to be ineffectual as a miscoding base, in contrast to 06-methylguanine (Loveless, 1969; Abbott and Saffhill, 1979), these methylating agents are comparatively weak mutagens. It should be noted, however, that they are almost as effective as the potent mutagen N-methyl-N-nitrosourea in causing chromosomal damage, as for example, in bone marrow of mice (Frei and Venitt, 1975). Furthermore, methyl methanesulfonate, although in strains of mice not genetically susceptible to development of tumors at the most sensitive site (the thymus in young adult mice) is, as expected, noncarcinogenic (Frei, 1971), despite its ability to methylate cellular DNA throughout all tissues of the mouse (Frei et al., 1978), can enhance the rate of appearance of thymic lymphoma in the genetically susceptible AKR strain (Warren et al., 1990). In order to interpret this finding, recourse had to be made to determinations of the genetic changes (in terms of DNA analyses) of genes relevant to the carcinogenic process. This type of analysis, often involving DNA base sequence determinations of oncogenes or tumor suppressor genes, in so far as it can be related to the chemistry of carcinogen-DNA reactions, has become referred to as the study of “mutational spectra.” In view of the apparent specificity of the promutagenic site of reaction of the methylating carcinogens, essentially confined to the 0 - 6 atom of guanine, with alkylation causing GC + AT transitions through an obvious miscoding mechanism, it was not surprising that the first definitive demonstration of a mutagenic spectrum came from analysis of DNA

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of a tumor induced by N-methyl-N-nitrosourea (Zarbl et al., 1985). This was a rat mammary tumor, and the base-substitution detected was, as expected, G + A, at base position 35,in codon 12 of the r a s H oncogene. This codon, as previously noted (Section 111, B), is the principal site of the limited number at which activation of the ras genes can occur, and is also involved in about one quarter of the thymic lymphomas induced by N-methyl-N-nitrosourea in AKR mice (Warren et al., 1990). In contrast, no ras gene activation was detected in spontaneous or methyl methanesulfonate-induced thymomas, which were associated with changes induced by murine leukemia virus (Warren et al., 1987). Evidently, this latter mechanism, dependent on integrations of provirus into the host DNA, can be accelerated by the type of DNA damage induced by methyl methanesulfonate and associated with cytotoxicity rather than mutagenicity. Although considerations of the details of the various specific reactions of the methylating carcinogens with DNA are thus seen to be essential for understanding the marked dependencies of their carcinogenic potencies on their mechanisms of reaction, this was obviously less true for the polycyclic aromatic hydrocarbons, where as previously noted (Section 11, D) carcinogenicity was correlated positively simply with ability to react with the target DNA overall, without dependence on the (at that time) unknown sites of reaction (Brookes and Lawley, 1964). Nevertheless, it was, of course, important to find out what these reactions were, and the key to this clearly lay in the relevant mechanism of metabolic activation. As also discussed previously (Section VI, D), ten years elapsed before this was demonstrated by Sims et al. (1974). For the carcinogen they investigated, benzo[a]pyrene, it was possible to isolate the proximate carcinogenic metabolites, a diol epoxide, and a prodigious number of studies on this and other hydrocarbons were thus launched (see the review by Dipple et al., 1984). In the interim, significant progress was made with the aromatic amines, because the proximate carcinogens were identified earlier as the products of N-oxidation (Cramer et al., 1960). Kriek (1965) showed that N-hydroxy-2-acetylaminofluorenceattacked the C-8 atom of guanine in DNA at moderately acid pH. The ultimate carcinogens derived from this proximate carcinogen (e.g., the N-acetoxy derivative), were then shown to give this reaction at neutral pH (Kriek et al., 1967). This was the first demonstration that a chemical carcinogen could, in fact, react at a site in DNA specifically associated with attack by typical electrophiles, such as the previously mentioned OH. radicals, generated in aqueous media by ionizing radiation, first detected by Hems (1958) (Section VI, B).

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However, further work showed that this activated aromatic amide could also react at the extranuclear N-2 atom of guanine in DNA (Westra et al., 1976), and because the resultant product persisted longer in rat liver in vivo than that from attack at C-8, it was hypothesized that it was more likely to be the source of cancer-initiating mutations. Meanwhile, similar progress was being made with the polycyclic aromatic hydrocarbons. Their main site of attack, through activated metabolites was also at N-2 of guanine. The first results suggested that this came from model reactive compounds which were not in fact proximate carcinogens in viva Brookes and Lawley, in unpublished work, found that the ultraviolet spectrum of a product from deoxyguanylic acid and 1,2-dihydronaphthalene oxide showed that only a product derived from aralkylation at the N-2 atom of guanine could account for the observed data (see Lawley, 1989). Dipple and co-workers continued along the same lines, but preferred to use as the aralkylating agent a reactive derivative of the carcinogen 7-methylbenz[a]anthracene. This choice was based on the suggestion by Miller and Miller (1967) that hydroxymethyl metabolites of methyl-substituted hydrocarbons could, by analogy with N-hydroxylated aromatic amines, be proximate carcinogens. Dipple et al. (197 1) therefore synthesized, as a model for ultimate carcinogens, following this hypothesis, 7-bromomethylbenz[a]anthracene. As previously noted, in Section VI, D, this speculation and various others concerning the metabolic activation of polycyclic aromatic hydrocarbons were effectively discounted by the work of Sims et al. (1974). Subsequently, their discovery that successive epoxidations were the key to solving this problem has been extended from their first example of benzo[a]pyrene to virtually all the carcinogenic hydrocarbons (for a review of earlier work, see Dipple et al., 1984). Jerina and Daly (1976) introduced the term “bay region” to denote that part of the molecular structure of hydrocarbons at which the ultimate site reactive towards DNA in vivo is generated, as a result of the second metabolic epoxidation process. I n view of .these complexities of the processes involved in metabolic activation of the hydrocarbons, it may seem at first sight somewhat fortuitous that the use of the “wrong” model compounds as reagents did in fact lead to the correct answer with respect to their sites of reaction in DNA. Dipple et al. (1971), found that the extranuclear amino groups of the DNA bases were the most reactive sites, and this was novel in the sense that it showed the archetypal carcinogens, represented by the polycyclic aromatic hydrocarbons, to differ from both the classical alkylating carcinogens such as the mustards, attacking ring-nitrogen atoms, and the aromatic amines, thought at the time to react at the C-8 atom, a

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principal site of attack by free radicals (but later, as noted, found also to react at other sites including N-2 of guanine). H. “ULTIMATE”CARCINOGENS: THEIR STRUCTURAL REQUIREMENTS INTERPRETED THROUGH PHYSICAL ORGANIC CHEMISTRY The question why, in fact, the ultimate carcinogenic electrophiles, despite their obviously very high reactivity, were able to penetrate to DNA o f t h e nucleus from their presumed cytoplasmic sites of metabolic generation had already been raised by Dipple et al. (1968). They considered the modes of metabolic activation that had been suggested at the time for the three main chemical classes of carcinogen: dialkylnitrosamines, aromatic amines, and polycyclic aromatic hydrocarbons. For the dialkylnitrosamines the ultimate carcinogens were considered to be alkyldiazonium ions. Alternatively, if they were envisaged to owe their reactivity toward the extranuclear O-atoms thought to be particularly important for generation of the promutagenic bases in DNA, 0 6 alkylguanines (Lawley and Thatcher, 1970),or 04-alkylthymines (Lawley et al., 1973), to react through Ingold’s S,1 mechanism, the ultimate carcinogens would be carbonium (or, more correctly, carbenium) ions. [It may be noted here that the isomerization of the n-propyl group in part to isopropyl, before reaction with O-atoms in DNA of N-n-propylN-nitrosourea, has been interpreted as showing involvement of the n-propyl cation through the S,1 mechanism (Morimoto et ul., 1983).] However, these cations are expected to be far too reactive to survive diffusion from their sites of metabolism within cells to nuclear DNA, and it appeared more likely that the proximate carcinogens would be monoalkyldiazonium hydroxides. For the aromatic amines, the conjugated forms of the N-hydroxylated metabolites would be envisaged to fulfill a similar role, the ultimate carcinogens being the nitrenium ions generated therefrom in the vicinity of the DNA. For the hydrocarbons, as noted, the nature of the proximate carcinogens had not been established at that time, and the choice of derivatives from C-hydroxylation, at methyl substituents, was later found to be inappropriate. Nevertheless, and admittedly fortuitously, the concept introduced gave what appeared at the time to be significant correlations between the carcinogenic potencies of methylbenz[u]anthracenes and their chemical structures. T h e basis was that the greater the stability of the ultimate carcinogens

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as cations, the more extensive would be their reaction with DNA, because the less stable cations would be more likely to suffer solvolysis immediately after the generation of the corresponding proximate carcinogens and would not survive to diffuse into the DNA. Some experimental evidence supported the concept. In mouse skin, the carcinogenic potency of hydrocarbons appeared to correlate positively with the overall extent of covalent reaction of the hydrocarbons with DNA for a given administered dose (Brookes and Lawley, 1964). Evidently there was no dependence on the type of reaction, as found for the dialkylnitrosamines and alkylnitrosoureas when compared with the S,2 alkylating agents such as methyl methanesulfonate. [It may be mentioned here that subsequent studies showed that, for a given inbred strain of mouse, the yield of tumors (thymomas) induced by single doses of alkylnitrosoureas or alkyl alkanesulfonates showed a positive linear correlation with the extent of alkylation at the 0-6 atom of guanine in the DNA of the target tissue, but not with the extent of DNA alkylation overall (Frei et al., 1978).] For the model hydrocarbon metabolites, the principal site of aralkylation, at N-2 of guanine in DNA (Dipple et al., 1971), was confirmed for the diol-epoxides as the true proximate carcinogens for most hydrocarbons (e.g., for benzo[a]pyrene by Osborne et al. (1976); see the review by Dipple et al., 1984) but not, it should be noted, for the most potent carcinogen of this type, 7,12-dimethylbenz[a]anthracene (Dipple et al., 1983), which reacts more extensively at N-6 of adenine. Thus the overall conclusion was that aralkylating agents derived from the hydrocarbons differed more in their overall extents of reaction with target DNA than in their sites of reaction. Secondly, the more stable cations would show less selectivity towards nucleophiles and therefore would be less likely to react with thiol groups of glutathione or proteins that possess greater nucleophilicity then DNA. This consideration essentially reiterates the correlations between reactivity of alkylating mutagens and mutagenicity found by Ehrenberg and co-workers as previously mentioned (Section VI, G). T h e underlying reason why the ardlkyl cations should show a wide spectrum of stabilities was sought in terms of the distribution of their positive charge through the molecules. This was susceptible to numerical expression through a simplified mode of calculation based on Huckel’s molecular orbitals introduced by Longuet-Higgins ( 1957) and Dewar (1969). Briefly, the more delocalization of charge throughout the carbocation, the greater its stability. As noted, the choice of significant modes of activation of the hydrocarbons was speculative, even though some degree of real correlation with carcinogenicity was achieved with the nitrenium ions known at that

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time to be the ultimate carcinogens derived from aromatic amines. More extensive correlations along similar lines were subsequently reported by Ford and Herman (1992) and Ford and Griffin (1992) showing good positive correlation between stability of the nitrenium ions and the mutagenicity of 30 aromatic and heteroaromatic amines. For the hydrocarbons, the increased knowledge of the modes of metabolic activation leading to DNA reactions stimulated much further work on analogous correlations between carcinogenicity and the role of electronic effects (see the review by Dipple et al., 1984). For the classical case of benzo[a]pyrene there was excellent agreement with theory in the sense that an extensive and symmetric distribution of the positive charge was predicted for the ultimate carcinogenic carbocation derived from the diol-epoxide proximate carcinogen. The effect of delocalization of positive charge in carbocations has been associated with an alternative unifying theory of reactivity in organic chemistry, that derives from the generalized theory of acids and bases due to G. N. Lewis (see Pearson and Songstad, 1967), i.e. that the aralkylating agents with more delocalization of charge will be “softer” and therefore able to react with weakly nucleophilic centers such as extranuclear amino groups of DNA bases (Moschel et al., 1979). Alkyldiazonium ions derived from alkylnitrosoureas and dialkylnitrosamines would give carbocations with charge necessarily localized at o r near their site of reaction (i.e. are “hard” in this classification), and characterized by reaction at extranuclear oxygen such as the 0 - 6 atom of guanine. This interpretation was supported by evidence on the distribution of relative reactivities of extranuclear oxygen and nitrogen atoms towards a series of aralkylating agents, and its dependence on solvent composition (Moschel et al., 19’79). However, as might be expected from attempts to account for relatively complex biologic phenomena, such as ability to initiate cancer, in terms of a single parameter, despite the broad positive correlations there remained notable anomalies. For example, one may quote the case of picene (benzo[a]chrysene), with a structure very similar to that of the generally accepted first discovered carcinogen, dibenz[a,h]anthracene. Although predicted by molecular orbital theory to be carcinogenic, five studies failed to reveal activity, the first being reported by Kennaway (1930). Sixty years later, picene was in fact shown to be a complete carcinogen by chronic application to mouse skin, but was confirmed as only a weak initiator, with comparatively low extent of binding to skin DNA (Platt et al., 1990). The reason suggested was that the extent of metabolism of picene to the proximate carcinogenic diol-epoxide was also comparatively low. Thus, whereas the relevant carbocation derived

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from the activated metabolite did indeed possess the electronic structure characteristic of a potent carcinogen, it was not formed in the amounts generally found with its structural homologues; when the diol-epoxide of picene was tested as a mutagen, it proved to be appropriately active as predicted by theory. Analogous dependence of carcinogenic activity on the extent of appropriate metabolic activation was held to account for the relatively weak mutagenicity (and presumably therefore tumor-initiating activity) of l-naphthylamine as opposed to its very potent isomer 2-naphthylamine, whereas molecular orbital theory predicts the opposite. Dipple et al. (1968) noted this anomaly, and suggested that the N-hydroxy derivatives themselves rather than the derived nitrenium ions might be the ultimate carcinogens. However, Ford and Herman (1992) preferred the alternative explanation that l-naphthylamine is more extensively metabolized in the aromatic ring by C-oxidation rather than by N-oxidation. Once again, therefore, the nitrenium ion from l-naphthylamine would be expected to be mutagenic and carcinogenic were it, in fact, generated metabolically. I. METABOLICACTIVATION OF CARCINOGENS AND PHARMACOGENETICS

These considerations are not merely of academic significance, but have proved to be of considerable importance for the pharmacogenetic aspects of carcinogenesis in humans. The best established instance refers to induction of bladder cancer, which, as previously noted (Sections 11, B and VI, E), has been unequivocally associated with industrial use of aromatic amines. Lower et al. (1979) reported that inherited lack of ability to acetylate aromatic amines was associated with susceptibility to bladder cancer; about one half of the population is “slow acetylators” (see Clark, 1985). Cartwright et al. (1982) found that in a population of bladder cancer patients known to have been exposed to aromatic amines in the dyestuffs industry 96% exhibited the “slow acetylator” phenotype. T h e correlation between metabolism and carcinogenesis was that efficient acetylation of aromatic amines precluded the alternative mode of conversion to the proximate carcinogens N-hydroxyamines. With respect to carcinogenesis by polycyclic aromatic hydrocarbons, although, as previously mentioned (Section IV, C), there is little doubt that these are the principal carcinogens responsible for skin cancer induction by tars and oils and (as will be discussed later), in tobacco smokeinduced lung cancer, it has proved difficult to implicate specific hydrocarbons in bladder carcinogenesis, unlike the unequivocal specification

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of 2-naphthylamine. Nevertheless, the well-established pathways of oxidative metabolic activation of hydrocarbons such as benzo[a]pyrene, that potentiate its reaction with DNA, and the supporting evidence for the involvement of a carbocation as ultimate carcinogen, have stimulated interest in the pharmacogenetic aspects of cancer initiation. T h e first evidence in this regard was reported by Kellermann et al. (1973) who found that ability to induce the enzyme aryl hydrocarbon hydroxylase was enhanced in patients with bronchogenic carcinoma, suggesting that individuals with lower ability to oxidatively metabolize carcinogenic aromatic hydrocarbons would be less likely to sustain cancer through smoking (or, from the lesser risk factor of atmospheric pollution in general). Subsequently, Ayesh el al. (1984) found that patients with lung cancer showed a preponderant ability to metabolize debrisoquin, an antihypertensive drug. As with the parallel case of aryl hydrocarbon hydroxylase, they deduced that the population falls into three groups in conformity with Mendelian inheritance, homozygous low metabolizers being the least cancer-susceptible group, with heterozygotes and homozygous high metabolizers being progressively more prone to induction of cancer. Several studies followed and some failed to confirm the associations claimed. Smith et al. (1992) used a DNA-based assay which was able to detect poor metabolizers of debrisoquin through three gene-inactivating mutations in the cytochrome P450 enzme involved (CYP2DG).They did not confirm the earlier suggestions from phenotype-based assays that lung cancer patients were over-represented in poor metabolizers. However, also using an analogous genotype-based assay, Hayashi et (11. (1992) did implicate a polymorphism in the cytochrome P450 involved in metabolism of benzo[a]pyrene, CYPl A l , in susceptibility to lung cancer. They also found that this was reinforced by inherited deficiency in an enzyme involved in the conjugation of reduced glutathione with epoxide metabolites of hydrocarbons, glutathione-S-transferase 1, which had previously been found by Seidegard et al. (1986) to confer susceptibility to lung cancer. T h e overall conclusion from studies of this type, which are developing into an important field of carcinogenesis research, is, as follows. Two types of enzyme are involved in processes directed towards detoxification of chemical carcinogens: (1) Phase I cytochromes that can, in certain instances as mentioned here, lead to the modes of metabolic oxidation that potentiate reaction of ultimate carcinogens with DNA, involving only a small proportion of the total metabolites, but sufficient, if specific mutations result, to initiate cancer; and (2) Phase I1 enzymes that complement these oxidations by conjugating the potentially reactive metab-

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olites, as for example to reduced glutathione, thus obviating their reaction with DNA as potentially a dangerous target. It now appears that inherited mutations in genes for either type of enzyme can affect susceptibility of individuals to cancer induced by chemical carcinogens that act as initiators of cancer (i.e., as somatic mutagens). VII. The Advent of Mutational Spectra: Attempts to Correlate Carcinogen-DNA Reactions and Carcinogenic Genetic Changes at the Molecular Level

A. ALKYLATING AGENTS As previously noted (Section VI, B), the first attempts to deduce which mechanism of mutation would result from a given type of reaction of a mutagen with DNA were the predictions that alkylating agents would cause GC + AT transitions (Lawley and Brookes, 1961). T h e suggested miscoding base, an ionized 7-alkylguanine, proved an inappropriate choice to induce this base substitution, probably because the transience of an ionized base rendered it inadequate to change the fidelity of a DNA-polymerizing enzyme system. As proposed by Loveless (1969) the fixation of the anomalous tautomeric form of guanine (in the Watson-Crick sense) could achieve this. This proposal has stood the test of time. With regard to methylating carcinogens it thus became clear that the typical S,2 agents such as methyl methanesulfonate were weak carcinogens because most of their reactions with DNA were of little consequence biologically (e.g., alkylation at N-7 of guanine), or were deduced to inactivate the DNA template (e.g., alkylation at N-3 of adenine; Lawley and Warren, 1976). Therefore doses which would induce sufficient of the promutagenic product, 06-methylguanine, would be so high as to be lethal to the animals under test; this was demonstrated by comparative studies of induction of tumors (thymic lymphoma) in mice by a series of alkylating agents covering a spectrum of chemical reactivities in relation to the extents of alkylation of the target DNA (Frei et al., 1978; reviewed, Lawley, 1990). Methylating carcinogens that react through the methyldiazonium ion, typified by N-methyl-N-nitrosourea or N-methyl-N’-nitro-N-nitrosoguanidine (Lawley and Thatcher, 1970) turned out to be particularly significant for fundamental studies of molecular mechanisms of carcinogenesis because they induce only a single major promutagenic base, 06methylguanine (about 7% of total methylation products; the alternative

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promutagenic pyrimidine 04-methylthymine amounts to only about one-tenth of this). Furthermore, the mechanism through which this base causes mutation is obvious as an example of the fixation of an anomalous miscoding tautomer according to the scheme for mutagenesis first proposed by Watson and Crick (1953b). As already mentioned (Section VI, G), N-methyl-N-nitrosourea was the first carcinogen shown to induce tumors (mammary, in rats) by “direct mutagenesis” (Zarbl et al., 1985), since the tumor DNA resulted from a GC + AT transition mutation at base 35 in rmH. In the two-stage mechanism of carcinogenesis induced by this methylating agent and its analogue, N-methyl-N’-nitro-N-nitrosoguanidine in mouse skin, the same mutation was found in papillomas, and in some carcinomas, indicating that ras-activation occurred during initiation of the tumors (Brown et al., 1993). This specific base-substitution was also quite often found in human cancers (Bos, 1989),and it could therefore be asked whether a methylating carcinogen, such as the environmentally ubiquitous diniethylnitrosamine, was involved as a causative agent. However, it was clear that if this were so, humans must be incredibly hypersensitive to this type of carcinogen. The tumorigenic response of mouse thymus, an organ known to be perhaps the most sensitive to tumor induction by methylating carcinogens, of which N-methyl-N-nitrosourea proved to be the most potent (Frei et al., 1978), had been quantitatively related to the extent of methylation of target DNA. The extents of methylation of human DNA, as estimated by immunoassays for 06-methylguanine, were generally so low as to be undetectable (i.e., less than the order of hundreds of molecules per genomic content of DNA per cell), whereas in mice over 10,000 molecules per cell would be required to cause detectable tumorigenesis. As previously noted (Section VI, C), the extent of alkylation of cellular DNA is not per se a quantitative indicator of biologic effect, because of the intervention of repair processes which effectively clear the template of damage before its replication. This was first found for crosslinking agents, such as mustards, which induce powerfully inactivating lesions that are removable by the same repair systems that deal with pyrimidine dimers given by ultraviolet radiation. Monofunctional alkylations of DNA at N-7 of guanine (the predominant reaction) did not appear to stimulate repair, reflecting their supposed relative ineffectuality as inactivating or promutagenic lesions, whether produced by mustards (Lawley and Brookes, 1965; 1968) or by methylating agents (Lawley and Orr, 1970; Lawley and Warren, 1976). However, it was found that other monoalkylations, including the generation of the powerfully miscoding base 06-methylguanine, were rap-

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idly removed from methylated DNA in Escherichia coli cells (Lawley and Orr, 1970; Lawley, 1970), although the most rapidly removed was the second most abundantly formed base 3-methyladenine. Lawley and Orr (1970) therefore suggested that enzymatic repair of 06-methylguanine would, in mammalian cells, counteract carcinogenesis due to methylating carcinogens. This was referred to by Lawley (1970) as a “first line of defence” against carcinogenesis. Subsequently, evidence accumulated in support of this view, notably recently when Dumenco et al. (1993) transferred a gene for 0 6 methylguanine repair from human cells into mice. The resultant transgenic strain exhibited much enhanced ability to repair this promutagenic lesion, around 180,000 molecules per cell in mouse thymus, compared with, in nontransgenic mice, only a few thousand molecules. T h e transgenic mice exhibited resistance to the carcinogenic action of N-methyl-N-nitrosourea with only about 10% responding to a dose which in nontransgenic mice would yield almost 100% tumors. These experiments provide excellent support for the previous deductions that carcinogens methylating DNA through methyldiazonium ions, such as dimethylnitrosamine, are unlikely to be as potent in humans as they are in mice. Human cells in vitro (peripheral blood lymphocytes) are approximately as efficient in repair of O6-rnethylguanine as is mouse liver in vivo which is, in turn, at least five times as proficient as mouse thymus. On the other hand, mouse lymphocytes in vitro are virtually inactive in this respect (Harris et al., 1983). Various studies have been made of the ability of human cells to repair 06-methylguanine, using biopsy material. These are generally based on the assumed mechanism for removal of 06-methylguanine from DNA, as first found for Escherichia coli, viz. the transfer of the methyl group from methylated guanine in DNA to an activated cysteinyl group of an alkyl-acceptor protein (often referred to as ‘W-methylguanine methyltransferease”), which is, unlike a conventional enzyme, thereby inactivated (Olsson and Lindahl, 1980). It would be expected that individuals with lower levels of this quasienzyme would have correspondingly higher levels of 06-methylguanine in their DNA, whether this be exogenously or endogenously induced. Measurements of @-methylguanine contents of cellular DNA have so far not correlated with assays of methyltransferase (see, e.g., Foiles et al., 1988). A possible explanation is that other enzymatic repair systems can act on 06-methylguanine (cf. Bishop et al., 1993). In Escherichia coli evidence implicated the classical nucleotide excision repair system for ultraviolet-induced damage in the repair of 06-ethylguanine (Warren and Lawley, 1980) and more recently of 0 6 methylguanine (Samson et al., 1988). Bronstein et al. (1992) found that

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excision repair of 06-ethylguanine occurred also in human cells. There is also a second line of defense against mutagenesis induced through 0”methylguanine in that postreplicative repair of mismatches between this miscoding base and thymine has been detected in human tumor cell lines (Sibghat-Ullah and Day, 1992). T h e studies on methylating carcinogens have therefore proved more significant with regard to elucidating the molecular mechanisms involved in carcinogenesis than, so far, for their immediate relevance to the etiology of human cancer. There is, however, no shortage of evidence implicating other alkylating agents as human carcinogens, principally because of their extensive use in cancer chemotherapy (see, e.g., a recent review, Venitt, 1993). As might be expected from the requirement to administer doses sufficiently high to cause reproductive cell death in tumor cells, relatively massive extents of alkylation of DNA in treated cancer patients have been reported; as examples, following a cancer chemotherapeutic dose of melphalan, 40,000 mol melphalan per diploid genomic content of DNA per cell in peripheral blood lymphocytes, and 300,000 mol per cell in a plasma cell tumor (Tilby et ul., 1991). These high in uiuo extents of alkylation in humans thus attain the levels at which cancer can be induced in mice. For a sensitive inbred strain (female RFM mice) at the mean carcinogenic dose following single injections of N-methyl-N-nitrosourea, 40,000 mol of the promutagenic 06-methylguanine were induced per diploid DNA content in the thymus (Lawley, 1989). Therefore, the alkylating agents used in cancer chemotherapy constitute the most numerous group of chemicals that are regarded as “carcinogenic” to humans “according to the criteria of the International Agency for Research on Cancer of the World Health Organisation” (see IARC, 1987). However, an element of tautology exists in this classification, in the sense that alkylating agents are now generally accepted as acting through direct chemical reactions with DNA in uiuo (see Secton VI, B, C) whether as cytotoxic agents, or as mutagens and initiators of cancer. Therefore, they must in general be classified as “probable” human carcinogens even if specific studies implicating individual alkylating agents are not (yet) available (see Venitt, 1993). Some evidence that cancer chemotherapy can produce mutations in an oncogene has been reported. Carter et al. ( 1990) found nine instances of activating mutations in r a P or rmN in peripheral blood lymphocytes of patients who had been treated with chemotherapy between 3 and 13 years previously. As these authors comment, the patients at the time the mutations were detected were hemologically normal, and “clones of mu-

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tant rus-bearing cells could therefore be detected prior to any overt sign of disease.” Terada et al. (1990) investigated r a P activation in childhood acute lymphoblastic leukemia. They found activating mutations in 2 out of 15 cases, and also in a relapsed case. T h e significance of the latter was that the specific site of base substitution, in both cases G -+ A, was different from that of the original; chemotherapy might have caused this second mutation. It is evident from the previous discussions contrasting the mode of reaction of two types of alkylating agent, the S,2 agents typified by dimethyl sulfate and by the mustards, as opposed to the alkylnitrosoureas and dialkylnitrosamines reacting through alkyldiazonium ions and, in certain cases by the S,1 mechanism through carboriium ions, that the established human carcinogens are mostly in the category of S,2 agents. Their ability to alkylate the 0-6 atom of guanine in DNA, associated with ability to induce GC + AT base substitutions through the direct miscoding mechanism, is therefore expected to be limited. Thus, although this promutagenic alkylation has been detected with mustard gas (Ludlum et al., 1986a) it constitutes a small proportion of total alkylations (about 0.1%). For some cancer-chemotherapeutic alkylating agents, incorporating I-nitrosourea), the N-nitroso group, such as BCNU (1,3-bis(2-chloroethyl)alkylation at 0-6 of guanine in DNA has been shown to be a major factor in conferring cytotoxic activity (Ludlum et al., 1986b). Their distinction from the classical mustards is underlined by the different DNA repair responses that they stimulate. T h e cytotoxicity of the chloroethylnitrosoureas is opposed by the alkyltransferase mechanism that removes 06-methylguanine; this alkyl acceptor protein does not react with the larger 06-alkyl substituent introduced into DNA by mustard gas (Ludlum et al., 1986a,b). These considerations are expected to be relevant to the types of mutation induced by the cancer chemotherapeutic alkylating agents. So far, not much attention has been paid to this question, although interest appears to be reviving relatively recently (see, e.g., a review on genetic activity of mustard gas, Ashby et ul., 1991). Melphalan has been evaluated as one of the most carcinogenic agents used in cancer chemotherapy, on the basis that, in survivors from melphalan-treated ovarian cancer, the 10-year cumulative risk of acquiring a leukemic disorder was at around 11%, or about twice that of cyclophosphamide-treated patients (Greene et al., 1986). T h e molecular analysis of melphalan-induced mutations has been investigated. Both

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adenine (Wang et al., 1991) and guanine (Finley Austin et al., 1992) have been found as sites of mutation. At the aprt locus of Chinese hamster ovary cells in culture, almost half of the base-substitution mutations induced by melphalan occurred at GNC sequences [i.e., a sequence where interstrand cross-linkage has been found to be potentiated by the aliphatic nitrogen mustard HN2 (Ojwang et al., 1989; Hopkins et al., 1991)J.Cross-linkages of guanines in DNA between N-7 atoms and of guanine N-7 to adenine N-3 have been detected by chemical analyses of melphalan-alkylated DNA (Osborne and Lawley, 1993). It appears, therefore, that evidence is accumulating that alkylating agents used in chemotherapy, by alkylating DNA in patients, can cause mutations which could be involved in the induction of secondary cancers or leukemias, but, as in experimental carcinogenesis, this of itself will not suffice to cause cancer unless appropriate promotional factors are operative; these are as yet not defined. T h e GC + AT base substitutions that are by far the predominant type of mutations induced by methylating carcinogens such as N-methylN-nitrosourea or dimethylnitrosamine (see, e.g., human fibroblasts in culture, Lukash et al., 1991), are also the most prominent type of mutation found overall in the TP53 gene of human tumors (Biggs et al., 1993; Caron de Fromental et al., 1992; Hollstein et al., 1991; Jones et al., 1991). However, they are equally predominant in the mutational spectrum of spontaneous germline mutations (see e.g., Giannelli et al., 1990). Therefore, it seems unlikely that GC + A T mutations are diagnostic for alkylation-induced cancer. T h e fact that these mutations occur predominantly at CG sequences makes it much more probable that they should be ascribed to hydrolysis of 5-methylcytosine in DNA, to yield the normal DNA pyrimidine thymine, which would obviously be more difficult to correct, since only mismatch repair enzymes could effect this. ‘The first experimental support for this mechanism of spontaneous mutation came from observations of the high rate found at this site for a strain of E . coli that, unusually for this species, biomethylates cytosine (Coulondre et al., 1978).

B. POLYCYCLIC AROMATIC HYDROCARBONS As previously discussed (Section IV, C), the discovery that benzo[a]pyrene was the principal carcinogenic hydrocarbon in coal tar prompted the question from its discoverer whether this compound would prove to be an important human carcinogen (Hieger, 196 1). Only relatively recently has it proved possible to give an affirmative answer, as

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a result of a train of experimental findings which might well have surprised its originator. Chemical analyses of human carcinogens such as tars and oils could not suffice, since they proved to contain several carcinogens apart from benzpyrene. The same was true for tobacco smoke which turned out to be the most important source of exogenous carcinogenesis. However, it became possible to detect the products of reaction of DNA in uivo in human material with the activated metabolites of the aromatic hydrocarbons and amines, now widely denoted as “bulky adducts.” Although these had been extensively studied in experimental animals through the use of radioactively labeled carcinogens, the current phase of intensive detection and quantitative estimation of these adducts could not begin until a methodology was developed that obviated the use of such labeling of carcinogens. This was discovered by Randerath and co-workers (Reddy et al., 1984). Essentially it depends on the chromatographic isolation of nucleoside diphosphates derived from 32P-labeling of enzymatic digests of DNA to give 3’-deoxyribonucleotides;the aralkylated deoxyribonucleotides are resistant to further digestion with P, nuclease and can therefore be selectively labeled and chromatographed by thin layer chromatography. T h e obvious advantages are that this is a postlabeling procedure (i.e. no radioactive carcinogens are required); 32P can be obtained at very much higher specific radioactivities than 3H o r 14C previously used to label carcinogens; and all adducts are detected irrespective of their precise chemical nature (although the method as generally practiced is best suited to aromatic hydrocarbons or amines, or other “bulky” substituents). This last feature of the method is, however, disadvantageous if it is required to positively identify a specific chemical carcinogen, since the chromatographic methods used, thin-layer chromatography, generally give a multiplicity of spots from human DNA obtained from tissues such as lung, where continuous exposure to environmental mutagens is inevitable. In order to specifically identify the chemical nature of these products, generally further chromatography, such as the use of high performance liquid chromatography (HPLC) is required (Pfau and Phillips, 1991). Studies along these lines have already become prodigious in number, and space permits here mention only of two, with regard to the etiology of smoking-induced lung cancer. Phillips et al. (1988) reported a positive linear correlation between the amount of adducts in lung DNA samples, determined by 32P-postlabeling, and the number of cigarettes smoked. Heavy smokers (40 per day) produced on the order of 2,500 adducts per

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genome, around ten times the levels found for nonsmokers. For heavy smokers the order of magnitude of the overall level of DNA modification in their lung cells thus approached the value of the mean tumorigenic dose for induction of papilloma in skin of a sensitive strain of mouse (SENCAR) by benzo[a]pyrene, of around 10,000 mol of the N-2 of guanine adduct per genome (Ashurst et al., 1983). However, it must be remembered that the experimental study used phorbol ester as a powerful promoter. Therefore, as previously discussed (Section 11, C), human lung may contain cells which are particularly susceptible to promotional factors, once mutated, as for example by ras activation. This latter is known to be involved in the initiation of skin cancer in mice (Brown et al., 1990), since papillomas induced by both aliphatic alkylating agents and aromatic hydrocarbons exhibit M S mutations, characteristic of the chemical action of the carcinogens on DNA. Alexandrov et al. (1992) devised a direct method for determining benzo[a]pyrene diol-epoxide-DNA adducts, in nontumorous parenchyma from lung cancer patients, by hydrolysis of DNA in 0.4M-HCI to yield benzo[a]pyrene 7,8,9,10-tetrols, which were isolated by HPLC and determined fluorometrically. T h e principal results showed that, in most cases, the major part of the bulky adducts found using the 32P-postlabeling method were accounted for as derived from benzo[a]pyrene; also, a positive correlation was found between the benzo[a]pyrene-derived adducts and aryl hydrocarbon hydroxylase activity. T h e latter finding wasjudged to be surprising, that in view of the multiplicity of cytochrome P-450 species that could be involved, CYP 1Al proved to be the one predominantly involved in mediation of adduct formation. These data provide an excellent answer to the question posed by Hieger (1961) as to whether benzo[a]pyrene would prove to be a significant contributor to the totality of causative agents in human carcinogenesis; evidently the chemical analytical data support the view that it is a quantitatively important specific chemical carcinogen involved in the action of cigarette smoking as the major exogenous source of cancer. T h e chain of events leading to lung cancer is thus extended to the promutagenic aralkylation of DNA in a target tissue in man. It can be further extended through considerations of mutagenic spectra, especially when these are compared for tumors in different organs. A priori, the demonstration that aralkylating carcinogens react predominantly at N-2 of guanine in DNA, in contrast to aliphatic alkylating agents (including epoxides; see Lawley and Jarnian, 1972) that react predominantly at ring-N atoms, gave no obvious indication that they would be powerfully mutagenic. With the aliphatic agents, mutagenicity

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proved to be specifically associated with those agents capable of more extensive reactions at extranuclear 0-atoms, 0 - 6 of guanine and to a lesser degree, 0 - 4 of thymine (Lawley et al., 1973). In these latter instances, obvious mispairings according to the scheme of Watson and Crick ( 1953b) could be envisaged, of alkylated guanine with thymine and of alkylated thymine with guanine. Aralkylation at extranuclear N-atoms (Dipple et al., 1971) did not suggest any obvious mispairing along these lines. However, studies of the mutagenic action of aralkylating agents [e.g., both of the 7-bromomethylbenz[a]anthracene of Dipple et al. (197 1) and the benzo[a]pyrene diol-epoxide of Sims et al. (1974)] soon showed that they were indeed potent in this respect. They proved to be very efficient mutagens since they could produce mutations (in the hprt gene of cultured Chinese hamster lung cells), at extents of aralkylation of cellular DNA that were low enough to cause negligible cytotoxicity (Newbold and Brookes, 1976). This outstandingly high mutagenic efficiency contrasted with their ability to induce lesions in DNA that were effective blocks to DNA synthesis, as shown by the action of these aralkylating agents on bacteriophage T7. At the mean lethal dose, about six aralkylations in DNA sufficed to inactivate, as with the cross-links induced by mustards, but the aralkylating agents were monofunctional, not difunctional, in their reactions (Venitt and Shooter, 1972; Shooter et al., 1977). T h e question thus arose of how to reconcile these, at first sight, contradictory modes of action: ability to inactivate, very efficiently, a DNA template, but also ability to induce mutations, apparently through the same types of aralkylation reactions, mainly at N-2 of guanine in DNA. It was found that mutations induced by the aralkylating agents were mainly the transversion type of base substitution. This was first shown by Eisenstadt et al. (1982) for mutations induced in the lac I gene of Escherichia cob. Fifty-six percent of the mutations from benzo[a]pyrene diol-epoxide were GC -+ TA transversions. Subsequently, initiating mutations induced by the hydrocarbon 3-methylcholanthrene, activating the rasHgene in mouse skin carcinogenesis, were shown to be mainly GC+ TA, with some AT+ TA transversions,whereas 7,12-dimethylbenz[a]anthracene gave predominantly AT + TA transversions (Brown et al., 1990). This difference reflects the greater tendency of the dimethylbenzanthracene to react at adenine in DNA, in contrast to the more general preference of hydrocarbon carcinogens for aralkylation of guanine. In line with their effectiveness as blocks to DNA synthesis, aralkylationinduced lesions in DNA respond to nucleotide excision repair [i.e., the repair system that removes ultraviolet light-induced pyrimidine dimers and cross-links (Venitt and Tarmy 1972; Dipple and Roberts, 1977;

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reviewed by Roberts, 1978)l. Evidently, mutations induced by aralkylating carcinogens also result, from inadequacy in the repair process as for other template-inactivating mutagens. T h e lesions in DNA that result from mutagens of this type have been classified by Strauss (1992) as “noninstructional,” on the grounds that they “cannot readily form Watson-Crick base pairs,” in contrast, for example with other promutagenic bases, such as OG-methylguanine, which is able to form a mis-pair with thymine, that he terms “instructional.” T h e studies by Strauss et al. (1982, 1986) on the replication of DNA templates containing noninstructional lesions by DNA polymerizing systems led to the generalization that the polymerase can insert a base opposite to such a lesion, thus bypassing unrepaired damage. But, the lack of appropriate hydrogen-bond formation according to the Watson and Crick (1953b) scheme potentiates the introduction of an anomalous base. These noninstructed insertions were found to be not entirely at random. In the majority of cases, adenine was the base favored by the polymerase. It will be noted that if the lesion was a thymine dimer, the most likely modification of DNA induced by ultraviolet radiation, insertion of adenine opposite the unrepaired lesion would not result in mutation. Strauss ( 199 1, 1992) refers to this principal source of mutations from the unrepaired noninstructional lesions as “the A rule.” As he points out, it does not account for every instance of mutation due to this causative factor, but it does account for the majority, and has proved useful in the interpretation of mutational spectra observed for the burgeoning data on specific base substitutions found in oncogenes and tumor suppressor genes with regard to their possible induction by carcinogens. Thus, in lung cancer, the evidence obtained points to benzo[a]pyrene as the most likely specific carcinogen involved (although, no doubt, there are many others). T h e spectrum of mutations in the TP53 gene of lung cancers shows that CC + TA transversions predominate, accounting for almost half of those observed. This contrasts with the data for colorectal cancer, where this type of base substitution is comparatively rare. Conversely, GC + AT transitions are predominant for colorectal cancer (around 80% of the total). As discussed in the previous section, these are thought to derive mainly from hydrolysis of 5-methylcytosine in DNA to thymine, a process studied in some detail by Rydberg and Lindahl (1982); see also Ehrlich et al. (1990). In this way a chain of evidence at the molecular level can be traced from ingestion of benzo[a]pyrene, through its metabolism and reaction with target DNA, to the misincorporation of adenine opposite N-2aralkylated guanine in the DNA template of a stem cell that ultimately,

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through some form of promotionally stimulated cell division, and further mutation(s), develops into a malignant cancer. This concept envisages that mutagenesis by the chemical carcinogen is the critical step in carcinogenesis, as deduced from interpretation of epidemiologic data (Stein, 1991), and also fits in with current knowledge of the pharmacogenetics of lung carcinogenesis (see Section VI, I). It leaves the reasons why human lung should be markedly susceptible to the induction of cancer by chemical mutagens out of consideration, except, in so far as required by the two-stage theory of carcinogenesis (Moolgavkar and Knudson, 1981), some factors giving efficient tumor promotion, indicated to be predominantly endogenous (Moolgavkar et al., 1989),can be deduced to operate. A comparison between the induction of lung cancer through cigarette smoking and skin cancer induced by chemical carcinogens, notably again with polycyclic aromatic hydrocarbons as the expected specific agents, would appear to be potentially instructive. Some of the data relevant to such a comparison are available. Exposure to the classical environmental sources of skin cancer is expected to result in formation in DNA of adducts derived from hydrocarbons. For example, Carmichael et al. (1991) found that used lubricating oils were able to induce DNA adducts in human skin maintained in short-term culture. Mutations in TP53 have been detected in squamous cell neoplasia of the skin, and in preinvasive lesions (Bowen’s disease) (Campbell et al., 1993),suggesting that these are early events; the mutations show clustering at pyrimidine dimer sites with C + T transitions predominating (i.e., according to the “A rule” previously discussed, are consistent with cytosine-containing dimers induced by sunlight as the source of mutation.) Therefore, mutations induced by hydrocarbons, which act as mutagens essentially in the same way as ultraviolet radiation through production of noninstructional lesions in DNA, would also be expected to be effective in this way. However, an experimental attempt to induce tumors in human fetal or adult skin, grafted onto the skin of nude mice, followed by promotion with phorwith 7,12-dimethylbenz[a]anthracene, bol ester, gave murine tumors only, in mouse skin tissue adjacent to the grafts (Graem, 1986). On the assumption that adducts were formed in the human DNA, this result suggests that human skin is resistant to tumor promotion. [Mouse skin is not invariably sensitive to promotion. T h e C57BL strain is resistant to both phorbol ester and to promotion by skin wounding (DiGiovanni et al., 1993).] It is therefore indicated that human skin, continually subject to the DNA-damaging action of sunlight, may be more resistant to tumor promotion than human lung. However, the origin of the widely observed

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phenomenon of organotropism (i.e., the marked differences in susceptibility to induction of tumours in different organs of different species that are observed even when the same level of modification of DNA by carcinogens has been induced) remains as yet almost entirely obscure.

C. AFLATOXIN B, T h e earliest associations between chemical carcinogenesis and human cancer came from the observations on local action of carcinogens on skin. As previously noted (Section 11, B), the discovery of chemical induction of bladder cancer showed that systemic action of carcinogens could be at least as important. A further significant indication of systemic chemical carcinogenesis came from observations that among the South African Bantus cancer of the liver was prevalent at unexpectedly high levels; for example, in workers medically examined in connection with their recruitment for employment in gold mines (Bergman, 1941). This report and others, showing high incidences in Southeast Asia led Kennaway (1944) to the view that the causative factor was not hereditary, but most likely to be a dietary carcinogen. T h e first clue to the nature of the principal specific carcinogen involved came from a doubtless unexpected source, when Sargeant et al. (1961) reported on the deaths of about 100,000 turkeys, after their being fed Brazilian groundnut meal that contained a powerful hepatotoxin. It emerged that the nuts were infected with the mold Aspergzllus ~ U V Z L Sa, source of several mycotoxins (Lancaster et al., 1961), of which aflatoxin B, proved to be both toxic and carcinogenic when tested in rats. T h e first suggestion that dietary mycotoxins could be an etiologic factor in liver cancer was made by Le Breton et al. (1962). Subsequently, Oettle ( 1965) deduced from epidemiologic studies that contamination of inadequately stored foodstuffs with toxigenic molds was the most likely source of the high incidence of liver cancer in certain areas of South Africa. Many more detailed studies followed implicating ingestion of aflatoxin B, as a causative agent, on the basis that positive correlations were found between aflatoxin content of foodstuffs and local incidences of liver cancer (see IARC, 1993). T h e early view of the mechanism of action of these mycotoxins was that they were powerfully complexed with DNA (Clifford and Rees, 1966), by analogy with the carcinogenic action of the antibiotic actinomycin D (DiPaolo, 1960). However, it became clear that a better analogy was with the carcinogenic aromatic hydrocarbons, because aflatoxin B, is activated by metabolic oxidation to an epoxide that reacts with DNA (Martin and Garner, 1977). T h e analogy is incomplete, since the

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proximate carcinogen is an aliphatic epoxide, and in fact this is to be regarded as the ultimate carcinogen, since its principal reaction, like that of aliphatic epoxides of simpler molecular structure, is at N-7 of guanine in DNA (see the review by Garner, 1978). This raises the question why aflatoxin B is such a potent carcinogen, since aliphatic epoxides in general are at most weakly carcinogenic; in fact, whether ethylene oxide is carcinogenic in man, despite the occurrence of industrial exposures, is still uncertain (see, e.g., Bisanti et al., 1993; Wong and Trent, 1993). The reason became clear when determinations were made of the extent of reaction of aflatoxin B ,, mediated by metabolism, in DNA of liver of rats (Garner and Wright, 1975). The carcinogen-binding index, i.e., the ratio of extent of alkylation, expressed for comparative purposes in units of kmol carcinogen per mol DNA-P, divided by the administered dose (mmol/kg), was over 30,000 (Croy et al., 1978). This explains immediately why the mycotoxin exerts hepatoxic action at very low doses (of the order of 1 pmol/kg), since the corresponding binding indices for carcinogenic hydrocarbons in skin of mice were about 150 for benzo[a]pyrene to about 300 for 7,12dimethylbenz[a]anthracene (Phillips et al., 1979), and for N-methylN-nitrosourea (referring to methylation of DNA at 0-6 of guanine) 18 for the thymus of the mouse (Frei et al., 1978). Evidently the epoxide metabolite owes its high specificity reactivity to its high affinity for complexing with DNA prior to covalent reaction. Subsequently, another specific feature of this reaction has been suggested to be significant for the mode of action of aflatoxin B,, both as a mutagen and carcinogen. In brief, this involves a remarkable instance of high probability of a mutagenic base substitution at a specific site (hot spot), in this instance a GC + T A transversion at the third base of codon 249 in the TP53 gene, observed in DNA of around 50% of hepatocellular carcinomas from areas geographically associated with the implication of aflatoxin B , as a dietary carcinogen (Bressac et al., 1991; Hsu et al., 1991). Several reports followed showing that mutations in TP53, and this specific base substitution in particular, are not commonly associated with liver cancers from other locations; for example, in a study of British cancers, only 2 out of 19 showed TP53 mutations and neither occurred at codon 249 (Challen et al., 1992). However, despite its limited significance from the epidemiologic point of view, this association between a specific human carcinogen and a specific cancer mutation, defined at the molecular level, is clearly destined to become a classical landmark in the historical progress of carcinogenesis studies. As might be expected from previous remarks, alkylation or aralkylation of DNA in a target organ has been deduced to be necessary, but not

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sufficient, for carcinogenesis by mutagenic chemicals (cf. Frei et al., 1978), and this appears to apply to aflatoxin B , since there is evidence that ingestion of the mycotoxin by itself is not always positively associated with liver cancer incidence. Cirrhotic damage to the liver has long been regarded as a prominent risk factor for tumorigenesis, but this is equally common in Europe o r the United States as in Southern Africa; Steiner (1960) was the first to note that a probably significant difference was the predominance of viral infectious hepatitis as a causative factor of cirrhosis among Africans. Eventually, hepatitis-B became established as the principal etiologic viral correlate with liver carcinogenesis in South Africa (Sherlock et al., 1970). In some studies, positive correlations between cancer incidence and hepatitis-B virus were established without a corresponding association with levels of dietary aflatoxin B, (e.g., certain areas in China, Campbell et al., 1990), but, in another, a positive correlation with aflatoxin B, and a multiplicative effect between level of ingestion of the mycotoxin and hepatitis-B virus infection was reported (Yeh et al., 1989). A current consensus (IARC, 1993) expresses the view that “although epidemiological studies show that hepatitis-B viral infection is intimately linked with the development of hepatocellular carcinoma in high-risk populations, the underlying molecular mechanism is unknown.” Molecular biologic studies have recently prompted a hypothesis to account for the supposed synergism between the chemical and viral carcinogens involved, that a protein coded for by hepatitis-B virus may complex with wild-type p53 protein, thus complementing aflatoxin B I-induced mutation of the second copy of this gene (Hsu el al., 1993).

,

EVALUATION OF MUTATIONAL SPECTRA D. OVERALL It will be evident from the preceding sections that current interest in mutational spectra of cancer-associated genetic changes, particularly in TP53, has already provided some of the most impressive evidence supporting the somatic mutation theory of carcinogenesis. This evidence relates in part to avoidable causes of cancer, exemplified by cigarette smoking and ingestion of mycotoxin from inadequately stored foodstuffs. However, the principal impact has been to establish that most cancer initiation cannot be attributed to exogenous mutagens, despite their acknowledged ubiquity in the environment, as shown by a vast array of data made available by the development of rapid screening tests for such mutagens, notably by Ames and his co-workers (Ames et d., 1975). During the subsequent decade, it emerged that, as a result of rapid screening of industrial chemicals which suggested the need for

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their testing as possible carcinogens in rodents, 212 compounds were classified as rodent carcinogens, about half the total number tested (Gold et al., 1989). Likewise, about the same proportion of a smaller number of naturally occurring chemicals also proved positive in these tests. Voices were raised challenging the interpretation of data of this type that mutagenic chemicals in the environment might constitute a major causative source of human cancer. As an example, reference was made to the comparative quantitative importance of an unavoidable environmental mutagen such as ultraviolet and solar radiation. Hereditary syndromes were known that conferred marked sensitivity to the effects of sunlight, including skin cancer in the disease xeroderma pigmentosum; for some forms of the disease, an inherited deficiency to repair ultraviolet-induced damage in cellular DNA was found (Cleaver, 1968). Since most exogenous mutagenic carcinogens are analogous to ultraviolet radiation, in that they induce noninstructional lesions, repair of chemical carcinogen-induced DNA damage would also be expected to be deficient in xeroderma pigmentosum patients. However, as German (1979) pointed out, this repair deficiency appeared to produce no additional cancers of internal organs, only of skin cancers. He therefore concluded that exogenous mutagens “do not regularly reach body tissues in significant amounts.” This view appears to be supported by determination of bulky adducts in DNA of people in general; with the use of the highly sensitive 32P-postlabeling technique, observed levels (e.g., in human blood lymphocytes) are comparatively small, of the order of 100 to 1000 molecules per diploid genomic cellular content of DNA (Phillips et al., 1986). Nevertheless, theoretical and experimental studies predict that continuous exposure to low levels of mutagens should cause initiation of cancer according to a linear dose-response relationship (see e.g., dialkylnitrosamines and carcinogenesis in liver of rats, Pet0 et al., 1984). This distinguishes mutagenic (genotoxic) carcinogens from nongenotoxic carcinogens, which are usually promoting, rather than initiating, agents (see, e.g., Butterworth and Slaga, 1987), and exhibit no-effect thresholds. Evidently the effective doses of environmental mutagens generally encountered must be assessed as low in comparison with the massive doses (“maximum tolerated”) used in conventional tests for carcinogenicity of chemicals. At these high doses, the contribution to carcinogenic effect of the linear low-dose portion of the dose-response relationship has given way to a “multihit” relationship, with the cytotoxic effects of the chemicals able to contribute to a promotional action (see, e.g., Ames and Gold, 1990; 1991); linear extrapolation from high doses would therefore lead to a more exaggerated estimate of the effect of low doses.

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The data from mutational spectra to some extent supplement these deductions. As discussed in the previous sections, the chemistry of the reactions of most exogenous chemical carcinogens through their metabolic activation results in the formation of noninstructional lesions in DNA, mainly in purines, that cause the transversion type of basesubstitution mutations. These are the minority in the totality of' mutations associated with human cancer, which as previously noted (Section VII, A) are mainly GC + AT transitions consistent with deamination of cytosine or 5-methylcytosine as the causative chemical modification of DNA. Transversions (most frequently GC + TA) are found to be more prominent in cancers of organs that are associated with exogenous mutagens, such as lung, and to lesser extents, bladder and esophagus. However, not all molecular lesions in DNA that cause transversions should be ascribed to exogenous carcinogens. A promutagenic base induced by oxidizing carcinogens, including x-rays, and also by OH. radicals thought to be generated by endogenous oxidative metabolic processes, 8-hydroxyguanine (Floyd, 1990) has been shown by molecular biologic studies to cause GC + TA transversions. Unlike 0 6 methylguanine, which has a miscoding frequency close to unity (Abbott and Saffhill, 1979), 8-hydroxyguanine in a DNA template forms a Watson-Crick mis-pair, with adenine, infrequently (at around 0.1 % to a few percent of replications, depending on the nature of the polymerase; Moriya, 1993; Wood et ol., 1992). Also, since 8-hydroxyguanine is one of the most abundant chemically modified bases, found in all samples of DNA in amounts exceeding 100,000 molecules per diploid genome (Malins et al., 1993; Olinski et al., 1992), it is not surprising that it stiniulates highly effective DNA repair systems, capable of removing the promutagenic base itself, and the adenine partner in mispairs it induces (Tchou and Grollman, 1993). Nevertheless, some GC + TA transversions found in cancer-associated mutations may be due to oxidative modification of DNA, rather than to exogenous mutagens that induce bulky adducts. A particularly intriguing question is raised by the observations that breast cancer, as assessed through mutational spectra, occupies a position intermediate between lung cancer (with about 40% of TP53 base substitutions found to be GC + TA transversions) and colon cancer (with virtual absence of GC + TA transversions); breast cancer has around 20%. T h e sole exogenous carcinogen known to cause breast cancer is X-irradiation. The outstandingly high sensitivity of the developing breast t o ionizing radiation is well illustrated by a study of chil1989). dren who received scalp irradiation for ringworm (Modan et d., This showed that girls in the age group 5 to 9 had a relative risk o f

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developing breast cancer of 12 times that of controls, following a dose to the breast calculated to be as low as 0.016 Gy; only in this age group, where presumably promotional factors associated with proliferation of radiation-initiated cells were at their highest, was there appreciably increased risk. From this and other studies also showing the specific risk to breast tissue in young women, Fentiman (1990) expressed the view that “radiation-induced mammary tumors may be more than a model for breast cancer; they may hold the key to the DNA damage responsible for the disease.” This theme has also been followed using the approach from studies of chemical analyses of DNA in breast cancer tissue is compared with that from adjacent noncancerous tissue. The first report found that the content of 8-hydroxyguanine in breast cancer was higher than in normal breast (Malins and Haimanot, 199 1). Subsequently, the outstanding difference was found to be that normal breast DNA showed a much higher ratio of content of the imidazole-ring-opened adenine base, 2,6diamino-5-formamidopyrimidineto that of 8-hydroxyguanine, when compared with breast cancer (Malins et al., 1993). T h e question remains therefore whether these three strands of data: ( 1 ) the unexpectedly high proportion of GC + TA TP53 mutations; (2) the high sensitivity of breast tissue in young women to X-rays; and (3) the changes in the distribution of bases in DNA thought to derive from the action of OH. radicals, are causally connected. Possibly the cells in which breast cancer can be initiated are particularly susceptible to the mutagenic action of OH. radicals, for example through relative deficiency in repair of oxidation damage. Inability to repair radiation-induced DNA damage has provided a classical instance of the concept that DNA is a critical target of carcinogens, evidenced by susceptibility to skin cancer in xeroderma pigmentosum, as already noted. Since, as previously pointed out, unrepaired solar radiation-induced thymine dimers would be less likely to cause mutations than those involving cytosine (according to the “A-rule”),the noninstructional lesions induced by this type of radiation are predicted to be C += T transitions. These could also be caused by deamination of cytosine, but two lines of evidence favor sunlight as the relevant mutagen. First, the C T transitions were rarely at CG sequences, unlike those in spontaneous mutations where hydrolysis of 5-methylcytosine was preferentially involved. Even more conclusive was the frequent occurrence of tandem mutations CC += TT (see, e.g., Brash et al., 1991, for squamous cell carcinoma of the skin); these would be expected to result from replication of DNA containing unrepaired cyclobutane-type dimers, or the so-called (6-4) photoproducts (Brash and Haseltine, 1982;

-

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Wang and Varghese, 1967) known to be induced at CC sequences by ultraviolet radiation. In summary, mutations of specific types have been associated with both exogenous chemical mutagens and with apparently unavoidable and ubiquitous sources of mutation, such as radiations and endogenous metabolically generated OH. radicals. From knowledge of the chemistry of the effects of these agents on DNA, it has proved possible to trace back a causative chain of events, through analysis of tumor DNA, to the nature of the most likely agent that initiated the tumor, or contributed to a second or subsequent mutation involved in the progression to malignancy. VIII. Summary and Conclusions

T h e first attempts to understand the causes of cancer were based on generalizations of what might now be termed a “holistic” nature, and hereditary influences were recognized at an early stage; these views survive principally through a supposed positive connection between psychological factors such as stress and diminished ability to combat the progressive development of tumors through some form of immunologically mediated rejection of potentially cancerous cells. While evidence for immunosurveillance is generally accepted, it is now widely regarded as almost wholly confined to instances where tumor viruses are involved as causative agents. T h e earliest theorists drew an analogy between the processes of carcinogenesis and of evolution; the cancer cells acquired the ability to outstrip their normal counterparts in their capacity for proliferation. This was even before evolution had been interpreted as involving a continuous succession of mutations. Evidence was already to hand before the end of the 18th century that exogenous agents, notably soot, a product of the “industrial revolution,” could cause skin cancer. Somewhat over 100 years later, another industrial innovation, the manufacture of synthetic dyestuffs, implicated specific chemical compounds that could act systemically to cause bladder cancer. Meanwhile, the 19th century saw the establishment of the fundamentals of modern medical science; of particular relevance to cancer was the demonstration that it involved abnormalities in the process of cell division. T h e commencement of the 20th century was marked by a rediscovery of the concept of mutation; and it was proposed that cancer originated through uncontrolled division of somatically mutated cells. At around this time, two further important exogenous causative agents were discovered: X-rays and tumor viruses. In the late 1920s,

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x-radiation became the first established exogenous cause of mutagenesis. The discoverer of this phenomenon, H. J. Muller, suggested that while mutation in a single cell was the primary causative mechanism in carcinogenesis, its generally observed logarithmic increase in incidence with age reflected a “multihit” process, and that multiple successive mutations were required in the progeny of the original mutants. He also recognized that the rate of proliferation of potentially cancerous cells would markedly influence the probability of their subsequent mutation. These considerations are essentially the foundation of the generally accepted view of carcinogenesis that now seems unlikely to be superseded. However, this acceptance did not come about unopposed. T h e analogy between carcinogenesis and evolution was disliked by many biologists because it embodied the concept that cancer was an inevitable consequence of our evolutionary origins. Opponents of the somatic mutation theory of cancer were able to derive support from the intensive studies of chemical carcinogenesis that began at around the same time as Muller’s discovery of the mutagenic action of X-rays. Although earlier theorists had generally classified what we now recognize as chemical carcinogens as acting through a physical process of “irritation,” the burgeoning advances in organic chemistry during the 19th century stimulated interest in the possibility that the carcinogenic action of the classical agents of that period, soot, tars, and oils, could be ascribed to specific chemical compounds, and the methods for isolating and identifying these were at hand. Kennaway, acknowledging the pioneering studies of Berthelot over half a century earlier, became convinced that these were polycyclic aromatic hydrocarbons. Joined in the mid-1920s by Hieger, Kennaway gave the first demonstration of carcinogenic action of a pure chemical compound of this type in 1930. Three years later, the isolation of the principal carcinogen from tar, benzo[a]pyrene, still the best known chemical carcinogen to the layman, was announced by Kennaway’s group. Soon, several other carcinogenic hydrocarbons were synthesized and the question arose of why this class of compound should, as appeared then, have the chemical features uniquely conferring carcinogenic potency. Structural analogies with the recently discovered steroids prompted Cook, the leading chemist of Kennaway’s team, to hypothesize a significant biochemical link between the action of carcinogenic aromatic hydrocarbons and hormones. This hypothesis became less attractive when chemical carcinogens of other molecular structural types were revealed during the later part of the 1930s, in particular aromatic amines, analogous to the carcinogens involved in the synthetic dyestuffs industry. However, their mode of

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action showed some analogy to that of hormones when the Millers found that the carcinogenic dye “butter yellow” was covalently bound to proteins of its target organ, liver of rats, as reported in 1947. In the meantime, the announcement was made that chemicals could act as mutagens. This was discovered by Auerbach and Robson at the beginning of the Second World War, but since the agent involved, mustard gas, was of military use, it was thought prudent to delay publication, thus the first report being made in 1946. T h e reason for testing this chemical was that its vesicant action resembled that of the known mutagen, x-rays. According to the somatic mutation theory of carcinogenesis, it should prove to be a carcinogen. Unfortunately for this theory, its activity in this respect in conventional tests was, at most, weak. This matched the weak, and generally discounted, mutagenic activity of the carcinogenic hydrocarbons. Furthermore in a conventional test of induction of skin cancer by these established carcinogens, mustard gas inhibited their action. T h e carcinogenic effect of the hydrocarbons could be enhanced by so called co-carcinogens, themselves noncarcinogenic, the most notable of which, croton oil, was discovered by Berenblum in 1941. T h e tumor-initiating action of the carcinogenic hydrocarbon turned out to be irreversible, that of the croton oil (later denoted at tumor promotion) being reversible, and involving some form of selective hyperplastic action. T h e decade of the 1940s saw significant advances towards an interpretation of the process of mutagenesis at the molecular level. Avery and co-workers showed that the genetic material was DNA, rather than protein. A few studies of the mode of action of mustard gas and its nitrogen mustard analog, introduced at the time as a cancer-chemotherapeutic agent, began to be made, involving DNA as a likely receptor because of the mutagenic action ofthis type of compound. In view of the high toxicity of these mustards, it was envisaged that their extent of reaction would be low, but might be detectable through the use of their radioactive labeling. Brookes and Lawley in 1960 showed that :%-labeled mustard gas gave the expected reaction at N-7 of guanine of nucleic acids in mice, and using similar methods with 1%-labeled dimethylnitrosaniine, Magee and Farber (1962) showed the analogous alkylation reaction in the target organ of this systemically acting carcinogen in the liver of the rat. T h e significance of DNA as a target for reaction of chemical carcinogens was further confirmed by parallel studies using the liver carcinogen 2-acetylaminofluorene by Farber and co-workers in 1962, and by the correlation between carcinogenic potency of a series of hydrocarbons and their extent of reaction in mouse skin by Brookes and Lawley in 1964. These chemical studies removed any obstacle to recognition of the

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concept that initiation of cancer, as previously deduced from epidemiologic considerations, should be equated with mutagenesis. Further studies have reinforced this interpretation. One area of significant investigation covered the biochemistry of how carcinogens could be activated through metabolism directed towards their detoxification but, in specific instances, leading to deleterious chemical modifications of DNA. Notable among these were the implication in this respect of N-hydroxylation of aromatic amines and amides by Cramer, Miller, and Miller in 1960, and that of epoxidation of polycyclic aromatic hydrocarbons by Booth and Boyland in 1949, leading to the specification of the requirement for two successive epoxidations by Sims and co-workers in 1974. Interpretations of mechanisms through which the observed chemical modifications of DNA by carcinogens could cause mutations derived from the molecular models for the structure and replication of DNA due to Watson and Crick (1953a,b), and began in the 1960s. T h e first satisfactory implication of a carcinogen-modified base in DNA as a source of miscoding was 06-methylguanine, as demonstrated by Loveless in 1969. T h e mechanism was obvious in terms of anomalous hydrogenbonding according to the Watson-Crick model, and resulted in GC + AT transition mutations. Confirmatory studies showed the validity of this concept in numerous instances of experimental mutagenesis and carcinogenesis. However, most carcinogens, including aromatic amines and hydrocarbons, did not conform to this type of mechanism, although their biologic action was predominantly mediated through induction of basesubstitution mutations. It emerged that chemical modifications that inactivate the DNA template, the so-called lethal lesions, must be subject to extensive enzymatic repair processes, before DNA replication in viuo can occur. Studies of DNA replication in nitro showed that unrepaired lesions, acting as noninstructional bases in the terminology of one of the pioneering investigators in this area, B. S. Strauss, can miscode in the Watson-Crick sense. Modified purines generally cause transversions, whereas modified pyrimidines cause transitions. These correlations have proved valuable for the current attempts to interpret the significance of cancer mutational spectra in terms of causative agents involved as human carcinogens. T h e first requirement was to identify the genes involved as receptors of mutagenic carcinogens. T h e necessary information came from studies of virally induced cancer that began in 1910, with the first reports on avian leukemia virus. This outcome was ironic in the sense that one of the co-discoverers of tumor viruses, Peyton Rous, wrote an article 50

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years afterward (in 1959) deploring what he regarded as misplaced emphasis on the somatic mutation theory of carcinogenesis. Twenty years later this theory was effectively clinched by the demonstrations by Weinberg and others that cancer could be transmitted through DNA isolated from cells transformed by a carcinogenic hydrocarbon, and a few years later that the gene involved was the cellular counterpart of an oncogene that had been isolated from a sarcoma-inducing retrovirus. The demonstrations that oncogenes could be activated through basesubstitution mutations were clearly in line with much previous work linking chemical reactions of carcinogens with DNA and their biologic effects. These links were strengthened by subsequent studies on tumor suppressor genes, particularly so far TP53, coding for a protein that is inactivated by carcinogen-induced mutations at a wider variety of sites than the more specific codons involved in activation of oncogenes. T h e tumor suppressor genes were discovered through inactivation of their protein products by tumor viral proteins. As a class, they had been shown to be significant in human carcinogenesis through epidemiologic studies of a rare inherited cancer of childhood, retinoblastoma. The discoverer of this phenomenon, A. G. Knudson, Jr, went on to generalize his observations into a comprehensive theory of carcinogenesis with the co-authorship of S. H. Moolgavkar in 1981. They could account for the salient features of the age dependence of cancer incidence, previously attributed to multiple mutations by Muller and developed by Nordling, to a requirement for two successive mutations in the development of malignancy. The first gives rise to intermediate cells which could manifest as benign tumors through clonal expansion of the first mutant cell and the second was required to confer malignant character. This theory embodies the features of two-stage carcinogenesis, discovered by Berenblum, and subsequently shown experimentally to apply to systems other than the classical induction of cancer in skin of mice used by the early investigators. It also draws attention to another consistently found feature of experimental carcinogenesis, that reactions of carcinogens with DNA known to be capable of causing mutations are necessary but not sufficient to result in cancer. In other words, the critical quantitative factor in carcinogenesis according to this model concerns the need for proliferation of the cells initiated by the first mutation, in order to provide a significant chance that the second mutation will occur. In some experimental systems this critical phase is termed promotion and can be affected by nonmutagenic chemicals. A further cause of cell proliferation that has been emphasized as significant is that mutagenic carcinogens are also cytotoxic agents, and themselves cause reparative cell division in target tissues.

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Observations on mutational spectra have shown that, in some organs, most notably in lung, exogenous chemical carcinogens inducing transversion mutations are indicated to be an important source of carcinogenesis. In particular, the classical carcinogen benzo[a]pyrene has been implicated as a constituent of the predominant cause of lung cancer, tobacco smoke. T h e converse is indicated for colorectal cancer: most mutations are deduced to be of spontaneous origin, and a predominant source of such mutations is hydrolysis of cytosine, and more particularly 5-methylcytosine, in CG sequences of DNA. Therefore, what can be termed promotional factors are in turn indicated to be causatively quantitatively more important than the action of environmental mutagens, and the promotional action may depend on dietary factors. Although intensely investigated, the links between diet and susceptibility to cancer, apart from its positive associations with inadequate intake of vitamin A or its precursors, continue to remain somewhat obscure. Dietary mutagens, of which the most extensively studied is the hepatocarcinogenic mycotoxin aflatoxin B found in foodstuffs containing molds due to inadequate storage, are obvious avoidable risk factors. Some known exogenous sources of cancer initiating mutations, such as both ionizing and ultraviolet or solar radiations, are ubiquitous. Endogenous mutations are also indicated to result from normal processes of oxidative metabolism, giving rise to apparently unavoidable extensive chemical modifications of DNA, apart from the hydrolytic reactions already mentioned. I n conclusion, it is difficult to see further fundamental developments in our concepts of carcinogenesis, since data relevant to all aspects ranging from chemical to epidemiologic of the action of carcinogens in causing cancer-initiating mutations are now available on a vast scale. The deduction that the majority of these mutations are caused by unavoidable chemical modifications of DNA appears to be inescapable. This makes understandable the view expressed throughout the historical development of carcinogenesis studies that the somatic mutation theory of tumor initiation contains an element of fatalism. Therefore, attention should be devoted to the second predominant factor in carcinogenesis, often termed tumor promotion, which may prove to lead, for example through modification of diet, to significant reductions in the incidence of cancer. REFERENCES

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FROM THE MELANOCYTE TO MELANOMA TO TUMOR BIOLOGY’ Wallace H. Clark, Jr. Department of Pathology, Harvard Medical School, and The Beth Israel Hospital, Boston, Massachusetts 02215; Pathology Services, Inc., Cambridge, Massachusetts 02139; and The Pigmented Lesion Group, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

I. An Introduction to an Essay on the Nature of Cancer 11. The Only Research Plan. T h e Present: Studies of Tumor Biology in Maine, at the Beth Israel Hospital, and at Harvard 111. The Melanocyte. T h e Years at Tulane University

IV. T h e Biologic Forms of Primary Cutaneous Melanoma and Levels of Invasion. T h e Melanoma Studies at the Massachusetts General Hospital, Harvard, and the Establishment of the First Pigmented Lesion Clinic V. Tumor Progression, the Dysplastic Nevus, and the Precursor State of Neoplasia. The Years in Philadelphia: Temple University and the Pigmented Lesion Group of the University of Pennsylvania A. Temple University and the Teaching of General Pathology B. T h e Pigmented Lesion Group of the University of Pennsylvania VI. Maine, the Beth Israel Hospital, Harvard Medical School, and Pathology Services, Inc. Back to the Future References

I. An Introduction to an Essay on the Nature of Cancer Several distinguished mentors have significantly influenced my life and work. Two of the living mentors do not know me nor do they know of my work. They are, however, as much my wise and trusted counselors as if I had been their formal student. One of these mentors is Stephen Jay Gould. I will return to his thinking and concepts as they apply to the nature of cancer but, at the outset, I will follow his guide in writing an essay and quote Montaigne’s letter to readers of his early essays. “I desire therein to be viewed as I appear in mine own genuine simple, and ordinary manner. . . If I had lived among those natives, which (they say) yet dwell under the sweet liberty of nature’s primitive laws, I assure thee I would most willingly have painted myself quite fully and quite naked” (Gould, 1993). An essay is a short literary composition, frequently about some common thing, that reflects the personal views of ’Supported by grants from the National Cancer Institute, USA: CA-58845 and CA-25298. I13 ADVANCES IN CANCER RESEARCH. VOL. 65

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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the author “painted quite fully and quite naked.” A primary cancer is a “common thing” and I should like to give the reader a personal view of how my present concepts of cancer arose and evolved. I have selected the literary form of an essay in order to bypass the sterility of scientific papers and monographs, where my views have, thus far, been presented. Scientific papers do not convey or clearly reflect how ideas arise, and they distort how ideas are tested for validity. Further, they reflect almost nothing of the personal agonies and ecstasies of inquiry into the mysterious. T h e standard scientific paper does not convey the unparalleled emotion of discovery. Sir Peter Medawar has considered the problem of the scientific paper and scientific discovery. He stated that, “The scientific paper in its orthodox form does embody a totally mistaken conception, even a travesty, of the nature of scientific thought.” T h e scientific paper “. . . is a fraud in the sense that it does give a totally misleading narrative of the processes of thought that go into the making of scientific discoveries” (Medawar, 1963). I should like to share with the reader my inner thoughts and feelings associated with work and scientific discovery in the field of melanocytic neoplasia. In turn, I should like to show how my observations and judgments as to the nature of these observations led me to study general tumor biology. Such sharing is not possible in most scientific papers of today for editors insist upon a form that precludes presentation of the actual nature of scientific endeavor and discovery. Superimposed upon the fraud perpetrated by the editorial form of today’s scientific paper is the widespread use of copy editors, a class of humanity whose function is to eliminate the last vestiges of personal human endeavor underlying scientific effort and discovery: the language of the scientist, himself. The manner and the matter really should not be separated. Mark Twain wrote to his publisher demanding that a copy editor be shot “without benefit of prayer” after discovering that his punctuation had been changed. Thus, the essay form permits me to tell you what I have actually done; what I think the resultant discoveries mean and will come to mean in tumor biology; and to share all of this with you in my own words. My words, after all, are the dominant means I have to communicate the interaction of my human nature with the phenomena of neoplasia and cancer. Any knowledge of a subject, such as a cell, a tissue, an organism, or cancer, is the product of the nature of’the investigator and the information about the subject being investigated. T h e total information-the entire body of facts-about a neoplastic system o r an organism is essentially infinite. Consequently, the particular information or aspect of a subject being investigated cannot be separated from the ability, training, and interest of the investigator. Some important principles underlying the interaction between investigator

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and investigations are illustrated by studies of cancer. The investigatorinvestigation principles largely determine the nature of new knowledge generated about a subject. The first aspect of cancer I decided to study was the pathology of a primary cancer. When I discuss this particular investigation, I will elaborate on the nature of the investigator as related to his investigations (Searle, 1990). It. The Only Research Plan. The Present: Studies of Tumor Biology in Maine, at the Beth Israel Hospital, and at Harvard

I have never had a life plan or a research plan. The foregoing statement means I have lacked some overarching ambition for my life and never dreamed of studying tumor biology until my later years. I have been blessed with the ability to formulate imaginative hypotheses and I have always wondered how things and people worked. The hypotheses and wonder at the mystery of living things were my incentives and these innate personal attributes bounced from one thing to another2 without a purposeful theme until quite recently. However, I now have a plan, an all encompassing plan. The story of the genesis of the only plan of my life informs my work on melanocytic neoplasia with perspective. My solitary plan, which has progressively dominated my thinking for the past seven years, emerged while I was sitting on a stalled train between Overbrook, Pennsylvania and Philadelphia, Pennsylvania. I enjoy trains, subways, and buses and, more or less dislike private automobiles, taxis, and limousines. Airplanes are uniquely loathsome to me. As a result of these preferences, I spent many years commuting on the “Paoli Local” between Malvern or Strafford, Pennsylvania and Philadelphia, Pennsylvania. I enjoy public transportation for it gives me time to read or affords the opportunity to talk with diverse people. Reading and people are a major part of my life. I gradually formulated reading rules. The dominant rule was: “Never read any science related to your work while on a train.” I rarely read novels, with major exceptions such as those of Wallace Stegner or Penelope Lively, so my train life was filled with people, philosophy, cosmology, the nature of time, anthropology, evolution and such. I was compelled to read when faced with offerings of J. T. Fraser, Isaiah Berlin, Richard C. Lewontin, and Stephen Jay Could (if Stephen 2 My bouncing took me from homicides on the New Orleans waterfront to the nature of zebra stripes in the Audubon Park Zoo, where I worked for pleasure as a pathologist and to cutaneous pustules of Egyptian mummy skin.

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Jay Could begins to write novels, I will read novels). There were rare exceptions to the idyll of my library train. T h e deadline of a grant application o r the urge to finish a manuscript immediately come to mind. It was not a rare event for the “Paoli Local” to stall. This particular morning in late 1986, I had broken my own train reading rules and was writing on the prognostic significance of patient and lesional attributes of malignant melanoma. T h e writing had been underway for some months and was to continue for more than two additional years before the paper was finally accepted for publication. The data were largely in hand and I believed, with more than a little conviction, that the statistical study of 343 attributes on 501 patients followed prospectively for > 8 years would give substantive answers to many problems in melanoma. T h e data had been carefully gathered and computerized for some 14 years and it was not hard to forgive myself for violating the train reading rules. T h e outcome of the study was not complex. Which attributes predicted disease-free survival for a minimum of 8 years, and which attributes predicted death due to metastatic melanoma? T h e work had been quite laborious over the years and I felt, with a naivete that ultimately became painful, that the results of the work would be useful and highly significant. T h e results were useful and highly significant, but the outcome did not approach my expectations. One group of patients would have attributes predicting survival and the other group attributes that would predict disseminated metastatic disease and there would be only a few cases in the gray zone of unpredictability. The totality of the properties of the two groups would become highly informative on the nature of cancer. T h e breakdowns on my train could be quite long, from time to time, and this particular stalling was one of the long ones. I began to think of the biologic significance of the attributes (the properties) that predicted metastasis from primary melanomas (Clark et al., 1989). 1. T h e capacity to grow in the epidermis and dermis (usually, the reticular dermis). At the time, this property was entered into the data base as the vertical growth phase of a primary melanoma. T h e property is still referred to as the presence of the vertical growth phase. A more descriptive term for the vertical growth phase is that of David Elder’s: tumorigenic melanoma. 2. A mitotic count 26/mm2. 3. T h e absence of tumor-infiltrating lymphocytes. 4. A tumor thickness of >1.70mm. 5. Site of the primary tumor on the trunk, head, neck, palms, or soles; a site category referred to as axial.

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6. Male patient. 7. Presence of regression somewhere within the primary tumor. For some years, I had thought of my work as being concerned with melanoma, but as I reflected on the biology of the properties of a melanoma associated with metastasis, I realized that the concept of “working on melanoma” was quite restrictive. Some subset of most primary cancers had the capacity to extent from one site to another, survive in the secondary site and grow at that site. Stated somewhat differently, some primary cancers have the capacity for metastasis and some primary cancers of the same type d o not have that capacity. Before I got off the train, it followed in my thinking that a cancer with metastatic capacity, regardless of type, must be similar to other cancers with metastatic capacity, regardless of type. Further, the mechanisms of metastasis must be similar in all forms of cancer. Before the subway ride to the University of Pennsylvania was finished, I felt as if I was in the midst of a personal epiphany. I was not “working on melanoma,” but on the properties of a primary cancer required for a cancer to metastasize. The properties of a primary cancer with a given behavior had to be similar in the different forms of cancer and the mechanisms of metastasis had to be similar even in different kinds of cancer. Therefore, at least some of the metastatic mechanisms operative in primary melanomas were general mechanisms applicable to all primary cancers. From that point it was a short and smooth mental transition to working on the development and biology of neoplasia; from the events at the beginning, to the precursor state, to primary cancer, to metastasis, and to metastasis from metastasis. I then developed a plan for the comparative study of neoplastic systems. I would study neoplastic development and progression by analogy between similar phenomena in different systems. I have followed this “only research plan” with ever greater excitement for these seven years and the details and some results of the plan are the final parts of this offering. For now, I must return to the beginnings that brought me to melanoma. 111. The Melanocyte. The Years at Tulane University

T h e beginning of the story is chosen somewhat arbitrarily. I could start with an affinity for optical systems and optical physics, beginning with hand lenses and extending to electron microscopes, but this would take us back to my teenage years and the essay would go quite beyond melanocytes, melanoma, and tumor biology, the task assigned to me by

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the editors." As the epidermal melanocyte is the parent cell of the melanocytic nevus and of primary cutaneous melanomas, my studies of that cell are a reasonable starting place. The melanocytic cell system has not always been recognized as a distinctive system. In 1949, Allan stated that putative melanocytes were transformed basal cells and did not differ in any essential respect from the average epidermal cell. Thus, melanomas came to be called melanocarcinomas and many workers came to doubt the existence of the melanocyte as a specific cell form, in spite of the elegant experimental embryologic studies of Mary Rawles and the meticulous anatomic studies of Pierre Masson. The melanocyte was not easily seen in routine histologic preparations of the skin, and it was not too difficult to be persuaded that Allan was correct in the matter. During the Korean war, while I was in the armed services, I studied the work of George Palade dealing with the fine structure of mitochondria and the endoplasmic reticulum. I was excited to attempt to use the resolving power and magnification of the electron microscope to solve the problem of the existence or nonexistence of the human epidermal melanocyte. My excitement should have been daunted by the fact that I did not have an electron microscope nor an ultramicrotome. I should have been further discouraged by not knowing how to use either instrument. T h e younger scientists of today may not realize that electron microscopes were present in only a few institutions until well after World War 11. Most of the institutions in the United States with electron microscope laboratories were the distinguished, large universities of the north and west. After my military service I returned to Tulane University in New Orleans. There was no electron microscope in the university, but Frank Low in the Anatomy Department of Louisiana State University did have an excellent electron microscope laboratory and patiently introduced me to the instrument and the methods of its use. In due course this experience permitted us to establish an electron microscope laboratory in the Pathology Department at Tulane. Biopsies of my own skin provided the study material. After some two years with Frank Low and one year in my own laboratory I began producing electron micrographs of acceptable quality for the time. Interpretation was another matter. For practical purposes there was no literature on the fine structure of the human epidermis. The fine structure of the hair bulb melanocyte had been described, but the one paper reporting electron microscope studies of the human epidermis failed to see any cell corresponding to the melanocyte (clear cell of Masson). That paper stated that the 9 I am grateful to the editors for the opportunity to discuss about the nature of cancer.

my work and thinking

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melanocyte was probably an artifact of light microscopy. Each day I would study the micrographs I had produced over the previous year. I could clearly see that there was a cell in the basal layer of the epidermis which had no tonofilaments nor desmosomes. It was quite different from the keratinocytes and I presumed it was a melanocyte. I did not see, however, any dendrites. The pictures did show, however, circular, anucleate structures with features similar to the cytoplasm of the basally located cells that had no desmosomes or tonofilaments. One morning I looked at the circular, anucleate structures I had seen for over a year and knew they could only be dendrites of melanocytes in cross section! The precipitous interpretation was an epiphany of discovery. It is remarkably difficult to see something that has not been seen or described previously and discover its nature. Such discovery is a unique and exciting experience. I wrote a paper with Dick Hibbs describing the human epidermal melanocyte as a separate cell system and submitted the paper to the Journal of Biophysical and Biochemical Cytology (now The Journal of Cell Biology), my excitement of discovery at a high peak. The paper was rejected stating that my interpretation of the “circular, anucleate structures” as dendrites in cross section was speculative and unwarranted. If others cannot accept your discovery it is hardly a discovery. My balloon of discovery had been deflated. Microscopy, especially electron microscopy, is pleasant and soothing to me. The morning I received the rejection of my paper, I put a grid with a newly cut section of skin into the electron microscope, searching more for a balm than a cell. The first image that came onto the screen was the nucleus of a melanocyte, its perikaryon, and in the same section plane, a dendrite that traversed the width of two keratinocytes and branched around a third keratinocyte. The structure of the dendrites was similar to the “circular, anucleate structures,” previously interpreted as cross sections of dendrites. I took several pictures of the dendrite extending from its perikaryon, printed them, selected one for the rejected paper, and then submitted a revised manuscript before the end of the day. The paper was accepted without further comment (Clark and Hibbs, 1958). As it turned out, our description of the fine structure of the human epidermal melanocyte was not the first such description. Unknown to us George Odland had also done electron microscopy of the human epidermis and clearly described the epidermal melanocyte. His excellent paper appeared one month before our paper was published. I learned several important things from “the discovery of the melanocyte.” It made no difference, at all, that our description was not the first. The complex process of observation and judgment leading to knowledge was not diminished by “not being first.” Whether one is

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“first” or not has nothing to do with research and learning. The essence of work is discovery and the act of learning. The generous and uninhibited sharing of your observations, learning, and knowledge is teaching. I began to think that competitiveness in science was a negative and destructive force. The subsequent years have reinforced that concept. The “discovery” of the human epidermal melanocyte through fine structural studies influenced much of my subsequent work and life. The electron micrograph of the melanocyte became the center of an exhibit on the fine structure of the epidermis presented to the American Academy of Dermatology by Dick Hibbs, Mildred Watson, and me in 1958. The exhibit led me to Tom Fitzpatrick and to Harvard Medical School and the Massachusetts General Hospital and to the melanomas of the Massachusetts General Hospital. I cannot describe my first studies of melanoma without telling of the excellence of the Department of Pathology at Tulane University School of Medicine. That department was a place of study, research, learning, and teaching. I regard the foregoing attributes as inseparably interrelated and cardinal to a university. Outside research funds were not abundant in the 1950s and our research and studies were driven primarily by curiosity concerning the mechanisms of disease. The study of disease mechanisms was central to our teaching, as well as our research. The influence and friendships of Tulane have remained with me for my entire life. I must mention three members of that department. Emmanuel (Manny) Farber, educated in biochemistry and medicine with further training in pathology, had a consummate interest in explaining the phenomena of a disease from its beginning to its end. He was meticulously critical of research, including his own. He is one o f the few scientists I have known whose interest in the nature of disease was paramount. His work was not paramount to him; The work that properly elucidated the nature of disease was paramount. Today, if one wishes to investigate cancer and appreciate the problems of the disorder one should study Farber’s work on hepatocarcinogenesis and reflect on his concepts of the beginnings of neoplasia (for example, see Farber and Cameron, 1980; Farber and Sarma, 1987; and Farber and Rubin, 1991). The late William (Will) H. Sternberg was the complete pathologist and a polymath, an artist and poet in his own right and knowledgeable in music. He taught me gross and microscopic pathology of human disease. Will had a marvelous sense of humor and was of unfailing good humor. Will’s opinion about a histologic diagnosis was the final opinion. Richard J. (Dick) Reed is a pathologist of the Sternberg school and of Will’s caliber. He was in the first class of medical students I taught. We have collaborated on many aspects of cutaneous pathology and

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melanocytic lesions and have an abiding friendship. Dick does not see pathology without thinking of disease mechanisms. The pathology department of Tulane of the 1950s and early 1960s was largely concerned with the study of the mechanisms of disease. IV. The Biologic Forms of Primary Cutaneous Melanoma and Levels of Invasion. The Melanoma Studies at the Massachusetts General Hospital, Harvard, and the Establishment of the First Pigmented Lesion Clinic

Thomas (Tom) B. Fitzpatrick and the late Dr. Benjamin (Ben-but I never addressed him other than Dr. Castleman) Castleman recruited me to the Massachusetts General Hospital (MGH) and Harvard Medical School. I had three responsibilities at MGH. I did general pathology, including rotations on the surgical and autopsy service. I developed a program in cutaneous pathology, and I established an electron microscope laboratory in the dermatology department. I had developed an interest in cutaneous pathology while in the armed services during the Korean War. Work in skin pathology, beyond electron microscopy, expanded while at Tulane and I now had the obligation to develop a full cutaneous pathology program at MGH and later taught cutaneous pathology at the Peter Bent Brigham Hospital. Dr. Walter Lever had done skin pathology at the MGH prior to my arrival. His work was as a diagnostic consultant to the Pathology Department, but a training program in cutaneous pathology was not formed prior to Dr. Lever leaving MGH to take the chair of dermatology at Tufts Medical School. In effect, the program I developed was new. Tom Fitzpatrick gave me free rein in my work and encouraged me in all facets of it. I was given space in the dermatology clinic and actually saw most of the patients that had a skin biopsy and studied the histology with dermatology and pathology residents in the clinic with the patients at hand. The correlation of the clinical presentation of the disease with the histology strongly reinforced my determination to never separate the study of pathology from the study of the whole patient. The organism is diseased and therefore the organism must be studied. In 1963, malignant melanoma was regarded as the most malignant of cancers. T h e diagnosis was thought by many, including some physicians, to be a virtual death sentence, but this was not the case, even then. The nationwide case fatality rate in the United States in 1960 was about 50 to 60%. I was interested in the pathology of melanocytic nevi and melanoma because of my fine structural studies of the human epidermal

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melanocytic cell system and my expanding responsibilities for skin pathology at MGH. I cannot remember, and I have made a significant effort to remember, what led to the primary hypothesis of my melanoma work. T h e hypothesis was simple. Are there gross and microscopic features of a primary melanoma that will predict whether a patient will die of disease? Medawar stated that hypotheses arise by inspiration and there is no logical way of devising them. He felt they were the subject matter of psychology (Medawar, 1963). I have already stated that I have been blessed from my earliest memories with a gift of questions. T h e hypothesis was just another one of those gifts. I am now sure that this hypothesis has driven much of my work on melanoma and cancer. My obvious expectation of the study would be the identification of gross and microscopic features of primary melanomas that predicted outcomes for patients with malignant melanoma. I have always enjoyed doing a study. The daily, physical act of gathering data, especially when it was gathered with a microscope, brought, and still brings me pleasure. T h e study of the melanomas of the Massachusetts General Hospital was a singular experience. It is stated that the formal discipline of surgical pathology began in the United States at MGH. This putative beginning of surgical pathology in the United States resulted in careful gross and microscopic descriptions and diagnoses written in beautiful and legible long hand in a large leather-bound book, similar to a ledger. The first report was dated January 1, 1898. With one assistant, we went through every pathology report of MGH from this date, the reputed beginning of surgical pathology, through May 31, 1968 and selected all reports with a diagnosis of malignant melanoma. There were some 1400 cases. T w o hundred nine of the reports with a diagnosis of melanoma formed the definitive study. Study cases were selected on the basis of the correct diagnosis, quantity and quality of the histologic material and a clinical follow-up of five years or more or follow-up until the patient died of metastatic melanoma. All clinical records were reviewed and these detailed and excellent records gave a remarkably clear picture of the disease as it was viewed in the first half of this century. T h e first group of 96 cases was reported in 1967 and the entire study group reported in 1969 (Clark, 1967 and Clark et al., 1969). T h e latter paper became a Science Citation Classic. The study contributed valuable information in three areas. First, it demonstrated that there are three biologic forms of melanoma. We had eliminated genital and plantar melanomas from our study and, thus, the important fourth biologic variant melanoma was not recognized until delineated by Arrington and Reed and their co-workers (Arrington et al., 1977). 'The use of superficial spreading melanoma, nodular melanoma, lentigo ma-

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ligna melanoma, and acral lentiginous melanoma (the biologic forms of melanoma) is valuable in histogenetic studies and especially useful in epidemiologic and etiologic investigations. Second, the work showed that histologic features of a primary cancer, such as levels of invasion, could be useful in predicting outcome. Alexander Breslow showed that thickness of a primary melanoma was a better single prognostic attribute than levels of invasion (Breslow, 1970). Thickness and levels of invasion are now but two of several attributes of primary melanomas of value in outcome prediction. Studies of multiple attributes of primary cancers and patients bearing the cancers are now extensively used in most neoplastic systems. Third, our melanoma studies at MGH led to the formation of the first Pigmented Lesion Clinic.4 The establishment of this clinic was done with Thomas Fitzpatrick. The clinic continues to this day and has served as a model for similar clinics throughout the United States and in other countries. I had many excellent students while I worked at MGH. I cannot mention all of their names, but I will not write an autobiographical essay about my work without telling you of one of them. Richard W. Sagebiel was and is a scholarly man. He encouraged me in times of depression and has continued to do so throughout the years. He has made many original contributions to the field of melanoma biology. His friendship and that of his wife, Daisey, have been a constant, warm, and welcome support.

V. Tumor Progression, the Dysplastic Nevus, and the Precursor State of Neoplasia. The Years in Philadelphia: Temple University and the Pigmented Lesion Group of the University of Pennsylvania A. TEMPLE UNIVERSITY AND THE TEACHING OF GENERAL PATHOLOGY Renato Baserga asked me to join his department at Temple University. In that department I had extensive teaching responsibilities in 4 I have no enthusiasm for claims that observations have primacy, such as, “This is the first report o f . . . .” T h e more I read the literature, the surer I am that precious few claims of primacy are warranted. Nothing in our melanoma papers of 1967 and 1969 was “first.” For example, lentigo maligna melanoma was described before the turn of the century by Sir Jonathan Hutchinson. Our studies did synthesize previous observations and presented them in a useful form, with reasonable proof of validity by appropriate knowledge of outcome of patients with malignant melanoma. I d o believe, however, that the Pigmented Lesion Clinic at the Massachusetts General Hospital was the first of its kind and this clinic and similar clinics have made valuable contributions to the understanding of nevi, melanoma, and the generalities of tumor biology.

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general pathology. I taught medical students the general pathology of neoplasia and this led me to the study of the principles of neoplasia. I began reading the work of the late Leslie Foulds. I think I have read the first 225 pages of his book on neoplastic development some 8 to 10 times (Foulds, 1969a). I have read parts of the book many more than 10 times (Foulds, 1969b). Foulds’ concepts were derived, in no small measure, from the study of mammary carcinogenesis and cutaneous carcinogenesis. I found his principles of tumor progression immediately applicable to human melanocytic neoplasia and to human cutaneous carcinogenesis. Later it became apparent to me that Foulds’ work is useful in the study of all forms of neoplasia. I started a Pigmented Lesion Clinic at Temple University and through that clinic studied two remarkable patients and their families. T h e study of both of these families was due to the interest of Michael Mastrangelo at The Institute for Cancer Research in Philadelphia. Michael presently works at Thomas Jefferson University. Michael saw a young woman who recently had a melanoma resected from her calf. He called me and said her moles were quite remarkable. He had never seen anything quite like them and asked me to see her. The patient’s name was Susan Betz.5 I had never seen a patient with such melanocytic nevi. They numbered well over 200 and were frequently >10.00 mm in width. T h e nevi varied greatly in size, shape, and color. Susan’s mother was with her and 1 asked permission to examine her mother. At the time I had no interest in and little knowledge of heritable aspects of melanoma or any other form of cancer. I had no conscious reason to request the examination of Susan’s mother. However, permission was granted. She had a melanoma on her left arm! I was astounded. Mike Mastrangelo felt that the heritable forms of melanoma were poorly described and understood. He convinced Mark Greene of the Family Studies Section of the Environmental Epidemiology Branch of the National Cancer Institute of the value of studying familial melanoma. This was the beginning of a productive collaboration with Mark Greene. Together we examined the Kruegerfi family about 4 weeks after I had examined Susan Betz and her mother. The examination of the Krueger family was precipitated by Dick Krueger,7 who insisted that She gave me permission to use her name and all aspects of her disease in both lectures and publications. Susan, who died of melanoma some years later, was interested in control of the mortality due to melanoma throughout her life. The nanie is used with permission. Dick is the index case o f the Krueger family. He died of metastatic melanoma a few years later. His insistence on the study of his family was a major contribution to understanding the phenotype of individuals at high risk for melanoma and his insistence saved his uncle’s life.

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his family had unusual moles. It was known that the family was affected by familial melanoma. Seven family members over 3 generations had 20 cutaneous malignant melanomas. Five family members had the disease prior to our examinations of the family as a group. The examinations were done on April 25, 1976, a cold, snowy Sunday afternoon, at the St. Francis Hospital in Trenton, New Jersey. No one, including an excellent local dermatologist, had any expectations of significant findings on cutaneous examination. In spite of having recently seen Susan Betz, I did not expect the work of a long day to be particularly fruitful. I did not know the meaning of Susan’s dramatic presentation and had mentally put her appearance aside for further study at a later time. I examined the skin of all 27 family members that Dick Krueger had persuaded to be checked and 1 was the official photographer of the day. I became excited after examining the first three family members. I had, by this time, worked in a Pigmented Lesion Clinic at the Massachusetts General Hospital and at Temple University for 11 years and I knew I had not seen patients with melanocytic nevi similar to those of most Krueger family members. The notable and dramatic exception was Susan Betz. Her cutaneous appearance now came vividly to mind. While none of the Kruegers had quite the array of Susan’s moles, the affected members were similar to her. I called in other observers as more and more family members who had quite unusual nevi were seen. The other observers did not share my excitement until late in the afternoon. The last patient, an uncle of Dick’s and the most reluctant participant in the study, had an obvious melanoma on his back!8 All observers were now stimulated. T h e last melanoma in the Krueger family was diagnosed by Dr. Margaret (Peggy) A. Tucker in 1993.9 One of the original families continues to be one of the most informative. T h e years at Temple led me back to the study of the principles of pathology, especially the principles of the pathology of neoplasia. Teaching medical students and residents continually focused my thinking on tumor progression. My work in the Pigmented Lesion Clinic directed my work to dysplastic nevi and these lesions, in turn, broadened my concepts from precursor lesions to the precursor state. The precursor state is that condition of an organ and organism that results in susceptibility to the development of cancer. I think that control of morbidity and mortality due to cancer is likely to come from 8 The lesion, although invasive, was still quite superficial and he is alive and free from disease as I write. g.Dr. Tucker succeeded Dr. Mark Greene in what is now the Genetic Epidemiology Branch of the National Cancer Institute. The studies discussed in this essay have been and are dependent upon collaboration with these fine workers and their Genetic Epidemiology Branch. The collaboration has been continuous for the past 18 years.

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understanding and managing the precursor state. Studies of the precursor state were to occupy much of my time at the University of Pennsylvania and there my thoughts gradually turned to the comparative study of different neoplastic systems, especially the precursor state of these systems.

B. THEPIGMENTED LESIONGROUPOF THE UNIVERSITY OF PENNSYLVANIA Albert Kligman and Walter Shelley brought me to the University of Pennsylvania and there the Pigmented Lesion Group reached full flower and continues to bloom.10 It is difficult for me to describe for you the daily life and endeavor of the Pigmented Lesion Group. Most of the staff, the patients, and the research data at Temple were moved to Penn. We were joined, from the beginning, by DuPont Guerry IV, Ralph Hamilton, Edward Bondi, whose position was later taken by Allan Halpern. A collaboration with the Wistar Institute already existed and this was strengthened by the move to Penn. T h e collaboration with Peggy Tucker and her Molecular Genetics Branch at the NCI also expanded. We had photographed the entire skin of every patient since my examination of Susan Betz in March, 1976. Prior to that all melanomas had been photographed as had much of the body surface of affected patients. After Susan, the photography was regarded as essential and was done in a standardized fashion. Gradually and with difficulty all of the data on the patients and their pathology was computerized. With time a large and carefully monitored data base on melanoma and nevi grew and became an important reference for the detailed study of a single form of human cancer. The work at Penn covered two decades and will be discussed under six headings. 1 . Thp Cumulative Experience of Seeing Large Numbers of Patients and the Study of the Pathology of Many Lesions The physicians of the Pigmented Lesion Clinic examined patients every Monday and the Clinic expanded rapidly and continuously at The members of the group, after the move to Penn, included David Elder (pathology), DuPont Cuerry IV (oncology and immunology), Ralph Hamilton (surgery), Edward Bondi (dermatology), Allan Halpern (dermatology), Rosalie Elinitsas (dermatology and pathology), Lynn Schucter (oncology), Marie Synnesvedt (data-base manager), Jean Thompson (nurse-practitioner), lssabelle Matozza (nurse-practitioner), William Witiner (photographer). The Wistar Group included, primarily, Meenhard and Dorothee Herlyn and their associates and graduate students.

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Penn. We routinely saw one or two patients with intact primary melanomas each week, and some weeks five or six patients with intact primary melanomas were observed. In addition, the clinic was something of a magnet for familial melanoma and we examined and followed many families affected by the disease. Sporadic melanomas and dysplastic nevi made up the bulk of our patient population, which grew to 80 or 90 patients per week. Patients with large congenital melanocytic nevi, with and without melanoma, were commonly referred to us and these patients were singularly instructive with regard to tumor biology. Experience with the pathology of all kinds of pigmented lesions grew in parallel with the clinical experience. We saw, studied, and cross-indexed all of the pathology originating from the patients of T h e Pigmented Lesion Clinic and saw many additional cases in consultation. It is to be remembered that the clinical and pathology experiences were computerized and all of the clinical observations of lesions were documented photographically. There were 343 clinical and pathology attributes per patient entered into the data base. The data could be browsed interactively and I learned much about melanocytic neoplasia and tumor biology, in general, perusing the data base. I could not have done this without the meticulous work and fussy cooperation of Marie Synnesvedt, who was in charge of the data base. She was in charge as a drill sergeant is in charge of a platoon. I use the word fussy, for Marie would not permit me to overinterpret data, as I am frequently tempted to do. After some 15 to 18 years and more than 20 years after my first clinical experiences with melanoma at MGH, I realized that extensive experience was an incomparable and perpetual teacher. No two melanomas, no two nevi, no two congenital nevi, and no two Homo sapiens are ever alike. T h e study of living systems requires compulsive concern with the recording and retrieval of data. Only then can classes of objects be studied comparatively for biologic meaning. The comparative studies of classes of objects is at the core of biologic investigations. The data base at Penn is now an encyclopedic reference source permitting the comparative study of classes of lesions and people having the lesional classes of melanocytic neoplasia. 2 . The Study of Cells Derived from Common Acquired Melanocytic Nevi, Dysplastic Nevi, and Melanomas

Cells derived from lesions representative of all the stages (lesional classes) of tumor progression have been studied. As a rule, tissue from all primary and metastatic melanomas and many melanocytic nevi of different kinds was submitted for tissue culture. The lesions and the concepts of melanocytic tumor progression are covered in these references

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from T h e Pigmented Lesion Group (Clark et al., 1976, 1984; Clark, 1991a, 1991b, 1994). The in vitro studies of the sequential lesions of tumor progression have shown that the distal lesions of tumor progression, radial growth-phase melanoma, vertical growth-phase melanoma, and metastatic melanoma have groups of antigens that reflect tumor progression (Elder and Herlyn, 1992; Lazzaro et al., 1993).Some aspects of the general biology of melanoma-associated antigens have been clarified. Some of the monoclonal antibodies identify a single protein which has been termed CD63 (Demetrick et al., 1992). The growth patterns and growth regulation of cells derived from the different lesions of tumor progression have been described (Graeven and Herlyn, 1992; Mancianti et al., 1993).In vitro studies have also shown that melanoma cells constitutively express multiple growth factor genes, but normal melanocytes do not (Rodeck et al., 1991a). Melanoma cells are dependent, in part, on basic fibroblast growth factor (bFGF), but melanocytes expressing the growth factor are neither transformed nor tumorigenic (Rodeck et al., 1991b). Studies of various oncogenes and antioncogenes have shown various abnormalities, but these have not been consistent in different melanomas. A similar statement may be made about chromosomal changes. These changes are more prominent with tumor progression, but do not consistently involve the same chromosomes in different primary and metastatic melanomas (Parmiter and Nowell, 1993).

3 . The Pigmented Lesion Study Group, the Concept of a University, and the Acquisition of Knowledge The Pigmented Lesion Study Group had lunch together every Wednesday and, as a result, began to carry out the primary function of a true university: the acquisition of knowledge. The entire group, including those largely concerned with in vitro studies, attended the lunches, which lasted from 1 to 3 hr. Virtually all conversations were based on clinical material, the clinical history, photographs, and pathology of one or more patients recently seen in the clinic. When the conversations were related to in vitro studies, the source material for these studies was related to a specific tumor stage in a specific patient. T h e clinical material constituted information and the conversations concerning its significance resulted in judgment. Information and judgment form knowledge, and knowledge is the ability to do, to make, to understand, and to explain. The interpretation of what we did at those conferences in these terms is largely retrospective and couched in the concepts of John Searle and Michael Oakeshott (Searle, 1990). I must emphasize that the starting point of the Wednesday conferences of The Pigmented Lesion Study Group was information derived from observations of some aspect of

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neoplasia as it actually occurred in a patient observed by members of the group. Decisions had not been made in advance as to the biologic significance of patient information. The conversations of the lunch revolved around the biologic significance of patient-derived information. The lunches gradually evolved into discussions of tumor biology based on information derived from the documented behavior of a tumor, in this instance some stage in melanocytic neoplasia manifested. The lunches fulfilled Oakeshott’s definition of a place of education (a university): . . they are serious; they are places of study; and they are detached, apart from the rest of the society” (see Searle, 1990). We were serious, we studied, and we were apart. The conferences had no mission, no defined goals. We detached ourselves, at times, from the theoretical constraints of grant applications and the compelling urgency of clinical decisions in an attempt to formulate knowledge about tumor biology. Obviously, some lunches were pragmatic and the whole process was not such a pristine exercise in learning as it appears to be in print. However, it was a major educational experience for me. The guide to learning was the disease itself, unencumbered by a priori decisions as to its nature. ‘I.

4 . The Study of Prognostic Attributes

At the pragmatic level, one project formed the central theme of our research. We had recorded 343 attributes per patient beginning September 1, 1972 and, except for an occasional person lost to follow-up, we knew the status of every patient. It was my firm expectation that the statistical analysis of the attributes would produce a predictive model with few errors. I have already listed at the beginning of this essay those attributes that proved to be of significant predictive value. The predictions were not as accurate as I had hoped, even though overall outcome predictions approached 90% (Clark, et al., 1989). There were other aspects to my disappointment that were fundamental. I had hoped the properties predictive of survival would give some clear picture of the nature of cancer when the properties were viewed as a whole for biologic significance. The study of 18 years did not unravel cancer’s Gordian knot. We then went back to each of the properties of demonstrated biologic significance and began our thinking again. New concepts arose that cancelled some of the disappointments. The radial and vertical growth phases of a primary melanoma are an important part of the new concepts (Elder et al., 1984; Guerry et al., 1993). T h e radial growth phase was a term selected to describe the evolution of a primary melanoma along the radii of an imperfect circle. Commensurate with this pattern of growth there was extension of cells across the basement membrane into the papillary dermis. As long as these cells did not actually show

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evidence of growth in the dermis, there were no metastases. Only when the cells acquired the property of growth in the dermis (the vertical growth phase or tuniorigenic melanoma), as opposed to extension into the dermis, did metastases occur and even then metastatic spread was not the rule. Other cancers have analogies to this situation. For example, carcinoma of the colon that only shows extension into the lamina propria (a structure analogous with the papillary dermis) has not been shown to metastasize (Clark, 1994). Analogies between developmental stages of different neoplastic systems gradually led us to the more formal use of analogy as a research tool. Stephen Jay Gould has shown that analogy is an effective method of research and it has become so for us (Gould, 1989). 5 . The Dysplastic Nevus and the Precursor State

With ever-expanding clinical experience, computerized data, and follow-up of all patients, the existence of a cutaneous phenotype at great risk for developing melanoma became obvious. We began to carefully delineate the properties of the phenotype at risk (Masri et al., 1990; Clark et al., 1990; Elder et al., 1993). Some of the patients had a clinical appearance as florid as that of our original patients (Clark et al., 1978). In others, the changes were niore subtle but recognizable. The work with the dysplastic nevus was intimately correlated with the studies of Mark Greene, Peggy Tucker, Alisa Goldstein, Sheri Bayle, and Nick Dracopoli at the National Cancer Institute and the Massachusetts Institute of Technology. Many patients with dysplastic nevi were members of familial melanoma families and research was directed toward genetic mechanisms through linkage analysis. Early studies indicated linkage on chromosome lp, but studies by others as well as the group at the NCI indicated linkage to chromosome 9. Some families, including the family of one of the original cases of florid dysplastic nevi, did not show linkage to either chromosome 1 or 9. The disorder shows genetic heterogeneity (Goldstein et al., 1993). Thus, as is the case with studies of the karyotype and studies of other genetic abnormalities, melanocytic neoplasia does not show genetic changes that are consistent from case to case. T h e dysplastic nevus, in addition to providing data indicating genetic inconsistency in melanocytic neoplasia, led us to study the precursor lesions and from the lesions to the precursor state of neoplastic systems (Clark et al., 1984; Clark, 1994). Foulds had already convincingly shown that the concept of a precancerous or prenialignant lesion was flawed. They are not “pre-” anything. Most early lesions in tumor progression were endstage lesions with no future; they were either indolent o r regressed (Foulds, 1969). T h e continuous photographic documentation of developing human primary melanomas showed that some arose from large

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precursor lesions (dysplastic nevi), others from small pigmented lesions, and still others from clinically normal skin. Clinically normal skin, clinically normal colon, and other clinically normal organs may harbor microscopic foci or atypical cells that could lead to overt cancer. An initiated organ is at risk for the development of cancer and the precise site or origin of that cancer may or may not be a manifest lesion. A precursor state is composed of all lesions and cells resulting from neoplastic induction. The ability to block progression from the precursor state to cancer, in our view, should be the linchpin of control of morbidity and mortality due to cancer (Clark, 1994). 6 . Other Neoplastic Systems and Tumor Biology I may now return to the stalled train. The years at Penn had been relentlessly informative through the continuous interaction of the same group of investigators with each other and an ever-expanding data base (the information base) descriptive of a single human neoplastic system. The events of neoplasia were similar regardless of the system o r the presence or absence of genetic change. Further, a given lesion (phenomenon), say a squamous papilloma, an adenoma, or a melanocytic nevus, was qualitatively similar in each system. The similarity existed even though chromosomal and genetic data were quite different or not demonstrable. Neoplastic systems could not be rigorously defined by extant hypotheses. I had also learned at Penn with the Pigmented Lesion Study Group something about learning and the university, or what I perceived a university should be. Central to my learning process was a concept of knowledge. Knowledge and information or data are not the same. Gradually armed with concepts of what I thought of knowledge, I wanted to spend my remaining years making judgments with information about different neoplastic systems that could lead to knowledgenew knowledge, if you will-about neoplasia and cancer. I decided to study tumor biology. Today’s medical school is barely recognizable as a part of a university, as it is trapped in data acquisition and service without thinking that such functions are not necessarily central to learning. I decided to leave the university in order to learn, to try to acquire knowledge; a sad state of affairs.

VI. Maine, the Beth Israel Hospital, Harvard Medical School, and Pathology Services, Inc. Back to the Future

Maine is one of the finest meetings of land and water on the face of the globe. I decided to go there to live and to study tumor biology. My wife, Patricia, was and still is of immeasurable help with my library and

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literature searches and listens patiently (occasionally impatiently) as 1 expound on my latest concept of the nature of cancer. T h e two of us were to be my idealized university. Fortune provided me with a major addendum to my plan. Terence J. Harrist asked me to be a consultant to his laboratory, Pathology Services, Inc. In this capacity I continued to see problems in cutaneous neoplasia. Each of the problems provided information that was useful in my thinking. Through Terry Harrist I began talking with Hal Dvorak (Harold F. Dvorak), the chairman of pathology at the Beth Israel Hospital. Hal’s major interest is cancer and he has formed a group of investigators, largely molecular biologists, who study and think about cancer biology. Many of the studies are in vitro and many are related to vascular permeability factor (VPF) and diverse aspects of tumor stroma. Hal and I meet every Friday morning for almost 2 hr. My Wednesday lunches at Penn are now Friday mornings with Hal Dvorak. The conversations range over all aspects of neoplasia. Hal and his group are well informed in the molecular biology of cells derived from neoplasms. Such an environment has been an ideal complement to my thinking on tumor progression and neoplastic developmental biology. My university expanded beyond Maine to Harvard (the Pathology Department of the Beth Israel Hospital) and to a large consultation laboratory, Pathology Services, Inc. The work has at its core the generation of knowledge about the nature of neoplasia and cancer. Shortly after my arrival in Boston, I attended a Gordon Conference on cancer and spent a good part of 2 days talking to Harry Rubin. Harry is a scholarly man, and he introduced me to many of his ideas concerning epigenesis and progressive state selection. I had already read a most remarkable paper of his on epigenesis (Rubin, 1990a). That paper precipitously led me into different ways of thinking about transfer of heritable information in living systems. Now, I was personally exposed to Harry’s ideas on cancer as a developmental disorder and on progressive state selection. I carefully studied his papers on these concepts and found they provided sound explanations of some neoplastic phenomena not provided by genetic concepts (Rubin, 1985, 1990b). I continue to study Rubin’s work. It has become an integral part of my thinking. If this were not enough, Harry introduced me to the work and thought of one of the remarkable minds of this century: the late Walter M. Elsasser. Elsasser, a distinguished theoretical physicist, spent much of his life thinking of life, specifically objective organismal life not inner life. He viewed organic life as a set of centers where the coordination of causal chains was totally lost in unfathomable complexity. The centers of coordination are organisms. Thus, an organism, in Elsasser’s view, is “. . . a dynamically stable coordination of organs and functions such that its

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interpretation along purely physico-chemical principles seems hard to conceive.” His concepts do not suggest any aspect of vitalism for he unequivocally states that the molecular mechanisms operative in organisms always obey the laws of quantum mechanics. He does not feel, however, that reductionism, which is incorporated into quantum mechanics, is sufficient to explain organismal life (Levins and Lewontin, 1985).11 Elsasser regards the genetic code as only one aspect of organisma1 life. He has a holistic view of life and with Weiss and others believes that hierarchical controls are constantly operative in organismal life. T h e Cartesian method (i.e., of breaking complex systems into its simpler parts and from the properties of the parts explain the properties of the whole) cannot be applied to the totality of organismal life. It follows that biology becomes a non-Cartesian science. Elsasser presented four principles which govern organismal life. They cannot be readily condensed for discussion in this essay, but the first of these principles, the principle of ordered heterogeneity, is partially descriptive of organismal life. The other principles are those that function to bring about organismal life and its ordered heterogeneity. Organismal life cannot be explained by the summation of properties derived from expression of its genes. In the words of Paul A. Weiss, “There is no step in development-or any process in a living system, for that matter-in which the genetic background is not intimately involved, but there is no phenomenon o r attribute of life, either, in which the genome would be solely instrumental as a kind of omnipotent autonomous ruler, in which capacity it has often been presented explicitly or by indirection.” Weiss’ Fig. 1 clearly illustrates the interpenetrating continuum, to use a term of Levins and Lewontin, characteristic of organismal life and representative of hierarchical control in an organism (Weiss, 1973). Levins and Lewontin discuss at length the Cartesian method and the interrelationship of parts and whole, much of which is applicable to Elsasser’s and Weiss’ concept of organismal life (Levins and Lewontin, 1985; Lewontin, 1989, 1992). Lewontin’s writings have been especially helpful to me and he has become a true mentor. As a result of my studies of Elsasser, Lewontin, and Rubin, I became quite excited about the nature of an organism and could readily view organismal life as ordered heterogeneity having complex hierarchical control mechanisms, only one of which was a genetic template. Elsasser, Weiss, Rubin, and Lewontin provide broad vistas permitting holistic views of organismal and cellular life, but none of these men presume to provide a complete alternative to reductionism. 1 1 The final portion of this monograph, entitled, “Conclusion: Dialectics” includes a valuable discussion of reductionism and the Cartesian mode of thought.

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Reductionism, especially that of monistic molecular biologists, fails to explain many phenomena of life. Holism may ultimately fulfill its promise of becoming an epistemologic revolution in biology, but that does not mean one cannot inquire into its possible mechanisms. Shortly before I left Penn I had developed some interest in stromal changes in lesions of the precursor state of melanocytic neoplasia. T h e interest in stroma was whetted even further by the hours of conversation with Hal Dvorak, who has been remarkably patient with me. His knowledge of wound healing, angiogenesis, vascular permeability, and the various proteoglycans of stroma (and many other things) was extensive. I began searching the literature weekly for articles on the extracellular matrix. I found an article by Lia Ettinger and Fanny Doljanski of immeasurable help in my thinking about organismal development, organismal life, and cancer. My view of the extracellular matrix would never be the same. These wonderful Israeli ladies have answered my queries in detail and permit me to think constantly about the dynamic continuum of epithelial-stromal interactions. In addition, they have also found the genetic concept unable to explain a developmental program. Ettinger and Doljanski give 1 1 recent references to articles "that develop alternative views to the generally accepted concept of a morphogenetic programme fully encoded in the genes." They add, ". . . very persuasive arguments regarding the inadequacy of the genetic metaphor have been presented. . ." (Ettinger and Doljanski, 1992). At a pragmatic level, Ettinger and Doljanski permitted me to think of the continuous interaction of cells and their extracellular matrix in organismal development and maintenance. As someone has said, biology does not stop at birth. An organism is under hierarchical control in development and maintenance and the result is a form of life characterized by ordered heterogeneity. A view of all of this infinite complexity as driven from the gene u p is an impoverished view of organismal life. What of neoplasia and its sometimes child, cancer?" In the broadest sense neoplasia lacks a well-delineated causal arrow. A hammer hitting a ' 2 There is no uniformity of definitions in the field of tumor biology. I have discussed this problem in a recent publication (Clark, 1991b) and have provided a short glossary in that paper. One should also see (Clark, 1994). For the purposes of this essay, neoplasia comprises all focal proliferative lesions, benign tumors, primary cancers, and metastases that may affect any given cell system. A cancer is a circumscribed area within an organ where therc is a continuing increase in the number of cells (growth has no time constraints or is temporally unrestricted) when compared with the surrounding cells. A cancer grows in two or more tissue compartments. Growth in this context is due, at least in part, to mitotic cell replication. T h e ability to give rise to a metastasis may or niay not be a property of a primary cancer. A primary cancer is a quite rare event even in an extant precursor neoplastic state.

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finger and producing a bruise is one of the few examples of a true causal arrow in disease and even this does not take into account the full concept of causation (Olsen and Overvad, 1993).13Neoplasia is almost the opposite. Causal arrows in neoplasia are as lost in complexity as they are in the parent organism. Even the valuable component cause model of Olsen and Overvad lacks the confounding complexity of time. Virtually anything or any agent can induce a precursor state, the first evident manifestation that neoplastic disease has begun. Many carcinogens are not mutagens and many mutagens are not carcinogens (see Strauss, 1992). Inert solid plastics will induce sarcomas (Bischoff et al., 1964). More than nine different chemical carcinogens will induce carcinoma of the rodent colon, but so will dextran sulfate after inducing ulcerative colitis (McLellan'4 et al., 1991; Tamaru et al., 1993). Even if one selects examples of experimental carcinogenesis where mutagenic carcinogens are used for induction, such as some studies of hepatocarcinogenesis, the carcinogens do not produce cancer as the first response to their injury. Over 75 carcinogens have been used in hepatocarcinogenesis. T h e initially manifest lesions are hepatocyte nodules, lesions that are a hallmark of the precursor state and lesions that are analogous with adenomatous polyps and squamous papillomas. Carcinoma may arise later and when cancers d o arise they are numerically rare when compared with the hepatocyte nodule of the precursor state. It follows that cancer is an uncommon response to a carcinogen. When cancer does emerge relatively frequently from a given precursor state, the high incidence is due to extensive inbreeding for the trait of frequent progression of a neoplastic system to cancer (Farber and Sarma, 1987). The precursor state may be regarded as a condition where organ maintenance is imperfect. If one regards an adenomatous polyp of the colon as a prototypic lesion of the precursor state, its very form suggests that it may be partially removed from the hierarchical controls over colonic form and function. With the polyp the developmental morphogenesis of an abnormal life form has begun. T h e cells of the polyp exist in a state different from the surrounding colonic mucosa and could well be one of the sites of progressive state selection of Rubin (Rubin, 1990b). Neoplasia is not a disease of a single-cell system, even though its most obvious and dramatic manifestations are seen in one cell system such as the parenchymal cells derived from an epithelium. However, the stroma of neoplastic lesions is I:3 The paper of Olsen and Overvad contains a valuable discussion of causation as a part of their exploration of the multifactorial etiology of cancer. 1 4 My correspondence and discussions with Beth (Elizabeth A,) McLellan concerning experimental colorectal carcinogenesis have been invaluable.

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altered and may be quite characteristic. This is seen in the precursor state where the extracellular matrix is significantly altered. One may note, for example, thickening of the basement membrane, unusual forms of fibroplasia, and angiogenesis. Occasional cancers emerge from the precursor state, but most organs affected by the precursor state remain without cancers. The emergent cancers may show progressive growth without competence for metastasis, but some cancers do have metastatic competence. Cancers show progressive disorganization, involving parenchyma and stroma. The latter is heterogeneous in appearance, in some areas showing an altered basement membrane, different patterns of fibrous tissue, and blood vessels, while in others the stroma of a cancer may be inconspicuous. Cancer is associated with the progressive loss of tissue maintenance related to progressive atypia of the parenchyma and progressive disorganization of its stroma. The very act of separating parenchyma from stroma is destructive of interrelationships and processes at the heart of the cancer problem. T h e parenchyma and stroma are reciprocally interrelated (Chung et al., 1991a, 1991b, 1993). Changes in the parenchyma are associated with changes in the stroma (Nara et al., 1991). The latter causes further alterations in the parenchyma and this, in turn, produces subsequent stromal changes. Reciprocal interactions are essentially unending (Bissell et al., 1982, 1987). The stromal influences include changes in gene expression, but such changes are but a part of an unimaginably complex whole (Lin and Bissell, 1993; Petersen et al., 1992; Streuli et al., 1993). In this view, cancer is the loss of ordered heterogeneity and the progressive supervention of disordered heterogeneity. Such progressive disordered heterogeneity is self-(dis)organizing and begins with induction of the precursor state (see Ettinger and Doljanski, 1992, for some discussion of self-organization in development; also see Edelman, 1988). Rowlatt discusses at some length a neoplasm as a focal self-perpetuating tissue disorganization (Rowlatt, 1993). Alteration of parenchymal-stromal interactions are first observed in the precursor state and continue through the neoplastic system to metastases. With progressive abnormality of the stroma it is to be expected that cytokine expression and storage will be altered. As cytokines control, in part, the remodeling of tissue (including the abnormal modeling attendant upon the neoplastic state), the neoplastic stroma could contribute through cytokines to the progressive self-disorganization of cancer. Further, the carbohydrate moities of the extracellular matrix potentially have a greater storage then the genome (Nathan and Sporn, 1991). The flawed parenchymal-stromal interactions are a prominent manifestation of the (dis)ordered heterogeneity characteristic of cancer and seem to be essential to this abnormal form of

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life, as, of course, normal parenchymal-stromal interactions are to the parent organismal life. Science devoted a number of articles to “Cancer” in the November 22, 1991 issue. The articles reflected the dominant concepts of cancer that exist today. One definition of cancer in that issue was, “Cancer may be defined as a progressive series of genetic events that occur in a single clone of cells because of alterations in a limited number of genes: the oncogenes and the tumor suppressor genes” (Solomon et al., 1991). Such a view gives genetic alterations both ontologic and etiologic primacy. Concepts of the nature of cancer that give due consideration to developmental biology, and neoplastic development and progression result in different concepts and definitions. Rowlatt, for example, says this about the beginnings of the disease, “Any transmissible stable alteration in behavior so that there is escape from any tissue-organizing mechanism which can cause a tissue mass will initiate neoplasia.” Rowlatt conceptualizes cancer as follows, “A neoplasm is a mass of tissue generated by cells capable of division which have acquired either permanent expressible heritable change or stable epigenetic change so that the same o r other cells no longer respond appropriately to one or more normal tissue organizing stimuli, chemical or physical, intracellular or extracellular, in the organism in which it occurs” (Rowlatt, 1982, 1993). At the beginning of this essay I said that investigations and investigators cannot be separated. Indeed, they are intimately related. I have spent most of a long professional life observing and recording the phenomena of neoplasia as seen in human melanocytic neoplasia. For reasons largely hidden in the mists of memories I did not bring to my observations of cancer prior concepts or assumptions as to its nature. I permitted the disease to lead me and assist in the formulations of hypotheses and definitions. I have not reached the present time with fixed concepts of the nature of cancer. The paradigms that satisfactorily explain some of what I have observed include developmental biology, progressive state selection, and progressive self-(dis)organization, partially related to flawed epithelial-stromal interactions. The investigator of cancer must explain the disease as it exists in man. He must attempt explanation of all of the phenomena without encumbering his investigations with decisions made in advance as to the nature of the disease (Clark, 1994b). Alfred North Whitehead gave us guides for scientific thought and endeavor and these seem especially useful for those who would study tumor biology (Whitehead, 1925). In formal logic, a contradiction is the signal of a defeat: but in the evolution of real knowledge it marks the first step in progress towardr a victory. This is one great reason for the utmost toleration

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vurzety of opinion. Once undfiirever, tliis duty of toleration has been sununi~dup i ~ thr i uiord\, ‘Let both grow together until the harvest.’ . . . I t is eclsy enough to f i n d a theory, l o g ~ a l l y harnionious and uiith important applications in the regions offiirt, provided that you are coiiteut to disregurd hay your evidence. . . . A n unflinching determincction to take the whole rvidrnce into account is the only method of preservation against the fluctuuting extrenies of ,fashiouuble opinion. This aduice seems so easy and is in fact so dtfficult to follow.” of

ACKNOWLEDGMENTS Dedicated to my colleagues, my students, my patients, and my wife, Patricia A. Clark. They are an integral part of my thought and work.

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Arrington, J. H. 111, Reed, R.J., Ichinose, H., and Krenientz, E. T. (1977). Am.,]. Surgzcal Path. 1, 131-143. Bischoff, F., and Bryson, G. (1964). Prog. Exp. Tumor Res. 5, 85-133. Bissell, M. J . , and Barcellos-Hoff, H. (1987).J. Cell. Sci. Suppl. 8, 327-343. Bissell, M. J., Hall, H . G., and Parry, G. (1982).J. Theor. Biol. 99, 31-68. Breslow, A. (1970). Ann. Surg. 172, 902-908. Chung, L. W. K. (199 I b). Cancer Metastasis Rev. 10, 263-274. Chung, L. W. K. (1993). Semin. Cancer Biol. 4, 183-192. Chung, L. W. K., Gleave, M. E., Hsieh, J., Hong, S. J., and Zhau, H. E. (1991a). Cancer Sun,. 11,91-121. Clark, W. H.,Jr. (1991a). Cancer Metastmis Rev. 10, 83-88. Clark, W. H. ( 199 1b). Brit. J . Cancer 64, 6 3 1-644. Clark, W. H., Jr., Elder, D. E., Guerry, D., IV, Braitman, L. E., Trock, B. J., Schultz, D., Synnesvedt, M., and Halpern, A. C. (1989).J. Natl. Cancer I m t . 81, 1893-1904. Clark, W. H., Jr. (1967). In “Advances in Biology of Skin. T h e Pignientary System” (W. Montagna, Ed.), Vol. VIII, 621-647. Perganion Press, Oxford and New York. Clark, W. H., Jr. (1994a). I n “The Science and Practice of Cancer Prevention and Control” (P. Greenwald, B. S. Kramer, and D. L. Weed, Eds.), 135-1 Clark, W. H., Jr. (199411). Actu Oncologicu, in press. Clark, W. H. Jr., and Hibbs, R. G. (1958). J . Biophys. Biorhern. Cytol. 4, 679-684. Clark, W. H.,Jr., Elder, D. E., G e r r y , D. IV, Epstein, M. N., Greene, M. H., anti van Horn, M. ( 1984). Hum. Pathol. 15, I 147- 1 16.5. Clark, W. H., Jr., From, L., Bernardino, E. A,, and Mihni, M. C., Jr. (1969). Cancfr Ra . 29, 705-726. Clark, W. H., J r . , Min, B. H., and Kligman, L. H. L. (1976). Cancer Re.r. 36, 4079-4091. Clark, W. H., Reirner, R. R., Greene, M., Ainsworth, A. M., and Mastrangelo, M. J. (1978). Arch. Derniatol. 114, 732-738. Demetrick, D. J., Herlyn, D., Tretiak, M., Creasey, D., Clevers, H., Donoso, L. A,, Vennegoor, C. J. G., Dixon, W. T., and Jerry, L. M. (1992).J. Natl. Cancer frwt. 84,422-429. Edelman, G. M. (1 988). “Topobiology: An Introduction to Molecular Embryology.” Basic Books, New York. Elder, D. E., and Herlyn, M. (1992). Pigment Cell Res. S2, 136-143. Elder, D. E., Clark, W. H., Jr., Elenitsas, R., Guerry, D. IV, and Halpern, A. C. (1993). In “Seminars in Diagnostic Pathology” (A. Cochran, Ed.), Vol. 10, pp. 18-35.

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THE ORIGINS OF THE SMALL DNA TUMOR VIRUSES Arnold J. Levine Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08540

I . Introduction A. Vaccines B. Cell Culture C. Poliovirus 11. T h e Viruses of Monkey Kidney Cells 111. T h e Origins of the DNA Tumor Viruses A. T h e Adenoviruses B. Simian Virus 40 IV. T h e Molecular Biology of the DNA Tumor Viruses V. Conclusions References

1. Introduction

A. VACCINES T h e credit for the “discovery” of viruses, first defined as infectious entities that can pass through a filter which normally retards bacteria, is most commonly given to Dimetri Ivanovsky in 1892 (Stanley, 1944; Lustig and Levine, 1992). However, the recorded history of viral diseases spans more than 2000 years with reports of smallpox originating in writings from India and Western Asia (Fenner et al., 1988). By 700 A D , this virus was described in the literature of Japan, Europe, and North America. T h e introduction of smallpox virus in the Americas, in the Caribbean (1507), Mexico (1 520), Peru (1524), and Brazil (1555), is documented by the terrible toll resulting from the infections of noninimune Amerindians and the extraordinary impact this disease had upon their history and survival (Hopkins, 1983). Similarly, the slave traders of the 16th and 17th centuries brought smallpox once again from Africa to the Americas and, 1 year after the arrival of the “first fleet” in Australia, the aborigines experienced their first serious epidemic of smallpox. This story of a virus, largely endemic in one portion of the world being introduced into a new territory with populations containing no

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Copyright Q I994 by Academic Pleas. I I K All rights of reproducth,~~ in any torin reaerved.

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immunity resulting in epidemics with high death rates, is a pattern that has repeated itself over the centuries. T h e development of practices to protect people from smallpox has been found in the Chinese literature as early as the 10th century (Fenner et al., 1988). Old powdered crusts from the skin pustules of diseased people were given to individuals free of the disease by intranasal insufflation. In India, Brahmins inoculated a powder made from dry crusts of infected patients into the scarafied skin of persons without disease symptoms. T h e Persians ingested the crusts of skin lesions from patients while the Turks inoculated fluid from the pustules into themselves (Fenner et al., 1988). There are two strains of smallpox virus, variola major which has a high fatality rate (20-2596 of infected individuals), and variola minor with a mortality rate of only 1 to 2%. The idea of using the variola minor strain as the first natural vaccine to protect against the variola major disease was adopted along with these immunization procedures. These practices were called “variolation” and they, by and large, worked to protect individuals but with some significant drawbacks. While the disease produced by these live viruses was frequently milder than variola major, there was still a 1 to 2% fatality rate. Nonetheless, the practice of variolation used in the Middle East was noticed by Lady Mary Wortley Montague, the wife of the British Ambassador to Turkey, and in 1718 she introduced it into England. It was widely employed in the British colonies for the next century (Fenner et al., 1988). In 1776 in Gloucestershire, England, Edward Jenner made his first set of observations noticing that milkmaids acquired a mild form of this disease from cows (variolae vaccinae or cowpox) and were then spared from getting smallpox even in times of epidemics in their community. By 1798, Jenner had introduced cowpox into people and showed that these individuals were protected from smallpox disease. The first scientifically tested vaccine and the use of a related animal virus attenuated for disease in humans are the real contributions of Edward Jenner (Jenner, E., reprinted, 1959). It is ironic that the present-day vaccine strain for smallpox is not related directly to the original cowpox virus. While vaccinia virus, used for vaccination to eliminate smallpox, is a closely related set of poxvirus strains, all of these viruses are quite distinct from their purported ancestor, the cowpox virus (Baxby, 1981; Fenner, 1990). Using this vaccine, the World Health Organization (WHO) in 1967 began a program to eradicate smallpox throughout the world. The lack of any known animal reservoir for this virus and an intensive effort in India and Pakistan to immunize all contacts surrounding each patient with smallpox was a successful strategy. In October 1977, the last recorded case of a natural infection with smallpox was detected in Ali Maoliri of

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Somalia (WHO, 1980). The endemic nature of smallpox, which was perpetuated by human to human passage, has been broken and a disease with over a 2000-year recorded history has disappeared. Thus, from 1798 to 1949, vaccines were developed for smallpox by Jenner, for rabies virus by Pasteur (1885), for yellow fever by Theiler and Smith (1937), and for influenza viruses (1940s). In each case, the sources of host material for these viral vaccines were infected animal organs or tissues (including the embryonated hens’ eggs) (Hilleman, 1992). Indeed, basic research with viruses during that time was considerably complicated by the need to have a colony of host organisms tethered to a laboratory in order to carry out virus infections. Throughout this period, it was only poorly appreciated that each organism used as a host for a virus, be it humans or cows (vaccinia), rabbits (rabies), or the chick embryo (yellow fever virus, influenza A and B viruses), brought with it a set of its own contaminating viruses which complicated the safety or efficacy of these early vaccines. B. CELL CULTURE

Unlike the plant virologists tied to their greenhouses and the animal virologists bound to the animal rooms, the study of bacteriophages, discovered by Frederick W. Twort ( 1915) and Felix d’Herelle ( 1917), provided these virologists with a host that replicated as a single cell organism. The development by d’Herelle of quantitative assays using limiting dilutions and a plaque assay gave meaning for the first time to the term concentration or titers of viruses. This in turn permitted the quantitation of the adsorption step of a virus to its host; the demonstration that a specific virus adsorbs to one bacterial host species but not a different host and the first measurement of a one-step growth curve by a virus in its host (Stent, 1963). These same experiments convinced d’Herelle that viruses must be particulate in nature and that each plaque was indeed the progeny of a single virus (d’Herelle, 1917). This was a considerable conceptual advance because Martinus Willem Beijerinck ( 1898) had previously studied the tobacco mosaic disease and had shown that the sap of infected leaves passed through a porcelain filter still transmitted this disease. He clearly showed that this “agent” had the ability to multiply (i.e., it could regain its strength after dilution.) Further, he demonstrated that the infectious agent only replicated in living, growing plant tissue, which he claimed made it more likely to be a living pathogen rather than a chemical substance. Finally, Beijerinck failed to see any infectious agents in the light microscope after filtration of the plant sap

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and so he termed the agent a “contagium vivum fluidum” (Beijerinck, 1898). This idea that viruses were liquids resulted in a 30-year debate about the chemical nature of viruses. Indeed, all through the 1920s, Jules Bordet, the Director of the Pasteur Institute in Brussels, held to the hypothesis that a soluble substance (such as an enzyme) touched the bacterial host, which in turn reproduced the viral substance (Stent, 1963). Not a bad hypothesis, if a bacteriophage had turned out to be a prion (Chesebro, 1990). All this debate aside, the value of having single cell cultures for investigating the nature of viruses was not lost on the virologists of that time. T h e problem was that the notion of growing animal cells outside the body o r in cell culture for any reasonable amount of time was commonly accepted as impossible (Pollack, 1973). However, Alexis Carrel of the Rockefeller Institute for Medical Research in New York challenged this notion (Carrel, 1912). Carrel kept an explant of chicken muscle tissue alive by continuously feeding the culture new medium (and possibly new cells as well). In his initial experiments he kept chick embryo cells alive and dividing for 85 days and spoke of it as “the solution of the problem of permanent life of tissues in vitro” (Carrel, 1912). Indeed, he eventually kept a tissue explant living longer than the chicken from which it came might have been expected to live. Carrel was indeed interested in “permanent life.” By 1948, Sanford, Earle, and Likely at the National Institutes of Health overcame the difficulty of culturing single animal cells (Carrel and others worked only with tissues o r clumps of cells) (Sanford et al., 1948). This paper described the first clones of mammalian cells in culture and a medium for the survival of single cells in a small capillary pipet. Harry Eagle (1955) worked out the optimal medium for growing single cells in culture and George 0. Gey and his colleagues (1952) in the Department of Surgery and Gynecology at the Johns Hopkins Medical School cultured human cells for the first time, starting with normal human epithelial cells and cells from cervical carcinomas (Gey, et al., 1952). They stated, “thus far, only one strain of epidermoid carcinoma has been established and grown in continuous roller tube cultures for almost a year” (Gey, et al., 1952) and HeLa cells were born. T h e availability of chicken, mouse, and human cells in culture ushered in the next era of virus research with animal viruses. C. POLIOVIRUS Epidemics of poliomyelitis were virtually unknown prior to the 20th century. T h e virus was endemic all over the world and populations were widely infected at an early age due to poor sewage systems and, in some

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regions, the common use of human excrement as fertilizer. This “oralfecal” route of poliovirus transmission and the high infection rate in a population ensured that mothers first provided passive immunity to their infants and that children often obtained their first exposures to poliovirus early in life so that active immunity began while passive immunity faded. Breast feeding yielded yet additional sources of antibody in the intestine of young hosts for this virus. Disease caused by this virus was rare and when it occurred, prior to the 20th century, it was on top of a high background of infant and childhood mortality. Because of this, poliomyelitis was not a major disease in the populations of the world before the 20th century (Melnick, 1990). It is quite paradoxical then that the first signs of epidemic poliomyelitis and increased paralytic disease occurred in the United States and the Scandinavian countries where improved sanitation was first accomplished in the 19th century. In areas where a high-quality water supply was installed and new alternative sources of fertilizers were used, children were born and grew up with little or no exposure to polioviruses. In the United States in the early 1950s, the peak incidence group for contracting poliovirus was children of elementary school age. T h e great majority of the poliomyelitis deaths, however, occurred in patients over age 15 (Melnick, 1990). The population was first exposed to poliovirus long after their infancy. T h e virus spread from person to person in an epidemic pattern in stark contrast to the previous endemic exposures of past centuries. The improved sanitation altered this disease from a rare and sporadic one to the epidemics of the summer seasons of the 1930s, 1940s, and early 1950s. Poliovirus enters the host via the oral route (i.e., food, water, oral secretions). The virus then replicates actively in the small intestine and the associated lymph nodes. If the host has been previously immunized (the strategy of the live Sabin vaccine), a vigorous immune response at this stage limits the infection. In the absence of such a response, the virus passes into the bloodstream and is spread throughout the body. Antibodies in the blood of immunized hosts (the strategy of the Salk vaccine) are the second line of defense. With no immunity, the virus replicates in a number of susceptible tissues including motor neurons of the central and peripheral nervous system (Melnick, 1990). One of the viruses that causes poliomyelitis was first isolated by K. Landsteiner and E. Popper as early as 1909 (Landsteiner and Popper, 1909). It subsequently became clear that three immunologically distinct poliovirus strains existed in the wild (Melnick, 1990). In what most virologists agree was the critical contribution to the development of the poliovirus vaccines, John F. Enders, Thomas H. Weller, and Frederick C.

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Robbins (1949) succeeded in replicating the Lansing strain of poliovirus in human explant embryonic tissues in culture. These experiments eliminated the then prevalent idea that poliovirus only grew well in neurons. This opened u p the possibility of developing a vaccine from viruses replicated in tissue culture. This discovery had a tremendous impact on the rapid development of additional vaccines (i.e., poliovirus, adenovirus, measles, mumps, rubella, varicella, and hepatitis A virus) (Hilleman, 1992), but it also ushered in a new era for the study of animal viruses at the molecular level. The use of Gey’s HeLa cells as a host to replicate poliovirus resulted in a quantitative plaque assay (Dulbecco and Vogt, 1953) and a one-step growth curve (Darnell and Levintow, 1960). The use of poliovirus RNA as a mRNA found in new structures called polysomes (Penman et al., 1963) which synthesized poliovirus capsid protein (Scharff et d., 1963) was first demonstrated using this experimental system. T h e detection of the poliovirus polyprotein which is then processed to produce capsid proteins and an RNA-dependent RNA polymerase (Baltimore and Franklin, 1962) which in turn synthesizes a double-stranded RNA replicative intermediate of poliovirus (Baltimore et d., 1964) created the new concept of RNA-dependent RNA synthesis. The impact of poliovirus on its host cell began the study on how a virus induced cell damage which was understood for the first time at the molecular level (Darnell and Levintow, 1960). Finally, the assembly and release of poliovirus from cells also provided insights into these processes occurring in v i m that were only poorly outlined before these experimental systems were developed (Luria and Darnell, 1968). Between 1949 and 1952, the rapid development of cell culture techniques and the growing list of permanent cell lines led both to the era of molecular animal virology and the rapid development of vaccines. In addition, several key experiments suggested that a vaccine for poliovirus would be efficacious. In 1952, Dorothy M. Horstmann (Horstmann, 1952) demonstrated that poliovirus enters the bloodstream during its incubation period in a cynomolgus monkey who was infected by the oral route. In addition, small amounts of antibody administered passively protected against paralytic disease in monkeys (Bodian, 1952), and in humans (Hammon et al., 1953). By that time it was clear that poliovirus would replicate only in human or monkey cells in culture. Several groups began developing poliovirus vaccines with Herald Cox and Hilary Koprowski leading a team at Lederle Laboratories in Pearl River, New York, working on a live attenuated vaccine (Koprowski et al., 1952). Albert Sabin was also developing a live attenuated vaccine (Sabin, 1957), and he was supported by the Sister Kenney Foundation. Basil O’Connor at the National Foundation for Infantile Paralysis which conducted an

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annual “March of Dimes” campaign supported a team of scientists including Jonas Salk who was pioneering a killed trivalent poliovirus vaccine (Salk et al., 1953). T h e problem remained that any vaccine would have to be grown in cells capable of producing hundreds of millions of doses. Primary human embryonic cells removed from embryos or fetuses, of the type described by Enders (Enders et al., 1949), could not be obtained in large enough quantities to support vaccine production for the entire population. In the early 1950s, continuous cell lines such as Gey’s HeLa were also rejected because they were derived from cancerous tissues (cervical cancer) and might harbor “human cancer viruses.” It would be shown many years later that HeLa cells have integrated into their DNA the genome of a human papilloma virus type 18 (Shah and Howley, 1990). This viral genome expresses two viral encoded oncogenes, termed the E6 and E7, which are required for continued immortalization of these cells in culture (Shah and Howley, 1990). T h e cell line produces no Papillomaviruses and it is unlikely that such viruses would ever contaminate a vaccine made in these cells. Thus, the only choice left for poliovirus vaccine production was monkey kidney cells. II. The Viruses of Monkey Kidney Cells

T h e use of monkey kidney cells on a scale of the size needed for vaccine trials rapidly led to considerable experience with these cells. Three species of monkeys were used for the production of poliovirus stocks and vaccines: rhesus monkeys (Macacca mulatta) from India; cynomolgus monkeys (Macacca cynornolgw) from the Philippines and Southeast Asia; and the African green monkey (Cercopithecus aethiops) from Africa. What was becoming clear, however, was that the cells of these monkeys harbored a large number of both latent and overt cytopathic and noncytopathic viruses. Because poliovirus replicated in the cells and the crude cell lysate was to be employed as a virus source for the vaccine, the batch-to-batch virus preparation was not reproducible and contained several diverse monkey viruses. The response at the time to this growing dilemma was to identify and classify as many of these monkey agents as possible. A growing list of Simian viruses (called SVI, SV2, SV3 etc.) were classified and Dr. R. N. Hull at Eli Lilly and Company became the keeper of the virus stocks and records for each new virus isolated from these monkey cells. Two goals rapidly took shape: (1) to identify and kill these viruses with antisera or chemical inactivation; or (2) to try to grow the poliovirus vaccine in monkey kidney cells that were tested for and free of these contaminating viruses. Because the Salk vaccine employed formalin to kill the poliovirus, it was also employed to

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hopefully kill all the endogenous viruses present in the monkey kidney cells. A constant watch for new monkey viruses was a critical part of poliovirus vaccine research and it appeared to be working reasonably well (Hilleman, 1990). Why were these potential problems permitted to persist into the vaccine trials? Part of the answer was the growing proportions of paralytic poliomyelitis each summer. Between 1950 and 1953, the last 3 years prior to the introduction of the Salk vaccine, there were about 21,000 cases of paralytic poliomyelitis in the United States each year. By 1960, after the use of the Salk vaccine, this was reduced to 2500 cases per year and, with the introduction of the Sabin vaccine in the United States in 1961, the number of cases fell to 465. By 1969, only 18 cases were reported (Melnick, 1990). There was considerable justification to proceed with monkey kidney cells in spite of their burden of viruses. Between 1954 and 1960, several pharmaceutical companies took over the poliovirus killed vaccine production, growing the virus in monkey kidney cells, inactivating it and the contaminating viruses with formalin (Wishnow and Steinfeld, 1976). Ill. The Origins of the DNA Tumor Viruses A. THEADENOVIRUSES Wallace P. Rowe, working at the National Institutes of Health in the first years of the 1950s, was attempting to isolate viruses that caused the common cold. At the time, it was a popular medical practice to remove the tonsils and adenoids from young children to minimize future infections. This meant that human tissue from these organs was readily available and Wally Rowe was studying the viruses that came from human adenoid cells in culture. The organ was placed in cell culture and medium and cells rapidly migrated out of the tissue to colonize the dish. These explant cell cultures would grow with time and a promising cell culture system looked to be developing from an abundant source of normal human tissue. As these observations were repeated, it was noted that the cells would degenerate and die at various times after the start of a culture. Two possible explanations were considered: (1) the cells had a finite life span in this medium and died after a few divisions (this was 8 years before Moorhead and Hayflick were to publish that normal cells do indeed have a limited life span (Hayflick and Moorhead, 1961);or (2) an infectious agent present in the adenoids was activated. It would then replicate in and kill these cells in culture. In the original paper describ-

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ing this observation by W. P. Rowe, R. J. Huebner, L. K. Gillmore, R. H. Parrott, and T. G. Ward (1953; “Isolation of a cytogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture”), Rowe and his colleagues discovered a new group of human viruses which they called AD viruses (adenoid viruses), and are now the human adenoviruses. These agents were to play a central role in eukaryotic molecular biology and tumor virology. At just about this same time, Maurice R. Hilleman was at the Walter Reed Army Institute studying an influenza respiratory epidemic in military recruits at Fort Leonard Wood, Missouri. He went out to several military bases to collect samples from soldiers with acute respiratory illness and to type the strain of influenza which was causing the epidemic. As these samples were tested, no evidence could be detected for the presence of influenza virus. Hilleman, with an airplane filled with samples of throat washings containing little o r no information about the influenza virus epidemic strain, was becoming quite concerned about the utility of his present research efforts. It was at this point that a new virus was consistently detected in all these samples and it was termed the RI agent (respiratory illness virus) in a publication by M. R. Hilleman and J. H. Werner (1954; “Recovery of new agent from patients with acute respiratory illness”). It soon became clear that the RI and AD agents were similar (Huebner and Rowe isolated the latent adenovirus serotypes 1 , 2 , 5 , and 6 while Hilleman isolated the epidemic serotypes 3,4, and 7). These viruses were then shown to be the cause of acute respiratory diseases such as atypical pneumonia and catarrhal fever in the armed forces. It was becoming clear that the adenoviruses were a previously unappreciated cause of considerable respiratory disease and time lost for the armed forces. Hilleman’s next goals were to prepare and test an adenovirus vaccine at Fort Dix, New Jersey, and other training facilities. This brought him straight to the question of what cells to use to grow this virus (Hilleman, 1968, 1979, 1990). The adenoviruses grew well in embryonic human cells which were in short supply and HeLa cells which were plentiful but derived from a cervical carcinoma. The human adenoviruses grew poorly, if at all, in primary monkey cells in culture. While the adenoviruses were to be adapted for excellent growth in monkey cells for vaccine production (Hartley et al., 1956), this turned out to require a contaminating monkey virus (which would come to be called Simian Virus 40 [SV40]). In one case, an human adenovirus which replicated well in monkey cells derived from a recombinant virus formed between the adenoviruses and SV40 (Lewis et al., 1969). The obligate helper function of SV40 for adenovirus replication in monkey cells, totally unexpected

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at the time (1954-1956), was not to be understood until another decade of research (1964-1966) (Rapp et al., 1964). These observations clearly demonstrate the impact of contaminating viruses on what appears to be a simple process of virus replication in a defined specific cell or tissue taken directly from an animal. With all these problems progressing at a rapid pace, the Armed Forces Epidemiological Board held a meeting on January 22, 1954, in Washington, DC, to determine which kinds of cells in culture should be employed in vaccine production of human viruses. Twenty-five scientists with diverse backgrounds managed to attend in spite of a terrible blizzard with snow drifts blocking traffic. For viruses such as poliovirus or the human adenoviruses, there appeared to be three choices of a host cell: ( 1 ) primary human cells in culture; (2) HeLa cells; or (3) primary monkey cells in culture. The problems were clear to everyone-the primary human cells were in too short supply, the HeLa cells derived from a human cancer, and the monkey cells harbored a growing list of viruses with ill-defined properties. The meeting was short (it lasted only 2 hr) and came to a remarkably concise conclusion “virus material grown on normal cells inactivated by formalin is acceptable for limited immunization trials” (Hilleman, 1979). This eliminated HeLa cells but the term “normal cells” was not defined and the growing ability to continually passage normal cells in culture was not addressed. This statement eventually became a rule of the U.S. Public Health Service Regulations for Biological Products (Biological Products, Public Health Service Regulations, Title 42, Part 43) which forbade the use of continuous cell lines for poliovirus production. All attempts to grow adenoviruses in monkey cells continually revealed the presence of endogenous monkey viruses and the preparation of a vaccine was rapidly becoming impossible. Just about this time, Werner Henle, at Childrens’ Hospital in Philadelphia, claimed he had developed a “normal” human cell line from embryonic intestine which had the unusual property of being a continuous cell line. An adenovirus vaccine (serotypes 3 , 4 , and 7) was readily prepared by Hilleman and his group in this cell line and trials in humans began on March 2, 1956 with six volunteers. While there were no adverse long-term effects of this vaccine, it was subsequently recognized that this was an early example of a HeLa cell contamination of the primary human embryonic cells in culture. There was eventually a complete replacement of the culture by HeLa cells as the host for these vaccine trials (Hilleman, 1979). This would not be the last time where nature steps in, despite the rules of man.

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B. SIMIAN VIRUS40

By 1957, Maurice Hilleman left the Walter Reed Army Institute and joined Merck, Sharp and Dohme Research Laboratories as the Director of Virus and Cell Biology Research. Born 38 years earlier in Miles City, Montana, he attended Montana State College and received his doctorate of philosophy in 1944 from the University of Chicago with specialization in virology. After taking a position at E. R. Squibb and Company (19441947) and the Walter Reed Army Institute of Research (1948-1957), he was well prepared for what would be a long and distinguished career pioneering vaccine development at Merck. Because of his past experiences with preparing vaccines in monkey cell culture, he decided that the highest priority in vaccine research ought to be the development of monkeys or monkey cells that were free of contaminating viruses. Eventually he wanted to make all Merck vaccines in monkey cells with no contaminating viruses. Through this period, Salk had reported the effectiveness of his formalin-inactivated vaccine in over 100 volunteers (Salk, 1953) and the National Foundation for Infantile Paralysis (the March of Dimes) sponsored a massive field trial enrolling 1.8 million school-age children (Paul, 1971). T h e quite successful results were reported on April 12, 1955, and the Public Health Service licensed several companies (i.e., Cutter Laboratories, Lederle Laboratories, Eli Lilly & Co., Parke Davis Corp.) to manufacture the vaccine. In spite of an early problem where aggregated virus particles failed to be uniformly inactivated by the formalin at the Cutter Laboratories in Berkeley, California (resulting in 204 vaccineassociated cases of poliomyelitis), the vaccine worked well and the incidence of disease fell in the United States. Hilleman began the search for monkeys with few or no viruses and, as part of this quest met with William Mann, then director of the Washington DC Zoological Park. Mann was knowledgeable about primates and how they were caught and shipped around the world. He pointed out that the rhesus monkeys captured in India were taken first to the trapper’s village and kept there (in contact with humans), until they were shipped to Delhi o r Calcutta. There the rhesus monkeys were kept in large “gang” cages housing 100 or more animals. I n the wild, the rhesus monkeys live in extended families of 10 to 50 animals forming a tight social structure. There is little or no contact between different family groups. In the gang cages of Delhi or Calcutta, diverse social groups were mixed for the first time and an infectious disease nightmare ensured. Viruses such as measles, which originated from human contact in

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the trappers’ villages, were epidemic in the gang cages of rhesus monkeys. As new monkeys were introduced to the cages, the older occupants would pass on this virus (Meyer et al., 1962). Thus, the rhesus monkeys from one location were sure to acquire the viruses of other monkeys in the gang cages or in the holding facilities used for vaccine and cell culture production. This was reminiscent of the introduction of smallpox virus in the New World o r the change of poliovirus from an endemic to an epidemic agent. Mann suggested that Hilleman go to Equatorial West Africa to obtain the African green monkey (Cercopithecus aethiops) and ship these primates through Madrid airport which had no other nonhuman primates in transit. From there the monkeys came to New York and then West Point, Pennsylvania, at Merck, minimizing all outside contacts. T h e African green monkey kidney cells proved to be quite free of endogenous viruses which had been classified in the past (i.e., Simian viruses 1, 2, 3, etc.). The most common source of monkey cells for preparing poliovirus vaccine at that time remained the rhesus and cynomolgus monkeys. They were plentiful, available, and inexpensive. Merck, like the other vaccine producing companies, made their vaccine for poliovirus, which was inactivated with formalin, in rhesus and cynomolgus monkey kidney cells. By 1960, Merck had joined the other companies in polio vaccine production and received its license to sell the purified poliovirus vaccine. Although the rhesus and cynomolgus monkey cells were tested and shown to be free of detectable contaminating viruses, B. H. Sweet and Hilleman made the curious observation that extracts of these rhesus cells, when incubated with the cells from African green monkeys, produced a characteristic cytopathic effect. The green monkey cells became highly vacuolated and died. It soon became clear that the rhesus and cynomolgus monkeys harbored a virus which did not cause any noticeable pathology in their own kidney cells, but this virus replicated in, and killed, the African green monkey kidney cells (Sweet and Hilleman, 1960). As they put it in their paper, “The discovery of this new virus, the vacuolating agent, represents the detection for the first time of a hitherto ‘nondetectable’ simian virus of monkey renal cultures and raises the important question of the existence of other such viruses.” This virus was named Simian virus 40. In their study, Hilleman demonstrated the presence of SV40 in his adenovirus vaccine seed stock used previously, three different serotypes of the Sabin polio vaccine strain were also contaminated with SV40, and normal rhesus and cynomolgus monkey cells all had SV40 in titers between 10-3.7 and 10-6.0 infectious doses. They tested for antibodies directed against SV40 in the military recruits who received the adenovirus vaccine or in children who received the Salk

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poliovirus vaccine. Nine of nine military recruits produced antibodies directed against SV40 only after they were vaccinated with the adenovirus vaccine. Five of seven children who received the Salk vaccine had antibodies directed against SV40, only after the vaccination took place (Sweet and Hilleman, 1960). Clearly, the poliovirus vaccine grown in rhesus or cynomolgus monkey cells was contaminated with a virus undetectable in these cells. While formalin treatment of SV40 inactivated the virus, reducing the titer by 104, SV40 present at doses higher than 104 infectious units appeared to resist killing by this treatment used in the Salk vaccine. SV40 arrived on the scene as a major contaminant of a successful vaccine widely used all over the world. In the wild only a small percentage of rhesus monkeys have antibodies directed against SV40 (about 10%)(Meyer et al., 1962). However, 69% of the rhesus monkeys arriving in the United States after stops in the gang cages had antibodies directed against SV40 (Meyer et al., 1962). Thus, the level of contamination of the poliovirus vaccine by SV40 was severe. Maurice Hilleman and his colleagues at Merck and Bernice Eddy and her colleagues at the National Institutes of Health began to study the properties of this new virus, SV40. Eddy was able to show (Eddy et al., 1961) that an extract of rhesus monkey kidney cells injected into newborn hamsters would cause tumors in these animals. By the next year, Hilleman’s group (Girardi et al., 1962) and Eddy’s team (Eddy et al., 1962) both showed that the agent responsible for causing these tumors was indeed SV40. At Merck, the group had inoculated SV40 into 151 newborn hamsters. Tumors arose at the site of injection with latent periods between 130 and 327 days in 42 hamsters. T h e first tumors appeared just three weeks after Merck and Hilleman’s group released the polio vaccine for use and 6 to 7 years after this vaccine had been developed, tested, and released by many different companies. Merck discontinued distribution of its new poliovirus vaccine and stopped its use immediately. Maurice Hilleman, Director of Virus and Cell Biology Research, Dr. Max Tishler, President of Research at Merck and John Horan, then the Vice President for Public Relations (soon to become the Chief Executive Officer of the company), took the train to Washington, DC to present this new information to Dr. Rodrick Murry, Chief of the Bureau of Biologics. The quandry faced by these individuals was considerable. The past 6- to 7-year history of the polio vaccine indicated that it was clearly efficacious, the epidemics of summer poliomyelitis were ended. At least in the short term, the vaccine was safe. T h e Salk vaccine used formalin which at the minimum reduced the infectious titers of SV40 in humans. The Sabin vaccine, with its live

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complement of SV40, was being extensively studied and tested in the Soviet Union and other countries between 1957 and 1961 (it was to be licensed by the Public Health Service in 1962) (Sabin, 1965). To that date, little or no safety or efficacy problems were detected. How could anyone remove these vaccines from the market and create a population of newborn children who would be susceptible once again to poliovirus and poliomyelitis? The resolution to these problems was swift and clear. Only African green monkey kidney cell cultures were to be used in the future for poliovirus production. They had been proven to be free of SV40. The poliovirus stocks employed for the Salk and Sabin vaccine strains were freed of SV40 (by limiting dilutions in the presence of SV40 antisera), and replicated in green monkey cells. Laboratory bred monkeys were developed and used as a source of monkey kidney cells and the monkeys and cells were tested for SV40. Merck never resumed production of the poliovirus vaccine, nor did they sell any of their stock of this vaccine. Many individuals who received the Salk and Sabin vaccines from 1953-1962 were exposed to SV40. A periodic follow-up of this population has shown no abnormal incidence of cancer. A few research scientists who had worked with large quantities of SV40 in the laboratory have died of cancer at a comparatively young age. Cancerous tissues from these individuals failed to show any SV40 DNA integrated into their tumor tissue (Howley et al., 1991a). Extensive studies with SV40 have shown that the viral DNA must integrate into a host chromosome and it must express a viral gene product, the SV40 large tumor antigen, to cause a tumor (Molecular Biology of the Tumor Viruses, 1973). When the SV40 DNA was integrated into the germ line of a transgenic mouse, the viral DNA was selectively expressed in the choroid plexus tissue of these mice. Within 3 months after birth, these mice all developed papillomas of the choroid plexus and died of an inherited tumor caused by SV40 DNA (Brinster et al., 1984). It is curious, therefore, that there is one report in the literature that SV40 DNA was detected in some human papillomas of the choroid plexus (Bergsagel et al., 1992). Whether this is a contaminant or a causal agent or even if this observation can be repeated needs to be tested with additional experiments. As the newborns of the 1953-1962 decade mature and grow old, it could well be difficult to find a statistical increase in the incidence of cancer presently observed in the population (one in three to one in four individuals will have cancer in their lifetimes). T h e presence or absence of SV40 DNA in a tumor may be the best marker for these studies. It should be pointed out that the evolution of humans and monkeys from a common ancestor has resulted in the continuing evolution of

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their endemic viruses along with the development of new species. Humans commonly harbor a virus closely related to SV40 called the BK virus. SV40 and BK viruses share about 80%homology in their protein sequences for the large tumor antigen and both viruses are capable of initiating tumors when they are injected into newborn hamsters (Shah, 1990). Humans, like rhesus monkeys with the SV40 virus, commonly acquire BK virus at a young age and it persists in kidney epithelium over the lifetime of the host. That humans harbor persistent viruses such as adenoviruses or the BK virus, which are able to initiate tumors in some animals, might have appeared to be a real clue to the origin of human cancers. However, the adenoviruses, BK virus, or SV40 virus have never shown a consistent, convincing close association with any human malignancy. T h e problems of using primary monkey kidney cells for the production of a vaccine widely introduced into the human population will not completely go away. In March of 1992, a reporter writing for the Rolling Stone Magazine suggested that the origins of the human immunodeficiency virus might arise from the oral poliovirus vaccine prepared from the kidneys of African green monkeys infected with simian immunodeficiency viruses. The vaccine specifically singled out in the article was one prepared by Hilary Koprowski at the Wistar Institute. It was given to a large population of adults and children in the Belgian Congo in the late 1950s. An independent panel of scientists commissioned by the Wistar Institute studied this problem. The committee was cochaired by Dr. Claudio Basilico of New York University School of Medicine and Dr. Frank Lilly of Albert Einstein College of Medicine, and it included Dr. Clayton Buck of the Wistar Institute, Dr. Ronald Desrosiers of Harvard Medical School, Dr. David Ho of the Aaron Diamond AIDS Research Center in New York, and Dr. Eckard Wimmer of the State University of New York at Stony Brook, School of Medicine. T h e committee concluded “It can be stated with almost complete certainty that the large polio vaccine trial begun in late 1957 in the Congo was not the origin of AIDS.” Nonetheless, the committee did recommend that available samples of the Wistar vaccine be tested for the simian immunodeficiency virus or inactive virus particles. Only one sample of the vaccine that may have been used in the Congo trials has been located to date. T h e panel did note that the earliest documented case of AIDS was found in a British seaman who appeared to have died from the consequences of his HIV infectian in 1959. He returned to Manchester, England, presumably with the virus, in early 1957 well before the start of the polio vaccine trials in the Congo. While it is therefore very unlikely that the origins of the human

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immunodeficiency virus derives from these events, the story points out the possibilities that one must consider in any biologics program of the magnitude of the polio vaccine. IV. The Molecular Biology of the DNA Tumor Viruses T h e human adenoviruses were discovered in 1953-1954 (Rowe et al., 1953; Hilleman and Werner, 1954), and SV40 was first identified in 1960 (Sweet and Hilleman, 1960). Over the next few years it became clear that SV40 (Eddy et al., 1962; Girardi et al., 1962) and some of the human adenoviruses (Trentin et al., 1962) could initiate tumors in hamsters when they were inoculated with these viruses as newborns. Several facts made these two virus groups very attractive for studying the question: What are the origins of cancer and how can a simple virus cause cancer? First, these viruses replicated efficiently in cell culture (SV40 in African green monkey kidney cells, adenoviruses in HeLa cells) and large amounts of these viruses could be obtained. This permitted a simple one-step growth curve to be followed in some detail (Ritzi and Levine, 1970) and, in the days before gene cloning, these viruses provided chromosomes with 8 genes (SV40) or 50 to 60 genes (adenoviruses) present in one intact DNA molecule. Second, the size of the SV40 genome (about 5200 nucleotides) and the adenovirus genomes (about 36,000 nucleotides) made it clear that only a few viral genes and proteins encoded by those genes were going to be involved in initiating cancer. These viruses came to be thought of as the simplest of model systems for understanding the origins of cancer in all animals. For these very same reasons-the simplicity of these viruses-it was clear that SV40 and the adenoviruses must rely on a large number of host cell functions and signals for the duplication of these viruses and even in their pathways to tumorigenesis. As such, these viruses were expected to lead virologists to important insights derived from the identification of cellular genes and molecules used by the virus and the cell to control gene expression and cell division. This reasoning came directly from the striking successes in elucidating the fundamental concepts of biology from the study of bacteriophages and their hosts. In the 1960s, many scientists were trained by working with the bacteriophages and they then entered the field of animal virology and applied the lessons learned with bacteriophages to the animal viruses. Third, the time was also right for large numbers of young investigators to look for an important, yet simple, experimental system to study the problems of cancer using animal viruses and mammalian cells in culture. This trend continued throughout the 1960s. By 1967- 1968, the DNA tumor virus group at Cold Spring Harbor Labora-

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tory had formed and by 1969, the first tumor virus meetings were held at Cold Spring Harbor (for the first few years, both DNA and RNA tumor viruses were to meet together, but as these fields grew in numbers and specialization, the meetings were divided). Curiously, the DNA tumor virus meetings were at first only focused on SV40, polyoma, and the adenoviruses. T h e Herpesviruses (Epstein-Barr virus), hepatitis B viruses, and papilloma viruses were not represented until later. The focus of the field was clear. Fourth, during the 1960s and 1970s a tremendous expansion of the medical sciences and molecular biology occurred in the United States. The funding opportunities (The War on Cancer and the expansion at the National Cancer Institute), the quality of the students attracted to the field, and the number of faculty positions opened up by major expansions of universities and research institutes all contributed to the development of a new era. What has come from this endeavor? What has the study of these viruses contributed to our fundamental knowledge of life processes? Has the study of these viruses given us new insights into the origins of human cancers? Did the field of virology move in the best direction by choosing SV40 and the human adenoviruses as model systems in the 1960- 1980 period? The raw material to provide some of the answers to these questions may be best and simply presented in several tables listing the experimental and conceptual advances made by using these viral agents. These tables have been divided into four areas of study: (1) the structure of viral DNA chromosomes (Table 1); (2) the transcriptional regulation of the DNA tumor viruses (Table 2); (3) post-transcriptional modification of mRNAs (Table 3); and (4) transformation and tumorigenesis (Table 4).As the lists of facts in these tables are integrated and analyzed, a clear picture with the answers to these questions surely emerges. T h e discovery of closed circular and superhelical DNA was made with polyoma virus DNA (Dulbecco and Vogt, 1963; Weil and Vinograd, 1963) and this was an extraordinary model for larger chromosomes from E. coli (Worcel and Burgi, 1972) to mammalian chromosomes (Laemmli et al., 1978). This structure of DNA resulted from the wrapping of the double helix about the cellular histones formed into nucleosomes (Fey and Hirt, 1975; Griffith, 1975), a packaging strategy used by these viruses and their host cells alike (see the Cold Spring Harbor Symposium on Quantitative Biology, 1978, Vol. 42). The chromatin structure of SV40 DNA was studied along side mammalian cell chromatin to help elucidate a fundamental set of observations concerning the packaging of DNA. Similarly, the replication of SV40 and adenovirus DNAs led to the identification of unique origins of DNA replication as well as the

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THE%IRUC.IURE

TABLE 1 VIRAL. DNA CHROMOSOMES

OF

1. SV40 DNA is a closed circular and superhelical

double stranded DNA (done with polyoma virus first) T h e first concept of closed circular DNA and superhelical turns 2. SV40 DNA is packaged in cellular histonesnucleosomes-removal of the nucleosomes from the DNA creates the superhelical turns 3. HzitdI1-Ill restriction enzyme map-the restriction map of-a DNA 4. EcoRl cuts SV40 DNA once

first

5. First recombinant DNA clones made by inserting SV40 DNA into lambda phage DNA 6. First mammalian cell expression vector-the insertion of lambda phage DNA into the SV40 late region 7. The first use o f site-specific niutagenesis as a tool

8 . T h e first identification of a eukaryotic origin of replication-SV40 9. The use of marker rescue to align the genetic and physical maps of SV40 10. The complete nucleotide sequences of SV40 DNA-the largest coniplete viral coromosome sequenced at the time 1 I . Adenovirus DNAs have inverted terminal repeats with a pIotein covalently bound to its 5’-dC:MP end 12. Adenovirus genomes show evolutionary similarities and diversity of chromosome structures and gene maps

Dulbecco and Vogt, 1963 wed and Vinograd, 196s Vinograd and Lebowitz, I966 Frearson and Crawford, I972 Fey and Hirt, 197.5 Griffith, 1975 Gerniond el d . , 1975 Danna et nl., 1973 Morrow and Berg, 1972 Mulder and Delius, 1972 Jackson rt al., 1972 Goff and Berg, 1976

Mertz and Berg, 1974 Shenk et al., 1976 Lai and Nathans, 1974a Danna and Nathans. 1972 Lai and Nathans, I974b Fiers e/ d ,1978 Keddy el al., 1978 Rekosh P / al., 1977 Garon et 01.. 1972 Green, 1970

demonstration of bidirectional DNA replication (Fareed et al., 1972), and the critical proteins involved in initiation and replication of DNA. These studies led to excellent in vitro DNA replication systems that still are among the best examples of how higher eukaryotic DNA replication proceeds. The recombinant DNA revolution of the 1970s had some of its earliest successes using SV40 DNA. SV40 DNA was first used as a simple defined substrate for restriction enzymes (Mulder and Delius, 1972;

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TABLE 2 THETRANSCRIPTIONAL REGULATIONOF T H E DNA TUMOR VIRUSES 1. SV40 and adenovirus transciption maps-the

2.

3.

4.

5.

6.

7.

8.

9.

first use of restriction maps to define transcription units T h e discovery of an enhancer element in SV40 DNA which regulates transcription. This provided the first concept for a distance and orientation independent DNA element regulating transcription. T h e recognition that the SV40 viral T antigen acts as a negative regulator or repressor of transcription by binding to the SV40 promoter region T h e identification of an SV40 promoter region which binds a specitic transcription factor (SP-I); this was the first eukaryotic transcription Factor recognized as being essential for transcription T h e recognition that an adenovirus E I A protein can positively regulate the level of viral and cellular transcripts by increasing the rate of transcription T h e SV40 enhancer element binds transcription factors (AP-l,2) with nucleotide specificity; this positively regulates transcription; this is the description of the first enhancer binding proteins T h e adenovirus niajor late promoter and the SV40 early enhancer-promoter become the paradigm for the development of an in uztio transcription system T h e promoter for polymerase 111 transcripts has two conserved recognition motifs internal to the gene (VA-gene) Adenovirus enhancer binding transcription factors can positively or negatively regulate gene expression (YY-I)

Khoury el nl., 1973 Sambrook et al., 1973

Benoist and Chambon, 198 1 Gruss el al., 1981

Reed el nl., 1976

Dynan and Tjian, 1983

Jones and Shenk, 1979 Berk el al., 1979 Nevins, 1981 Lee et nl., 1987

Weil el

a/..

1979

Fowlkes and Shenk, 1980

Shi et nl., 3991

Morrow and Berg, 1972). T h e first restriction enzyme map was constructed with SV40 DNA (Danna and Nathans, 1971). Some of the earliest DNA cloning experiments used SV40 DNA into lambda DNA or human beta-globin genes into SV40 DNA to construct the first mammalian cell expression vector (Jackson et al., 1972). Indeed it was the discussion of these very experiments which led to a temporary moratorium of all recombinant experiments to determine if such recombinant molecules might be dangerous. The development of SV40 as a

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ARNOLD J. LEVINE

1. The discovery of splicing of inKNA with the adenoviruses; this was the first description of RNA splicing i n any system 2. The development of S-I mapping provides the

structure of niRNAs 3. The adenovirus late transcript is made as one continuous KNA molecule and KNA processing regulates the type of mRNA and protein made and its levels 4 . The nucleotide signal for cleavage and polyadenylation of a RNA molecule is first identified in SV40 and proves to he general for all host and viral mRNAs 5. T h e adenovirus ElB-55Kd protein can regulate the transport of host and viral mRNA from the nucleus to the cytoplasm

Berget el al., 1977 Chow rt al., 1977 Berk and Sharp, 1978 (SV40) Berk and Sharp, 1977 (Adeno) Fraser et nl., I979

Fitzgerald and Shenk, I981

Pilder et nl., 1986

model experimental system in the 1970s broadened the base for recombinant DNA technology from the bacteriophages and prokaryotic plasmids to eukaryotic viruses and vectors. SV40 DNA continued to be a model to develop the new tools of site specific mutagenesis (Mertz and Berg, 1974; Shenk et al., 1976; Lai and Nathans, 1974a), marker rescue in eukaryotic cells and eventually the complete sequencing of a simple mammalian virus (Fiers et al., 1978; Reddy et al., 1978). This new found information and technology provided the first transcription maps and led to the fundamental discoveries of segments of DNA that were employed as regulatory signals for transcription in eukaryotic cells. The identification of enhancer elements that acted at a distance, in an orientation independent manner and function both 5' or 3' to a gene, was a totally new concept described first with SV40 and not anticipated from the studies in prokaryotes (Gruss et al., 198 1). Promoter elements that were positively regulated by transcription factors (SP-1) or negatively regulated by viral gene (SV40 T antigen) products rapidly filled in the picture. This led to the discovery of positive (AP-1, 2) (Lee et al., 1987) and negative (YY-I) (Shi et al., 1991) transcription factors that act at the enhancers of genes. When AP-1 was shown to be composed of two oncogene products, jun and fos, the functions of oncogenes became clear from the fundamental studies of transcription using SV40 DNA (Bohmann et al., 1987). Similarly, the functions of the adenovirus oncogene product E I A in regulating transcription of viral and cellular genes provided new insights into the

TABLE 4 TKANSFOKMATION A N D TUMOKICENESIS 1. SV40 and portions of the adenovirus DNA can

integrate into the chromosomes of the host; cancers arise with single, clonal viral DNA integration sites 2. Only a selected portion of the viral chromosome expresses in cancer cells; this produces a set of viral proteins in each cancer cell; a host can respond by producing antibody directed against these viral tumor antigens 3. Some of these viral tumor antigens are also tumor specific transplantation rejection antigens eliciting a T-cell rejection response 4. T h e viral encoded tumor antigens are required to establish and maintain tumors in animals or transformation of cells in culture 5 . T h e SV40 large tumor antigen binds to the cellular p53 protein; the p53 tumor suppressor protein was discovered in this fashion 6. T h e adenovirus oncogene EIB-55Kd protein binds to the p53 protein 7. T h e SV40 large T antigen and the adenovirus EIA protein bind t o the cellular Rb tumor suppressor protein 8. A detailed genetic analysis of the transforming genes and domains of SV40 T antigen and the adenovirus oncogenes find three regions critical to transformation; binding sites for Rb, p53 and a third protein, p300 9. T h e adenovirus genome encodes a set of gene functions designed to modulate down the immune response of the host a. VA-KNAs block the ability of interferon to act upon translation b. T h e E3-19Kd glycoprotein binds to the class I major histocompatibility antigen and immobilizes it in the endoplasmic reticulum This reduces the level of class I antigen presenting molecules on the surface of infected cells c. The adenovirus EIA gene product reduces the level of class I MHC molecules made by the cell d . The adenovirus E 3 proteins act to block the ability of tumor necrosis factor (TNF) to act on infected cells 10. AP-I, a transcription factor found to bind to SV40 DNA is composed of fos and jun oncogene products

Sambrook et al., 1968 Sambrook et nl., 1980

Sambrook et al., 1974 Gallimore et al., 1974 Huebner et al., 1963

Tevethia, 1990 Tevethia and Rapp, 1965 Tegtmeyer, 1975 Brugge and Beutel, 1975 Osborn and Weber, 1975 Martin and Chou, 1975 Linzer and Levine, 1979 Lane and Crawford, 1979 Sarnow et nl., 1982 DeCaprio et a/., 1988 Whyte et nl., 1988 Srinivasan et al., 1989 Yaciuk tt al., 1991 Zhu et al., 1992

Kitajewski et d.,I986 Persson et al., 1979

Burgert el al., 1987

Friedman and Ricciardi, 198X

Gooding et nl., 1988

Bohmann et al.. 1987

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ARNOLD J. LEVINE

mechanisms of transcriptional regulation and transformation (Jones and Shenk, 1979; Berk, et al., 1979). The field of transcriptional control and regulation in eukaryotic cells has now elucidated a large number of basal transcription factors acting at the promoter or modulating transcription factors acting as the enhancer elements of a gene. Progress in this area has been made by purifying each protein component of the transcriptional machinery and cloning the gene that encodes this protein. This exercise has been made possible by using the adenovirus major late promoter and the SV40 early enhancer-promoter elements as the model system to study transcription in vitro. In this field, SV40 and adenoviruses became paradigms for our present day understanding of gene regulation by combinatorial use of transcription factors. Just as in DNA structure, packaging, replication and in the area of gene cloning, the small DNA tumor viruses contributed more than their share to understanding fundamental concepts in transcription. Perhaps one of the biggest surprises of the recombinant DNA revolution was the elucidation that many eukaryotic genes were composed of exons and introns and, unlike the clear lessons with prokaryotes, the eukaryotic gene structure was not colinear with the protein product. The studies with SV40 and the adenoviruses provided early clues to this unusual arrangement of genes. That the SV40 large and small T and t antigens shared N-terminal sequences but different C-terminal sequences needed to be explained. That a deletion in the middle of the SV40 large T-antigen gene did not change the size of the protein (a deleted intron) needed to be explained (Shenk et al., 1976). But nothing made the growing evidence for split gene segments so clear as the discovery of RNA splicing and the rearrangements of transcripts into mRNA first shown with the adenoviruses (Berget et al., 1977; Chow, et al., 1977). This remarkable observation added a new concept and a new level of cellular control. The study of mRNA structure using heteroduplex mapping or S-1 mapping made it clear that the adenovirus late transcripts use RNA splicing to regulate efficiency of translation, control the relative levels of mRNA, and possibly regulate the transport of RNA to the cytoplasm (Berk and Sharp, 1977). T h e signals for intron-exon boundaries, splicing and polyadenylation of primary transcripts, were first worked out with these viruses (Fitzgerald and Shenk, 1981). T h e mechanisms for splicing RNA molecules were elucidated, in part, using these viral systems. This whole field of post-transcriptional RNA regulation has its origins in virology (methylation and capping of the 5' ends of mRNA came from the Reovirus systems [Furuichi et al., 19771). Clearly then, many new fundamental principles of gene regulation in

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mammalian life forms derive from a study of the mammalian viruses, and SV40 and adenoviruses in particular, have contributed greatly to our present information base. But it is also the case that the viruses in general, and SV40 and adenoviruses in particular, have also helped to lay the foundations for our modern understanding of the origins of human cancer. It is now appreciated that neoplasia in humans arises from a series of mutations in two sets of genes in the cell-the oncogenes and the tumor suppressor genes. The great majority of the oncogenes have been identified, cloned, and studied because of their insertion into retroviruses or the insertion of retroviruses into chromosome locations near an oncogene. The tumor suppressor genes were first postulated to exist from studies using somatic cell genetics (Harris eta!., 1969) as well as epidemiologic studies of children with retinoblastomas (Knudson, 1971). The retinoblastoma susceptibility gene was cloned from cells using a comparison of normal and mutant (deleted) alleles in these tumors (Friend et al., 1986). The other major tumor suppressor gene product, p53, was first detected by its ability to bind to the SV40 oncogene product, the large T-antigen (Linzer and Levine, 1993; Lane and Crawford, 1979). It was then shown to bind to an adenovirus oncogene protein, the E1B-55Kd protein (Sarnow et al., 1982) and the human papillomavirus oncogene product, the E6 protein (Werness et al., 1990). T h e DNA tumor viruses gave us p53 and the p53 gene has recently been shown to be the single most common target for genetic changes in human cancers. Seventy-five percent of colorectal cancers, 50% of lung cancers, and 40% of breast cancers suffer mutations in this gene. Families with inherited mutations at the p53 locus develop high incidences of cancer often at young ages (Levine, 1992). Patients with cancers containing p53 mutations often have a much poorer prognosis for treatment responses and five-year survival times, when compared to individuals with cancerous cells containing the wild-type p53 protein (Lowe et al., 1993). T h e mechanisms of action and functions of the Rb protein, which binds to the adenovirus E l A protein (Whyte et al., 1988), the SV40 large T-antigen (DeCaprio et al., 1988) and the human papillomavirus oncogene product, E7 (Dyson et al., 1989), have been largely elucidated by studying these viral proteins and their impact on Rb (Levine, 1993). The DNA tumor viruses continue to be important in the study of the tumor suppressor genes and their products.

V. Conclusions Thus, it appears clear that the small DNA tumor viruses studied so intensively between 1960 and 1993 have contributed out of proportion to their size, to a fundamental body of knowledge about eukaryotes.

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From the perspective of the early days where searches for new viruses and vaccine development led to the discoveries of the adenoviruses and SV40, no one could have predicted the central role these viruses would play in both basic biology and oncology. Such is the very nature and essence of science. T h e history of the discoveries of and studies with SV40 and the human adenoviruses teach us once again the powerful role of basic fundamental scientific research in showing the way to the applications of medical needs. This history also shows us how basic and applied research goals can go hand and hand to produce good science. ACKNOWLEDGMENTS T h e author thanks M. Hillenian and H. Meyer for stimulating conversations, and Kate James for help in preparing this manuscript.

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RETROVIRUSES AND WILD MICE: AN HISTORICAL AND PERSONAL PERSPECTIVE Murray B. Gardner Department of Pathology, School of Medicine, University of California, Davis, Davis, California 95616

I. Introduction 11. Trapping of Wild Mice and Establishment of Aging

Populations in the Laboratory 111. Spontaneous Cancer, Lymphoma, Motor Neuron Disease, and Type C

IV. V. VI. VII. VIII.

IX. X.

Retrovirus (MuLV) Infection in Aging Wild Mice A. Cancer-Resistant Low MuLV Populations B. Lymphoma-Paralysis Prone High MuLV Populations Pathogenesis of Lymphomas Pathogenesis of MuLV Neurologic Disease Nongenetic Control of Type C Virus and Associated Diseases in Wild Mice Genetic Control'of Type C Virus in Wild Mice by Introduction of the Fv-lb MuLV-Resistance Allele from Inbred Mice Genetic Control of Type C Virus in Wild Mice by Natural Segregation of the Fv-4 Ecotropic MuLV-ResistanceAllele Type B Mammary Tumor Viruses in Wild Mice Lessons Learned References

I. Introduction

T h e chance of a lifetime came my way 25 years ago when I was given the opportunity to help with the search for RNA tumor viruses in humans and animals, as part of the mission of the Virus Cancer Program (VCP), sponsored by the National Cancer Institute under the banner of President Nixon's Cancer Crusade (Rettig, 1977). By accepting this challenge, my career changed dramatically overnight and I was able to participate in an exciting scientific endeavor. At that time (1967), I was in the Department of Pathology at the USC School of Medicine and was studying whether Los Angeles ambient air pollution (i.e., smog) was carcinogenic to mice. For five years I counted pulmonary adenomas in different strains of laboratory mice housed in between the inbound and outbound lanes of several major Los Angeles freeways, some exposed to the ambient air and some breathing filtered air. The results indicated that smog was nontumorigenic for mouse lungs (Gardner, 1966; Gardner

169 ADVANCES IN CANCER RESEARCH. VOI.. 65

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et al., 1970). I did enjoy the teamwork that went into this effort and most importantly, it brought me to the attention of Robert Huebner, one of

the leaders of the VCP, who suggested that I might like to explore the role of smog and other environmental and host factors in the activation of latent RNA tumor viruses, not just in mice but also in other animals and humans. As he explained to me, RNA tumor viruses (now called retroviruses) caused cancer in several species of animals under natural conditions (in contrast to many DNA tumor viruses such as polyoma virus, adenovirus, and SV40 which caused cancer only under experimental conditions), and it was important to find out if these RNA tumor viruses also existed in humans. Moreover, Ludwig Gross had shown in the early 1950s that viruses of this type could be passaged vertically, presumably as infectious agents, from one generation to the next, in lymphoma prone AKR laboratory mice (Gross, 1951a, 195 lb). Aniazingly, it became evident by the late 1960s that in certain lymphoma or breast cancer prone inbred mouse strains, these RNA tumor viruses were transmitted equally by egg and sperm, presumably as genetic elements or “virogenes” (Rowe, 1973; Bentvelzen and Daams, 1969). I n uiuo, these inherited (endogenous) virogenes became activated early in life to produce infectious virus particles which spread cell-to-cell in the animal host, and eventually led to cancer. These malignancies consisted of either lymphoma [murine leukemia (MuLV) or Type C virus] or breast cancer [murine mammary tumor virus (MMTV) or Type B virus]. In vitro, each individual AKR mouse embryo cell that was initially free of infectious Type C virus could be induced to release such virus by aging or treatment with a mutagen. Even inbred mice with low lymphoma incidence had similar endogenous virogenes that could be activated to produce complete virus particles and cause lymphoma. Added to this was the ability of Type C leukemia viruses of chickens and mice to rescue defective “oncogenes” in the form of sarcoma (or rapidly oncogenic) virus genomes in cell culture and in viuo. Based on these lines of evidence, Huebner and Todaro (1969) put forth the Viral Oncogene Hypothesis which suggested that inherited Type C and Type B RNA tumor virogenes and associated oncogenes in all mammalian species, including humans, might provide the “seeds” for cancer. The hypothesis suggested that latent virogenes and oncogenes could be activated independently during aging or after exposure to chemical or physical carcinogens. Howard Teniin suggested the related protovirus hypothesis, which featured a central role for the enzyme reverse transcriptase and posited that information exchange from DNA to RNA and back to DNA was a

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normal cellular process involved in cell differentiation (Temin, 1974). Mutation aberrations in the process might create retrovirus particles or the genetic information for cancer. The protovirus hypothesis differed from the viral-oncogene hypothesis in that it implied that the information for virus and cancer was synthesized de novo rather than being inherited as stable genetic elements. Common to both hypotheses was that the potential for cancer and retrovirus production were inherent within the cell genome and independently controlled; therefore, cancer could occur in the absence of complete virus particles, as appeared to be the case in humans. The viral oncogene and protovirus hypotheses provided much of the scientific impetus prompting this intensive search for RNA tumor viruses. My task was to organize a research team at USC, go after these RNA tumor viruses in nature, find out if they existed in humans, companion animals or wild mice, and to understand the natural history of these viruses. Ultimately, if RNA tumor viruses could be identified and isolated from humans, either as exogenous and/or endogenous agents, an RNA tumor virus vaccine might become feasible as a vaccine against cancer. The search for Type C and Type B RNA tumor viruses (these were not called retroviruses until after the discovery in 1970 of the enzyme reverse transcriptase encoded by the viral genome) in wild mice was especially relevant because, as mentioned above, much of the VCP research effort was predicated on the idea that latent inherited virogene activation was responsible for initiating leukemogenesis (e.g., the AKR mouse model) (Rowe, 1973), or breast carcinogenesis (e.g., the GR mouse model) (Bentvelzen and Daams, 1969). Noteworthy, of course, was that these observations of murine malignancy were made in laboratory mice that earlier in this century had been highly inbred for genetic studies on coat color, radiation resistance, and tumor transplantation but had coincidentally developed a high incidence of lymphoma or breast cancer (Strong, 1978). One obvious line of investigation thus became to determine the nature and function of RNA tumor viruses in wild mice (Mus musculus), the progenitor of laboratory mice. For these studies, wild mice were considered a better surrogate for humans than laboratory mice. Both humans and wild mice are outbred, unexposed to laboratory adapted viruses, but exposed to similar environmental factors. Did such viruses exist in outbred, feral, or wild mice, and did these viruses contribute to cancer in these animals under truly natural conditions? If so, what was the natural history of viral cancer and what could be done to control the viruses and associated malignancy? These questions provided me with a wonderful 12-year adventure in

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biology. From my previous training in medicine and pathology, I had no knowledge about wild mice, much less other animals (e.g., wild rats, domestic cats, zoo primates), and I knew nothing about RNA tumor viruses. Thanks to the largess of the VCP and Dr. Huebner, I was allowed to build an interdisciplinary research team at USC to study viral oncogenesis in wild mice. I learned from colleagues including Earle Officer, Suraiya Rasheed, Vaclov Klement, Martin Bryant, Robert Rongey, Pradip Roy-Burman, Bijay Pal, Howard Charman, Paul Arnstein, Edan Yan, Brian Henderson, Malcolm Pike and John Casagrande. Robert McAllister at Children’s Hospital in Los Angeles gave me much help and added credibility in virology. I was also greatly helped and encouraged by my Chairman, Hugh Edmondson, M.D., who together with his wife, Dorothy, purchased a building for this research group. We learned a lot from our hunt for retroviruses in humans, rats, cats and other animals, and discovered a truly fascinating new biology of Type C and Type B RNA tumor viruses in feral mice. The inconvenience of leaving the conventional laboratory mouse model was amply rewarded by the adventure of trapping feral mice and by the discovery of new scientific knowledge. Novel Type C viruses, new diseases, new genetic resistance factors, new unexpected natural history features all became apparent, and these findings differed in many respects from those previously made in inbred mice models (Table 1) (for summary, see Gardner, 1978; Gardner and Rasheed, 1982). Our results indicated that although all wild mice contained ample amounts of endogenous Type C and Type B virogenes, these inherited sequences seemed to contribute little, if anything, to disease; the onus was entirely placed on exogenous Type C and Type B viruses. Surprisingly, a few wild mice completely free of endogenous (or exogenous) MMTV virogenes were identified (Cohen and Varmus, 1979). A colony of these mice bred in the laboratory has been very useful for segregating and determining the function of endogenous MMTV alleles from inbred mice (Cohen et d., 1982; Morris, et al., 1986). Our results demonstrated the well-worn adage that natural history can best be studied outside the laboratory environment and away from animals that have been extensively manipulated by man. I will now summarize the major findings of our research on Type C and Type B retroviruses of wild mice performed during 1978-1980 and will compare these results with those found in laboratory mice and humans. This will provide a unique retrospective view of the accuracy of this animal model for predicting the natural history of the first Type C human retrovirus, the human T-cell leukemia virus (HTLV), which was not detected until a decade later (1980) (Poiesz et al., 1980; Yoshida et al., 1981).

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TABLE 1 TYPE C RWROVIRUS~S I N FERAL MICEI N SOUTHERNCALIFORNIA: MAJOR FINDINCS 1. Populations studied are normal outbred Mu7 mzwculut dome.tliclw, the progenitors of

inbred mice. 2. Type C viruses, regulating genes, associated diseases and natural history features are distinct hetween the prototype high lymphoma strains of laboratory (AKR) and wild (LC) mice. 3 . Spontaneous tumors occur very infrequently in most populations of wild mice and only in old age. 4. Exogenous Type C viruses are not detectable in most wild mice but can he isolated from some lymphomas occurring in old age. 5 . In certain feral mouse populations (e.g., Lake Casitas), MuLV is highly expressed, transmitted by milk, and associated etiologically with an increased incidence of’ lymphomas and neuromotor hind limb paralysis. 6. MuLV-associated natural lymphoma and paralysis in wild mice are amenable to prevention by specific immunologic and genetic measures. 7. A strong dominant ecotropic MuLV restriction gene, Akvr-ZR, is polymorphic in LC wild mice. 8. Endogenous (inherited) MuLV related DNA is present in many copies (30-50) hut expression is powerfully repressed in most wild mice. 9. Endogenous virogenes play no known role in lymphogenesis or the neuroniotor paralysis of feral mice. 10. Chemical tumorigenesis is independent of infectious MuLV expression in wild mice.

II. Trapping of Wild Mice and Establishment of Aging Populations in the Laboratory T h e “story behind the story” of how we obtained and studied wild mice has its own intrigue. Locating populations of feral mice in Los Angeles County and environs was not easy at first. The Rodent Control Division of the Los Angeles County Health Department was unable to help, and assured us that it would be very difficult to find wild mice in any number within their jurisdiction. First, we had to make sure that we could distinguish one kind of rodent from another; we were looking for M w musculus, a mouse of Old World origin and the progenitor of the laboratory mice used in cancer virus research. These mice live in a commensal relationship with humans relying mainly on grain for their sustenance. In this sense, they are man’s oldest “house pet.” In reading of their folklore (Keeler, 193I), I was amazed at how the lowly house mouse had served mankind from antiquity as a subject of worship, fear, entertainment, and knowledge. We can, of course, thank these creatures for much of what we know about mammalian genetics and RNA tumor viruses. By contrast, many other rodents such as deer mice (Peromyscus

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maniculatus) and field mice (Apodemis agrarius) of New World origin were not of interest to us because they apparently lack endogenous or exogenous retroviruses. These mice live further removed from human habitation and have not been inbred and genetically characterized to the extent of Mus musculus. Wild rats (Rattus mttus and Rattus nor-vegzcus) also are out there, particularly around sewage. These animals are also of Old World origin and they harbor endogenous Type C retroviruses, one of which we isolated and characterized (Rasheed, et al., 1978). However, wild rats are very difficult to work with in the laboratory and we found no evidence that their Type C virus was linked to cancer. After many false leads, we eventually located about 15 different widely separated populations of Mus musculus in the Los Angeles area; these were mostly squab and duck farms, egg farms, horse racetracks, and a bird seed factory, all providing an ample source of grain. T h e mice were studied in their natural habitat, occasionally bred (with difficulty) for 1 to 2 generations in the laboratory and subjected to various experimental procedures to induce latent retrovirus expression. From 1968 to 1978, about 10,000 wild trapped mice were housed singly in mason jars and allowed to live out their “natural” lifespan in separate “leisure world” communities in the laboratory at USC Medical School while under observation for spontaneous tumors or other diseases. The mice were autopsied when sick or moribund, tissues were examined microscopically and samples collected for Type C and Type B retrovirus assays. The details of how these mice were housed, fed, screened for indigenous pathogens, and examined for retrovirus activity are covered elsewhere (Gardner et al., 197la,b, 1973a, 1976a, 1980a; Rongey et al., 1973, 1975). In collaboration with Steve O’Brien at the NCI, we also confirmed that these wild mice were as outbred and polymorphous for gene-enzyme markers as other previously studied feral mouse populations (Rice et al., 1980). The trapping and surveillance of wild mice constituted a new technology in itself, mostly because of the ease with which they could escape when being handled and their proclivity to bite and fight. We were fortunate to have John Estes on our team because he had learned these skills while trapping wild mice in Harlem tenement houses for Huebner, Rowe, and Hartley’s polyoma virus studies in the 1950s (Rowe et al., 1961). As discussed in the next section, wild mice from one particular trapping area, a squab farm near Lake Casitas (LC) in southern Ventura County, were of special interest because of their high prevalence and level of MuLV infection, associated increased predilection to lytnphorna with leukemia and independent fatal lower motor neuron disease. Ironically, public health issues created some of the greatest obstacles to work-

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ing with this interesting mouse population. The LC squab farm, indeed all of the squab and duck farms at which we trapped mice, were in the business of raising food for human consumption. Squab are fledgling pigeons served as a culinary delicacy in Chinese restaurants. Because of their commercial nature, these farms had to pass public health inspection once a month and which included certification that they were “rodent-free operations.” On account of this stipulation, we had to carry out the trapping of these mice in a surreptitious manner. This was particularly important at the LC squab farm because we trapped several hundred mice there every few weeks. At first, the mice were captured singly in Sherman traps, but later we picked them up by hand from the squab nesting boxes and grain bins late at night after stunning them with the bright light of a miner’s helmet. We paid high school students $0.10 per live mouse. T h e mice were delivered in large garbage containers early in the morning to a predesignated pick-up spot, where they were transferred to a van with covered windows that delivered them to their “leisure world” homes. During a decade of this clandestine operation, we managed to keep this enterprise out of the newspapers, although we certainly published the unique retrovirus characteristics of the LC mice in scientific journals. Ill. Spontaneous Cancer, Lymphoma, Motor Neuron Disease, and Type C Retrovirus (MuLV) Infection in Aging Wild Mice

A. CANCEK-RESISTANT Low MuLV POPULATIONS Most of the aging populations of wild mice that we studied, about 12 in number, were resistant to spontaneous cancer; only a few tumors occurred late in life (Gardner et al., 1973a). The total tumor prevalence was 9.5%, mostly occurring after two years of age. The majority of tumors were lymphomas localized to spleen or lymph node. T h e only other tumor types observed were pulmonary adenomas, hepatomas, fibrosarcomas, and myeloid leukemia. Typical of these wild mouse populations in Los Angeles County were those located at a squab farm in Bouquet Canyon, a birdseed plant (Hartz Mountain) in downtown Los Angeles, an egg ranch at Munneke, and a squab farm in Soledad Canyon. The cumulative total mortality and cumulative specific tumor mortality as calculated by life table methods for these representative cancerresistant aging populations of wild mice in comparison with the lymphoma-paralysis prone Casitas population of wild mice are shown in Figure 1 (Gardner et al., 1976a). Other common pathology found in the

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aging mice included inflammation of the liver portal tracts (cholangitis) due to biliary obstruction from the dwarf tapeworm (Hymenolepis nana) and glomerulosclerosis of the kidneys. Neurologic disease did not occur in these particular mice. No amyloidosis was found. No microscopic abnormalities were found in about 25% of the moribund or dead mice. I n most (about 80%) of the sporatic lymphomas occurring in these aged wild mice, MuLV core (p30) antigen was detected by complement fixation (CF), and Type C particles were observed by electron microscopy (EM) (Gardner et al., 197 la). Virus was not demonstrable in the other tumor types or in spleens in normal aging mice. MuLV p30 antigen and Type C particles were detected in only a few sarcomas induced by 3-methylcholanthrene in older mice. The mice were also very resistant to x-irradiation induced tumors. Indigenous polyoma virus infection, noted particularly in the Bouquet Canyon mice, did not cause any increase in spontaneous tumors (Gardner et al., 1971b). Treatment with antilymphocytic sera caused an activation of latent cytomegalovirus and death but no appreciable activation of latent MuLV or polyoma virus (Gardner et al., 1974). These findings indicated that the feral Mw m w culus were generally very resistant to spontaneous tumor development and that detectable Type C MuLV was strongly repressed and of low frequency in most populations. However, we did confirm that MuLV particles and core antigens were detectable in some aged wild mice in their spontaneous lymphomas or chemically induced sarcomas. T h e MuLV that was isolated from the lymphomas o r sarcomas of these low MuLV cancer resistant mice was of two host range classes, amphotropic and ecotropic, both distinct from but related to MuLVs of laboratory mice. T h e same two classes of mice were isolated more readily from the high MuLV cancer prone mice described next. T h e amphotropic viruses were remarkable because of their wide in uitro host range for mouse and other species’ cells including human (Rasheed et al., 1976; Hartley and Rowe, 1976). Little did we dream that 20 years later these amphotropic viruses would be useful vectors for human gene therapy (Miller, 1992). The amphotropic viruses had a different interference and neutralization pattern and receptor from all of the laboratory mouse MuLV strains (Gazdar et al., 1977). The ecotropic viruses grew FIG. 1 . A. Cumulative total mortality from all causes; B. Cumulative total mortality excluding deaths from tumors and paralysis; C. Cumulative incidence rate for all tumors; D. Cumulative incidence rate for lymphoma; E. Cumulative incidence rate for carcinoma, excluding hepatoma; F. Cumulative incidence rate for hepatoma; G. Cumulative incidence rate for lung adenoma; H . Cumulative incidence rate for sarcoma; I. Cumulative rate for paralysis. [Reproduced with permission from Gardner et al. (1976a).]

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only on murine cells and were in the same interference and neutralization class and used the same receptor as the ecotropic MuLV of laboratory mice (Rasheed el al., 1977; Oie et al., 1978; Sarma et al., 1967). Apart from in vitro host range, these viruses could also be distinguished in culture because only the ecotropic virus induced syncytia on XC cells. By immunologic and biochemical analyses, both amphotropic and ecotropic classes of wild mouse MuLV were remarkably uniform although they did show some microheterogeneity (Bryant et al., 1978a, 1978b; Lai et al., 1982; Pal et al., 1973, 1975). Interestingly, both classes were more closely related to the Friend, Moloney, and Rauscher strains than to the AKR strain of lab mouse MuLV (Barbacid et al., 1979). Both classes of virus were fully competent, occurring independently of each other, although often intermixed. Both classes of virus were N-tropic (i.e., they replicated better on NIH Swiss than Balb/C embryo cells). Experimental transmission of biologic clones of virus to newborn, susceptible ( F z J - ~ ”laboratory ) mice showed that both classes, amphotropic and ecotropic, were only weakly lymphogenic, inducing <30% tumors after a latent period of 2 1 2 months (Gardner, 1978; Gardner and Rasheed, 1982). The viruses were not synergistic in vivo. T h e ecotropic virus from the low MuLV population of wild mice did not induce the neurologic disease characteristic of the high MuLV paralysis prone LC mice (see below). Xenotropic viruses, infectious only for cells of a foreign species, were only infrequently isolated from wild mouse cells and mink cell focus forming (MCF) ecotropic-xenotropic recombinant viruses, characteristic of AKR inbred mice, were not recovered from the wild mice o r their tumors (see below). At first, based on the AKK lab mouse model (Rowe, 1973; Huebner and Todaro, 1969), we suspected that the MuLVs of wild mice were transmitted endogenously (i.e., as inherited virogenes). Based on foster nursing studies (Gardner et al., 1979), and later confirmed by more precise molecular hybridization data (Barbacid et al., 1979; O’Neill et al., 1987), we soon learned that both amphotropic and ecotropic MuLV were transmitted in the low as well as the high MuLV populations as a purely exogenous, congenital milk-borne infection. Because of the neonatal infection, the viruses were recognized as “self” and the mice were specifically immune tolerant to them (Klement et al., 1976). In retrospect, after our first two years of this project, we could conclude that wild mice, in general, if spared death from predators, fighting, starvation, or infectious disease, were long lived (>2 years), were cancer resistant, and had only a low incidence and level of Type C MuLV expression. However, a few such wild mice that had apparently acquired a congenital infection with MuLV were prone to develop lymphoma in

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later life, well after the age (approximately 1 year) that they would likely die under natural conditions.

B. LYMPHOMA-PARALYSIS PRONEHIGH MuLV POPULATIONS Fortunately, we eventually discovered three interesting populations of wild mice situated in widely separate locations: a duck farm at La Puenta, a grain mill in Norwalk (both in Los Angeles County), and a squab farm near Lake Casitas (LC) in southern Ventura County (Figure 2). These populations were characterized by a high prevalence and level of amphotropic and, to a lesser extent, ecotropic MuLV infection throughout life, and the frequent occurrence of lymphoma at a younger age (Gardner et al., 1973c, 1976b). Most surprisingly, these MuLV-infected mice also often developed a fatal lower motor neuron neurologic disease with hind leg paralysis which usually existed without lymphoma (Gardner et al., 1973b; Gardner, 1985). The LC population was most thor-

FIG. 2. Lake Casitas squab farm. This farm is located in a rural part of southern Ventura County. It consists of 19 sheds, each containing 17 pens. Each pen houses 30 pigeons and their offspring. The wild mice live in the nesting boxes under the straw matting.

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oughly studied between 1970 and 1980 and provided most of the knowledge about MuLV in wild mice. Major features of the LC wild mice are listed in Table 2. The incidence of lymphomas in LC mice was ten times greater than that observed in the low MuLV expressor mice (Figure 1) (Gardner et a!., 1976a). After about one year of age, 15% of aging MuLV-infected LC mice eventually developed lymphoma, 10% developed paralysis, 2% had both diseases, and 5 % had epithelial tumors (Le., breast and liver carcinomas, lung adenoma). After one year of age, about 2% of the surviving mice came down with lymphoma per month until they were all dead by about 36 months of age. By contrast, all the mice destined to get the paralytic disease did so by about 18 months of age. The carcinomas occurred sporadically after about 2 years of age. T h e lymphomas uniformly arose in the spleen and were of pre-B or null cell origin (Bryant et a/., 1981). The tumors were comprised of stem cells lacking classic T and B cell markers. An occasional lymphoma cell line developed pre-B cell markers. The MuLV was integrated monoclonally in the tumor cells. Systemic lymph nodes and bone marrow involvement with leukemia were evident but, in striking contrast to the AKR lab mouse model, the thymus was not involved. The neurologic disease was characterized by high levels of MuLV in the central ner-

TABLE 2 MAJOR TYPE C RETROVIRUS FEATURES OF LC WILD MICE

I . Most (-85%) LC mice are congenitally infected via milk with amphotropic MuLV: ecotropic MuLV is also present in much lower prevalence. They have lifelong systemic infection with viremia and specific immune tolerance: the spleen is the major virus “factory.” 2. MuLV-infected LC mice are prone to pre-B-cell lymphoma and a lower motor neuron paralysis, both occurring after 1 year of age. The total incidence of these diseases is 530% of the viremic mice. 3. Both amphotropic and ecotropic viruses causes the lympomas; the ecotropic virus is uniquely paralytogenic. These viruses are exogenous in origin. The endogenous virogenes are strongly repressed and recombination with exogenous viruses does not occur in wild mice. 4. Lymphomas result from MuLV activation of cellular oncogenes. 5. Paralysis is caused by nonirnmunogenic virus injury to lower motor neurons. 6. MuLV-infected LC mice can be converted by foster nursing to an uninfected population free of lymphoma and paralysis. 7. LC mice which escape congenital infection (-15%) remain free of infectious MuLV, lymphoma, and paralysis, but can be converted back t o high virus phenotype by exogenous infection with indigenous MuLV. 8. Fv-4, a defective endogenous Type c provirus, segregates as a dominant gene in L(; mice and restricts infection with the exogenous ecotropic MuLV.

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vous system (CNS), a spongiform noninflammatory histopathology with gliosis and loss of anterior horn motor neurons, located mainly in the lumbar spinal cord (Gardner et al., 1973b; Officer et al., 1973; Andrews and Gardner, 1974) (see later). The disease, called “spongiform polioencephalomyelopathy,” had the clinical features of a lower motor neuron disease. It presented as a hind leg tremor leading to flaccid hind leg paralysis (Figure 3), motor unit atrophy of skeletal muscle and death. Virologic studies of LC mice showed the same two MuLV classes, amphotropic and ecotropic, as found in the low MuLV cancer-resistant wild mice (Gardner et al., 1976b). However, in the LC mice, both viruses were more prevalent and higher titered than in the cancer-resistant wild mice. The amphotropic MuLV was by far the most prevalent, being present in approximately 85% of LC mice whereas the ecotropic MuLV was found in only 120% of these mice, mostly in those with or destined to develop the neurologic disease. On experimental transmission to newborn NIH Swiss laboratory mice, the purified amphotropic virus

FIG.3. MuLV-paralyzed wild mouse. The neuroparalysis manifests as a lower motor neuron type of hind leg paraparesis with profound hind quarter atrophy.

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induced only pre-B cell lymphoma in 130% recipients after 12 months, similar to the amphotropic virus of the low MuLV wild mice (Gardner et al., 1978). In contrast to the ecotropic virus from the low MuLV expressor wild mice, the LC ecotropic MuLV, including a molecular clone (Jolicoeur et al., 1983), induced the paralytic disease as well as pre-B cell lymphoma. In high MuLV LC wild mice, as in the low MuLV wild mice, the amphotropic and ecotropic viruses were transmitted congenitally via milk leading to lifelong persistent systemic infection with high titered viremia (104- 106 infectious unitdml), and resultant immune tolerance (Gardner et al., 1979, 1976b; Klement et al., 1976). However general immunity, vigor, and reproductivity were not impaired in the viremic mice. B-cell areas of the spleen were the major site of virus replication early in life. Sexual transmission of MuLV did not appear to occur among LC mice. However, sexual transmission in both directions readily occurred when infected LC mice were mated with highly susceptible (e.g., C57L, NIH Swiss, IRW) laboratory mice (Gardner et al., 1976b; Portis et al., 1987). Transmission of MuLV by milk, sex, and blood (which would have occurred had these mice received blood transfusions or shared intravenous needles) were remarkably ominous harbingers of what was to happen with the transmission of the human retroviruses, HTLV and HIV. We found no serologic or virologic evidence that the amphotropic MuLV was infectious for humans or squabs who were in close contact with the wild mice, or worked with their retroviruses in the laboratory (Gardner, 1973~). About 15% of LC mice escaped congenital infection with either amphotropic or ecotropic MuLV and remained free of infectious MuLV and related lymphomas or paralysis throughout their lifetime (Gardner et al., 1980b). In this respect, they closely resembled the more common cancer resistant, low MuLV populations of wild mice. However, these MuLV-free wild mice could be converted back to high virus phenotype by exogenous infection with amphotropic MuLV. Remarkably, it is also possible through foster nursing on MuLV-free lab mice to eliminate infectious MuLV from the infected LC mice, and thereby, convert them into a long-lived disease resistant population. It had never been possible to do this with the lymphoma prone AKR inbred mice because their ecotropic virus was transmitted primarily as inherited virogenes. As in all laboratory mice, the LC and other feral mice contained numerous copies of noninfectious, defective, endogenous MuLVrelated proviral DNA (virogenes) in their genome (Kozak and O’Neill, 1987). These defective virogenes were thought to be the evolutionary relic of ancient infections with exogenous MuLV. T h e pattern of these

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virogene sequences could be used to draw evolutionary lineages between different mouse subpopulations. Using this approach, the LC mice were later shown to be hybrids between M. m. domesticus of Western European origin and M. m. castaneus from southeast Asia (see below). Although most of the inherited MuLV virogenes in laboratory and feral mice have no known biologic function, we shall see shortly a remarkable example in LC mice of a useful function (i.e., Fu-4R gene). In AKR and related lymphoma prone inbred mouse strains, some of these endogenous, noninfectious MuLV genes recombine with endogenous, infectious (ecotropic) MuLV to give rise to highly oncogenic MuLV (Hartley et al., 1977). This oncogenic recombinant MuLV transforms mink cells (MCF virus) and induces the thymic lymphomas characteristic of these inbred mice. A similar recombination phenomenon frequently occurred when amphotropic or ecotropic viruses from feral mice were passed through inbred mice (Bergeron et al., 1991, 1992; Holmes et al., 1985; Rasheed et al., 1982, 1983). This recombination phenomena led to viruses with increased oncogenicity, different cell tropisms, and different cell surface receptors. However, as mentioned above, similar recombination events were not evident in wild mice. In nature, the amphotropic and ecotropic MuLV strains behaved as entirely exogenous, genetically stable viruses. They did not detectably recombine with endogenous virogenes in the feral mouse genome and they remained only weakly oncogenic. If we had used modern molecular technology, it is, of course, possible that evidence of recombination might have been found in the wild mouse lymphomas at the proviral DNA level but, if so, there was no apparent effect on the biology of the tumours or recovered viruses. IV. Pathogenesis of Lymphomas T h e naturally occurring null o r pre-B cell leukemias in L C mice, caused by the amphotropic and ecotropic MuLV, presumably resulted from the same molecular mechanism, (i.e., activated proto-oncogenes), as described later in laboratory mice (for summary, see Askew et al., 1993). T h e molecular tools to study this question in the naturally occurring lymphomas of LC mice were unavailable in the 1970s. In the 1980s, others showed that passage of LC ecotropic MuLV in laboratory mice did indeed activate certain cellular proto-oncogenes (e.g., Fli-I, Myb, and E v i - I ) and also recombine with endogenous virogenes (Bergeron et al., 1991, 1992; Holmes, et al., 1985; Weinstein et al., 1986). In contrast to the spontaneous lymphomas of LC mice, all of the same null cell type and occurring only in old age, laboratory mice inoculated with LCMuLV sometimes developed myeloid or erythroid leukemias at a young

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age, the result of recombination with endogenous virogenes. However, as emphasized earlier, viral recombinants were not detected in the naturally occurring LC mouse lymphomas or other tumor types based on the biologic properties of the virus isolates from the tumors or tumor cell cultures. V. Pathogenesis of MuLV Neurologic Disease

Major features concerning the pathogenesis of the naturally occurring and experimentally induced MuLV spongiform encephalopathy are listed in Table 3 and are previously reviewed (Gardner, 1985). In the late 1980s, other investigators showed, by using viral recombinants between amphotropic and ecotropic MuLV of LC mice or between paralytogenic and nonparalytogenic ecotropic MuLV strains, that the neurologic disease was primarily a property of the viral envelope (Paquette et al., 1990; Rassart et al., 1986; Lynch et al., 1991). In addition, subtle changes in the viral LTR of different MuLV strains or recombinants also accounted for variations in the latency, incidence, severity, and distribu-

THEWILD MOUSEAS

A

TABLE 3 NEUHOrROPI(: k T R O V l K U S

MODEL

1. Paralysis is a naturally occurring disease in certain feral mouse populations that express

high levels of indigenous ecotropic MuLV. 2. Latent period o f natural disease is 12-18 months of age and about 10% of the total population is involved. 3. Under natural conditions, ecotropic (and amphotropic) viruses are congenitally transmitted, persist as lifelong vireniia, and evoke no detectable immune response; passive immunization of newborns and removal of spleen at an early age prevent paralysis. 4. Experimental paralysis induced in newborn, susceptible hbordtory mice by biologic and molecular clones of wild mouse ecotropic virus in similar pathologically and virologically to the natural disease except for a higher incidence (2100%) and shortened latent period (1-2 months). The experimentally induced paralysis correlates directly with ecotropic virus level in central nervous system tissues; the ecotropic virus may also induce lymphoma. 5. T h e central nervous system pathology is a spongiform encephalopathy involving the lower spinal cord, brain stem and cerebellum, the result of direct virus injury with no inflammatory component. Virus replicates in endothelial cells, oligodendroglial cells, and microglia. Infection of anterior horn neurons is controversial because of difficulty in distinguishing exogenous from activated endogenous MuLV. Damage to and loss o f oligodendroglial cells and anterior horn neurons leads to primary and secondary demyelination and reactive gliosis. 6. A similar disease is induced in newborn laboratory mice or rats with closely related strains of Friend or Moloney MuLV.

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tion of pathology between different regions of the CNS in the experimentally induced disease. Most critical to the experimental disease induction was a high dose of the neuropathogenic, ecotropic MuLV strain given in the newborn period whereas the route of inoculation was immaterial. Virus infection of the CNS via cell-free viremia must be accomplished during the first few days of life (Czub et al., 1991; Lynch and Portis, 1993). Some degree of postnatal development of the CNS was required for infection to occur but, after 10 days of life, resistance was complete. This age factor is probably due to a combination of decreased susceptibility of CNS cells to infection and acquisition of immune competence. MuLV specific class I restricted cytotoxic T lymphocytes from older laboratory mice that resisted infection after exposure to ecotropic MuLV could passively confer resistance to newborn mice (Robbins and Hoffman, 199 1). Productive infection of CNS endothelial cells occurred usually without accompanying morphologic o r functional defects. However, the site of CNS endothelial cell infection did not necessarily determine the site of later neuropathology. Although the pathology in most models was most severe in the anterior horn grey matter of the lower spinal cord, damage was also present in the brain stem and deep cerebellar gray matter or, even rarely in cerebral and cerebellar white matter. Demyelination occurred probably secondary to damage to motor neurons as well as damage to oligodendroglia, but the relative contribution of these processes to demyelination is uncertain. I n general, there was a close association between virus level in specific regions of the CNS and the characteristic spongiform change in these same regions. Most recently, a mild form of the same spongiform appearing encephalopathia with gliosis was induced in transgenic mice expressing only the env gene of Cas-Br-E MuLV under control of its own LTR (Kay et al., 1993). Because of the low level of transgene expression, the affected cell types could not be detected by Northern blot or by in situ hybridization. However the results suggest that the neurologic disease can occur in the absence of virus replication and that the env gp70/p15E complex is sufficient to induce disease. T h e mechanism leading to the spongiform encephalopathy and neuronal loss is not understood (for summary, see Wiley and Gardner, 1993). Recombination between exogenous and endogenous MuLV genes apparently is not involved in the pathogenesis of this CNS disease. Some investigators believe that MuLV envelope expression may be neurotoxic while others suggest that abortive infection of neurons with failure of normal envelope processing to take place may lead to neuronal cytopathology. Detection of MuLV in motor neurons by electron microscopy

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(Figure 4),immunohistochemistry, or in situ hybridization has been an infrequent finding and, based on the specificity of the probes used, it has been impossible to distinguish between activation of endogenous (inherited) virogenes and exogenous infection. Only abortive viral replication without detectable env protein synthesis was apparently found in neurons of laboratory mice infected in midgestation or at birth with CasBr-E (Sharpe el al., 1987). Equally plausible is the neurotoxic effect of cytokines released by infected glial, microglial, or endothelial cells. Such cytokines could damage cell membranes and lead to the leakage of fluid and resultant spongiform change. This particular pathologic feature is very similar to that caused by prions. The absence of scrapie associated fibrils in the brains of mice with Cas-Br-E encephalopathy or of activation of endogenous MuLV in the brains of mice with scrapie (Hoffman et al., 1982) does not eliminate the possibility that a common molecular mechanism underlies the pathogenesis of both disease processes. T h e answer to the riddle may lie in a better understanding of the MuLV neurologic disease model.

FIG.4. Electron micrograph of' anterior horn motor neuron from a paralyzed wild mouse. A cytoplasmic vacuole contains numerous budding arid free aberrant-appearing Type C virus particles. I t has not yet been possible to determine whether these particles are endogenous o r exogenous in origin.

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VI. Nongenetic Control of Type C Virus and Associated Diseases in Wild Mice

Insofar as congenitally infected viremic LC mice were immune tolerant, it was not possible to actively immunize them with inactivated whole amphotropic or ecotropic MuLV vaccines (Klement et al., 1976). However, it was possible to largely eliminate the virus by passive immunization or animal husbandry methods. Repeated serotherapy of newborn LC mice with goat antisera against the LC ecotropic MuLV markedly reduced the titer of ecotropic virus in the progeny of viremic mothers, lowered the incidence of lymphomas, and completely prevented the development of paralysis (Gardner et al., 1977; Henderson et al., 1974). However, since the amphotropic MuLV of these mice was not neutralized by this antisera, some treated animals did develop lymphomas later in life. Because the spleen is the major virus “factory,” it was possible to significantly reduce the serum MuLV titer, prevent the neurologic disease, and reduce the incidence of lymphoma by splenectomy at 6 weeks of age (Gardner et al., 1978). Infected laboratory mice also transmitted the LC ecotropic virus by milk. In recent years, antiviral treatment of the infected pregnant mothers with 3’-azido-3‘-deoxythymidine (AZT) prevented the establishment of infection in the newborn pups (Sharpe et al., 1987). T h e most dramatic means of nongenetic control of MuLV in the LC mice was by foster nursing them on nonviremic laboratory mice; it was possible to virtually eliminate these viruses in this way (Gardner et al., 1979). Conversely, it was possible to introdu1:e the LC MuLV into uninfected but susceptible lab mice (e.g., NIH S viss, IRW, C57L) by sexual contact with or foster nursing on viremic LC mice (Gardner et al., 1979; Portis et al., 1987). These infected lab mice then developed the lymphoma and paralytic disease that were typical of naturally infected LC mice and transmitted the viruses via milk to their progeny. Selective breeding of nonviremic LC mice, which constituted about 15% of the total population, led to a subpopulation of wild mice that remained free of infectious MuLV throughout their lifetime and did not develop the associated lymphoma or paralysis (Gardner et al., 1980b). These nonviremic mice were susceptible, however, to the amphotropic MuLV when it was introduced by needle inoculation or nursing on viremic LC mothers. The coexistence in nature of this noninfected subset of LC mice and their ability to remain uninfected into old age indicated that horizontal transmission of MuLV, including sexual, did not occur among unrelated wild mice under natural conditions and confirmed the

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absence of activated endogenous MuLV appearing as infectious virus in the aging uninfected wild mice. VII. Genetic Control of Type C Virus in Wild Mice by the Introduction of the FV-7b MuLV-Resistance Allele from Inbred Mice

Before Type C virus (MuLV) was discovered in wild mice, it had been shown that this type of virus was subject to genetic control in inbred laboratory mice (Lilly and Pincus, 1973). The gene in lab mice primarily responsible for this control was called Fu-I because it restricted the growth of Friend MuLV. Two alleles, Fv-In and Fu-Ih, present in different strains of inbred mice, restricted B-tropic of N-tropic MuLV growth. B-tropic and N-tropic MuLV were defined by their relative susceptibility to growth in Balb/C or NIH Swiss cells, respectively. This dominant gene effect was exerted after viral entry during the process of reverse transcription. All of the wild mouse ecotropic and amphotropic viruses were found to be N-tropic and all of the LC wild mice tested were monomorphic for the Fu-17’ genotype. Therefore, the Fu-1 locus was fully permissive for the infectious MuLV present naturally in these wild mice and differences in prevalence of ecotropic or amphotropic MuLV in the LC wild mice could not be accounted for by segregation of the Fu-I alleles, as seen in lab mice. Incidently, the unique SC-1 cell line that lacked Fu-I gene expression and thus was susceptible to all MuLVs of laboratory and feral mice was derived from a wild mouse embryo from Bouquet Canyon (Hartley and Rowe, 1975). It was possible to show by experimental cross breeding that the Fu-1 allele could block the growth of the N-tropic LC-MuLV because the F1 progeny of crosses between viremic LC wild mice and uninfected C57B1 mice (Fu-Ib) were completely free of any infectious Type C virus, even after nursing on the infected LC mothers (Gardner et al., 1976~).Backcrosses of the FI hybrids to the LC parental strain showed that his virus resistance segregated with the Fu-16 allele from the C57B1 parental strain. VIII. Genetic Control of Type C Virus in Wild Mice by Natural Segregation of the Fv-4 Ecotropic MuLVResistance Allele

As noted earlier, the LC wild mice were monophoric for the Fu-I?l genotype and their amphotropic and ecotropic MuLV were N-tropic. Therefore, this locus could not account for the control of ecotropic MuLV in these animals. Surprisingly, another dominant gene called Fv-4

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was found to control replication of ecotropic MuLV in these mice. This gene was distinct from other MuLV restriction genes (e.g., Fv-2 and Fv-3) described in laboratory mice. When Fv-4 was first discovered in LC mice, it was called Akvr-I because it restricted replication of the AKR endogenous ecotropic MuLV and prevented lymphoma in AKR x LC F1 hybrids (Gardner et al., 1980~).Later, we showed that Akvr-I was allelic and identical in sequence on chromosome 12 with the Fv-4 dominant resistance gene which was first described as preventing exogenous infection by N- and NB-tropic Friend MuLV in Japanese wild mice ( M . m. rnolossinus) and the derivative G inbred line (Suzuki, 1974; Odaka et al., 1981; O’Brien et al., 1983). The major features of the Fv-4 gene are shown in Table 4. We discovered the Fv-4 (Akvr-I ) resistance gene serendipitously while cross-breeding LC mice with AKR inbred mice (Gardner et al., 1980~). Three patterns of MuLV viremia were observed in the F1 progeny of the individual AKR X LC crosses: at 2 months of age, either all of the F1 progeny were viremic, all were nonviremic, or about 50% were viremic. T h e viremia was entirely due to AKR ecotropic MuLV because only LC males or nonviremic LC females were bred to the AKR mice and we had already determined that infectious MuLV was transmitted only by viremic LC females. In F1 backcrosses to AKR mice and in F2 progeny, viremia was present in about 50% and 25%, respectively, thus indicating the segregation in LC mice of a dominant gene capable of strongly blocking the expression of the endogenous ecotropic MuLV inherited from the AKR parent. T h e dominant MuLV-restrictive allele was called Fv-4R and the recessive allele, F v - ~ The ~ . Fv-4R restriction effect was long lasting (>18 mo) and associated with prevention of lymphoma in the AKR X LC F1 progeny. Restriction was generally stronger in vivo

TABLE 4 ECOTROPIC VIRUSRESTRICTION GENEFv-4

IN

WILDMICE

1 . Polymorphic in LC wild mice; allele frequencies conform to the Hardy-Weinberg equilibrium. Determines susceptibility or resistance to ecotropic MuLV induced neurologic disease and lymphoma in individual LC mice. 2. Dominant, fully penetrant, strong blocking effect upon expression of ecotropic virus; segregates as a single locus in F2 and backcross animals. 3. Allelic and sequence identical to the same gene on chromosome 12 in Japanese wild mice ( M w molossinw). Gene originated in Asian wild mice ( M w castanew). 4. Represents a defective, truncated endogenous MuLV provirus expressing an ecotropic MuLV related glycoprotein that blocks the ecotropic virus receptor by an interference phenomena.

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then in uitro and stronger in hemopoietic cells than in fibroblasts (Rasheed and Gardner, 1983b). Fu-4 (Akur-I) strongly blocked cell-tocell spread of all exogenous and endogenous infectious ecotropic MuLVs (i.e., N-tropic, B-tropic or NB-tropic) both in vivo and m uitro. All ecotropic MuLVs use the same receptor encoded by the Rec-1 locus on mouse chromosome 5 and thus would be interfered with by binding of the Fv-4 encoded gp70 to this receptor (Sarma et al., 1967). This ecotropic MuLV receptor has recently been cloned and sequenced and its normal function shown to be that of a di-basic amino acid transporter (Albritton et al., 1989; Kim et al, 1991). The homologous human gene has also been cloned (Yoshimoto et al., 1991), and both mouse and human genes are activated in rapidly proliferating cells (Yoshimoto et al., 1992). Fu-4R does not, however, block amphotropic MuLV which uses a different receptor encoded by a gene on chromosome 8 (Gazdar et al., 1977). T h e cellular receptor for amphotropic MuLV was quite recently cloned and revealed homology to the receptors for gibbon ape leukemia virus and feline leukemia virus, subgroup B (Miller et al., 1994). Expression of the Fu-4R locus in uninfected cells was associated with the presence of an ecotropic MuLV-related envelope glycoprotein of about 70,000 kD (gp70) on the cell surface (Ikeda and Odaka, 1983). Hirt analysis of Fu-4R resistant cells showed no proviral DNA after ecotropic virus challenge whereas the same cells did show proviral DNA after challenge with amphotropic virus (Dandekar et al., 1987). The block to infection at the cell surface level presumably occurred by receptor interference. Using an AKR ecotropic enu probe, the Fu-4R gene was cloned and shown to be a truncated proviral genome containing a small segment of pol, the entire enu gene (gp70) and a 3' LTR (Dandekar et al., 1987; Kozak et al., 1984; Ikeda et al., 1985). Sequence analysis showed that the Fu-4R gp70 was 70% related to the AKR ecotropic gp70, 58% similar to endogenous xenotropic and MCF envelope sequences, but 90% related to the LC ecotropic gp70. The protein encoding sequence of the Fu-4R allele, cloned from LC mice, was identical to that of the Fu-4R gene from M . m. molossinus (Dandekar et al., 1987). Southern blot hybridization showed that M . m. molossinus, M . m. castanew (wild mice from southeast Asia), and LC mice each contained the same Fv-4" provirus (Kozak and O'Neill, 1987). M . m. molossinus contained a single Fu-4R provirus whereas LC mice and M . m. castaneus each carried several addi~ ~ Moreover, M . m. castaneus and LC mice tional copies of the F u - gene. shared two specific Fu-4 proviral integrations. Therefore, the Fu-4 resistance gene was probably introduced into M . ni. molossinus and LC mice by natural interbreeding with M . m. castaneus during this past century. LC feral mice are thus hybrids between M . m. domesticw from Europe and

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M . m. castaneus from southeast Asia. Opportunities for such interbreeding would have occurred in the last century via the transpacific shipping trade or immigration from Asian countries. ‘The observed frequency of the Fv-4 resistance allele in LC mice randomly trapped at the squab farm was 56% which does not vary from expectation of the Hardy-Weinberg equilibrium (Gardner et al., 19804. T h e probable frequency of LC mice that contain at least one Fv-4 restriction allele is 80% and all of these mice would, therefore, be resistant to the LC ecotropic MuLV and associated paralysis and lymphoma. They would not, however, be totally resistant to lymphoma because they might still be congenitally infected with amphotropic MuLV. T h e 20% of LC mice not inheriting this resistance allele would be considered homozygous for the susceptibility allele (i.e., F V - ~ and ~ . ~ would ) be vulnerable to ecotropic virus congenital infection and the associated diseases later in life. Inheritance of F V - would ~ ~ thus have a protective value on the survival of individual LC mice. Although manifested after the onset of breeding, evolutionary conservation of this locus might be expected because of its beneficial effect on survival and reproductive life span. In summary, this fascinating wild mouse model (summarized recently in Gardner et al., 1991) has taught us that susceptibility or resistance to lymphoma and a slow neurologic disease, both developing after midlife (>1 year of age) are determined, not only by exposure at birth to maternally transmitted ecotropic-MuLV but, ultimately, by inheritance of an ecotropic-MuLV resistance gene (Fv-4R) segregating in this outbred population. Similar interference genes found in laboratory mice (Bassin et al., 1982) and chickens (Robinson et al., 1981) confer resistance to Type C viruses and prevent development of the associated diseases. Recently, Fv-4 transgenic mice were made and found resistant to Friend MuLV infection because of the inherited viral envelop mediated interference phenomenon (Limjoco et al., 1993), in essence duplicating in the lab mouse what nature had already done in the Fv-4R wild mice in Asia and southern California. Interestingly, the F V - positive ~ ~ wild and laboratory mice are perfectly healthy suggesting that the ecotropic MuLV receptor may be a nonessential gene in mice. IX. Type B Mammary Tumor Viruses in Wild Mice

In contrast to low or high prevalence of infectious Type C MuLV in different populations of wild mice, the Type B mammary tumor virus (MMTV) particles and antigen were equally prevalent in all of the populations examined (Rongey et al., 1973, 1975; Fine et al., 1978). M M T V

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was present in the milk, lactating mammary gland, and spontaneous breast tumors of about 50% of feral mice but only a few of these animals (51%) ever developed breast tumors after about one year of age (Gardner et al., 1980a). Thus, in nature the MMTV is only weakly tumorigenic for its natural feral host. T h e MMTV of feral mice was also only weakly tumorigenic in Balb/C laboratory mice. By molecular hybridization, most wild mice contained a few copies of MMTV-related proviral DNA in their genome (Cohen and Varmus, 1979). However, a few LC wild mice were identified that carried no MMTV DNA in their genome and a breeding colony of these M M T V proviral DNA-free mice (WLC-0) was established in the laboratory (Cohen et al., 1982). This WLC-0 colony was useful for showing that M M T V is not required for normal mammary gland development and lactation or for the chemical induction of certain kinds of breast tumors, i.e., ductular carcinomas (Faulkin et al., 1984; Gardner et al., 1985). However the more common alveolar type of breast cancer did not occur in these M M T V DNA negative mice and the genesis of this tumor presumably required MMTV as a cofactor. T h e WLC-0 mice have been very useful for making genetic crosses to segregate specific M M T V proviruses from lab mice, such as Mtv-2 from GR mice, in order to analyze the effect of single MMTV alleles on breast tumor phenotype and to better understand the MMTV superantigen interaction with the developing immune system (Morris et al., 1986; Ferrick et al., 1992). As a model for human breast cancer, the wild mouse MMTV may have little relevance because infectious Type B viruses have not been found in human milk (Cardiff and Gardner, 1983). T h e protooncogenes (Znt-I and Int-2), often cis-activated by MMTV infection of laboratory mice (Nusse and Varmus, 1982), have not been shown to play a significant role in human breast cancer. Nor is there strong evidence to suggest that the MMTV-related endogenous retroviral related sequences present in the human genome (Medstrand and Blomberg, 1993) are involved in human breast cancer. X. Lessons Learned

In retrospect, we can now see how accurately the natural history features of Type C retroviruses found in wild mice predicted the natural history of the human Type C retrovirus, HTLV, discovered a decade later. T h e similarities (Table 5) far outweigh the differences (Table 6) (Gardner, 1987). This is not so surprising when one considers that wild mice, like humans, are exposed to natural evolutionary forces and exhibit the maximum genetic heterogeneity endowed to the species. Thus, it is understandable why the wild mice might portray more accurately

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TABLE 5 SIMILARITIES BETWEEN LEUKEMIA VIRUSES I N WILDMICEA N D HUMANS 1. Regional and familial clustering of virus and specific type of lymphoma or leukemia. 2. Low pathogenicity, long latent period, persistent infection with viremia. 3. Viruses are exogenous and horizontally transmitted mainly by milk. Maternal congenital transmission, venereal spread, and blood transfusion are the only known routes of natural virus spread. 4. T h e viruses are unique, stable, replication competent and seldom, if ever, undergo recombination with endogenous host-cell sequences. 5. Viruses integrate monoclonally in leukemia cells. 6. Viruses are neurotropic as well as lymphomagenic.

than inbred mice the natural spectrum of host retrovirus interrelationships. Remarkable similarities between leukemia viruses of wild mice and humans included the regional and familial clustering of virus with a specific type of lymphoma, the purely exogenous nature of the virus and its congenital transmission by milk. Based on the AKR mouse model, we certainly did not expect MuLV in wild mice to be transmitted primarily by milk, resembling more in this respect the M M T V of laboratory mice. Not to be forgotten, however, was the discovery soon after the initial isolation of Gross MuLV in AKR mice, of yet other immunologically distinct strains of MuLV. Rather than representing activated latent, genetically transmitted viruses like the Gross MuLV, these other isolatesbearing the names of their discoverers, e.g., Friend, Moloney, Rauscher, Graffi-were derived from “foreign” tumor cells, e.g., Erlich ascites, that had been passaged for many decades as transplant tumors in various mouse strains (Gross, 1970). These MuLVs were considered “pasTABLE 6 DIFFEHENCES BETWEEN LEUKEMIA VIRUSES

IN

WILD MICEA N D HUMANS

I . HTLV-I is more t cell restricted and induces a T-cell leukemia. Wild mouse MuLV is more B-cell tropic and induces a null or pre-B-cell leukemia. 2. HTLV-I infection appears more latent and cell associated with a lower level of viremia. 3. HTLV-I usually induces an immune response, whereas wild mice are immunologically tolerant to their congenitally acquired leukemia virus. 4. HTLV-I can directly transform T-lymphoid cells in vztro. HTLV-I has a transactivating (TAT) gene. Wild mouse MuLV transforms pre-B-cells by cis activation. 5. HTLV-I may be associated with general immunosuppression. 6. Virus restriction by receptor interference at the cell receptor level (e.g., Fv-4) has not yet been identified with HTLV.

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senger” viruses picked up by the transplant tumor cells at some point in their in vim passage history. Although capable of inducing leukemia after experimental inoculation of newborn mice, these viruses were thereafter transmitted exclusively by exogenous nongenetic means, mainly by mothers’ milk (Law and Moloney, 1961; Buffet et al., 1969). Here, then, was the precedence for what we found in the wild mice. Understandably, however, these other MuLVs were considered an experimental artifact compared to the “natural” genetic transmission of MuLV in AKR laboratory mice with “spontaneous” lymphoma. Overlooked to same extent was the critical role of intense inbreeding in selecting for stable inheritance (homozygosity) of infectious MuLV and other susceptibility genes that “stacked the deck,” allowing for uniform development of thymic lymphomas. T h e realization that genetic transmission of infectious MuLV was indeed a laboratory artifact was certainly highlighted by our discovery that infectious MuLV in low and high-leukemia populations of outbred feral mice in southern California was transmitted by an entirely nongenetic milk-borne route. In this respect, and also by immunologic and genetic comparisons, the infectious MuLV of feral mice more closely resembled the Friend, Moloney, Rauscher virus group than the Gross-related MuLV group. Apparently, the Friend, Moloney, and Rauscher MuLVs and the wild mouse MuLVs represent members of a separate MuLV lineage that have been carried as solely exogenous viruses for a long time. In contrast to leukeniogenesis in the AKR mouse model, which featured genetic transmission and recombination among endogenous MuLV sequences (virogenes), the endogenous MuLV sequences in the wild mice, although present in amounts equal to that of lab mice, did not appear to play a similar role. In the wild mice, as later found with H‘TLV in humans, recombination with endogenous virogenes apparently seldom, if ever, occurs, at least as far as can be detected. ’The Type C retroviruses of wild mice and humans appear remarkably stable with little genetic variation over time. Both viruses are well adapted to their hosts. We found no change in the natural history of MuLV in the LC wild mice between 1970 and 1980 and it was still apparently unchanged when we revisited the LC farm in 1993 during an unsuccessful search for lentiviruses in these animals. T h e conclusion appears inescapable that the recombination phenomena o r insertional mutagenesis in the germ line, typical of Type C retroviruses in inbred laboratory mice (Soriano et al., 1987; Spence et al., 1989; Stoye et al., 1988; Tsichlis and Lazo, 1991) is mostly, if not entirely, an artifact of inbreeding. Under natural conditions, the genome of outbred Mus musculus and humans presum-

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ably protects against this kind of life threatening event. Perhaps this defense mechanism also accounts for our inability to ever recover oncogenes in the form of sarcoma viruses from wild mouse sarcomas, even from those that were infected in vivo with MuLV. Thus, one major lesson learned from the wild mouse MuLV model that appears to apply also to humans is that endogenous virogenes, although present in the genome, are not oncogenic, at least as far as known today. Another important lesson learned was that certain endogenous virogenes in wild mice such as Fv-4R might have a useful function by preventing infection with related exogenous retroviruses. Presumably, the Pz1-4~allele represents the truncated relic of ancient infection with ecotropic MuLV in the ancestral Asian wild mouse (Mus castaneus) and it is carried today by progeny wild mice in Japan and southern California (LC squab farm) (Gardner et al., 1991). By physiologic mimicry of retroviral interference, the Fv-4 gene has provided an adaptive strategy that protects its LC mouse carriers from an ongoing lethal epizootic. T h e development of viral protection by the expression of truncated (defective) endogenous retroviral genes has been observed in chickens and laboratory mice and the phenomenon of inherited viral envelope mediated resistance is also seen with other classes of viruses and even in plants. Gene therapy experiments have taken advantage of this phenomena to engineer resistance to natural pathogens in commercially important crops (Abel et al., 1986; Tumer et al., 1987). Perhaps a similar retrovirus interference phenomena will be discovered with further deciphering of the human genome sequences that contain many copies of retroviral related genes. T h e highly specific integration patterns for endogenous retroviruses suggest that they may yet be shown to have beneficial functions in humans (Taruscio and Manuelidis, 1991). Also gene therapy may someday use such envelop genes for therapy of human retroviral infections. The Fv-4 gene has not been found in laboratory mice because the rather narrow genetic base from which the inbred lines were derived did not include Mus castaneus from Asia (Strong, 1978). This observation and similar examples in animal and plant breeding illustrate the point that new genes useful for genetic engineering may best be identified among the natural wealth of genetic diversity in wild species. These studies pointed out the all-importance of genetic make-up rather than environment in determining the natural history of retroviruses in wild mice. The factors that put the individual LC wild mouse at risk to Type C retroviral disease depended on exposure to the virus in maternal milk and inheritance of the Fv-4 resistance or susceptibility

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allele. T h e local environment where the mouse lived was inconsequential and there was no detectable effect on tumor incidence or retrovirus expression from exposure to Los Angeles smog. T h e value of animal husbandry and immunologic methods for control of exogenous Type C retroviruses was highlighted by the ability to confer resistance to the N-tropic MuLV of wild mice through introduction of the Fv-1B allele by cross-breeding with C57B1 inbred mice; by the ability to switch off the AKR or related ecotropic MuLV of laboratory or wild mice by introduction of the Fv-4 resistance allele; by the dramatic elimination or reintroduction of exogenous MuLV by foster nursing of wild mice; and by the prevention of the neurologic disease from passive immunization or splenectomy early in life. Avoidance of breast-feeding in humans is now proving to be remarkably effective in lowering the incidence of HTLV infection and associated diseases in Japan (Hino and Doi, 1989). T h e wild mouse clearly pointed the way. T h e spontaneous lymphomas in wild mice and the lymphomas experimentally induced by LC MuLV in laboratory mice provide a useful model for childhood acute lymphoblastic leukemia and for study of the earliest steps in B-lymphocyte differentiation in vitru. Molecular analysis of the MuLV cis activated proto-oncogenes in the naturally occurring lymphomas in wild mice might yield other yet unidentified cellular proto-oncogenes of relevance to human cancer. Finally, we learned from wild mice that Type C luekemia viruses could be neuropathogenic as well as lymphogenic. This was the first indication that the same, genetically identical Type C retrovirus could cause a degenerative neurologic disease as well as lymphoma, a precedence for what was found with HTLV a decade later. Although the MuLV-induced CNS disease of wild mice is not identical to the HTLV or HIV induced neurologic diseases of humans, some of the intermediate steps in pathogenesis may well be similar. Further study of this mouse model may also help us understand common mechanisms of cell membrane damage that underlie the spongiform encephalopathies of unconventional viruses (i.e., prions). One of the special benefits of being in on the discovery of this wild mouse neurologic disease has been the pleasure of getting to know other investigators working on the model over the next 20 years (Table 7). It is now clear, of course, that the closest animal models for HTLV are not MuLV in wild mice but, instead, the bovine leukemia viruses and simian T-lymphotropic viruses (for summary, see Gardner, 1990). However, in the 1970s, the time was ripe to compare the laboratory mouse model with the wild mouse model of retroviral leukemogenesis and the results were remarkably informative and unanticipated. I feel very for-

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TABLE 7 INVESTIGATORS DOINGRESEARCH O N THE MuLV SIWNCIFORM ENCEPHALOPA~HY Location('

Investigators.

USC/UC Davis

Officer, Rasheed, Rongey, Bryant, Estes, Henderson, Dandakas, Cardiff, Morris, Cardner

Scripps Institute

Oldstone, Elder, Dixon

UC San Diego

Wileyb, Nagra

Johns Hopkins/University of Wisconsin

Brooks, Swarz, Johnson

University of MarylandINCI

Hoffman, Robbins, Bilello, Kuscetti, Masuda

NIH

Hartley, Morse, Frederickson, Kozak, O'Brien

Montreal

Jolicoeur, DeGrosseillers, Paquette, Robitaille, Rassart, Kay

Rocky Moutain Laboratories

Portis, Lynch, McAtee, Czub, Chesebro

University of Illinois

Zachary

University of Texas

Wong, Yuen

Dana-Farber Institute

Ruprecht, Sharpe Kai, Furata

Japan

b

List not all inclusive. Curwnl nddrru: University of Pittsburgh

tunate to have had the opportunity to participate in this research endeavor, to enjoy the sense of discovery and accomplishment, and to share the intellectual excitement with my friends and colleagues. ACKNOWLEDGMENTS This work was supported by NCI contracts as part of the Virus Cancer Program of the 1970s. 1 am indebted to all of my colleagues at USC, UC Davis, NCI and elsewhere who did so much of the research summarized here. I thank Robert Malone for his editorial help and Donna Chan for preparation of the manuscript.

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Bassin, R. H., Ruscetti, S., Ali, I., Haapala, D. K., and Rein, A. (1982). Virology 123, 139151. Bentvelzen, P., and Daams, J. H. (1969).J. Natl. Cancer /rut. 43, 1025-1035. Bergeron, D., Poliquin, L., Kozak, C. A., and Rassart, E. (1991).J . Virol. 65, 7-15. Bergeron, D., Poliquin, L., Houde, J., Barbeau, B., and Rassart, E. (1992). Virology 191, 661-669. Bryant, M. L., Pal, €3. K., Gardner, M. B., Elder,J. H., Jensen, F. C., and Lerner, R. A. (1978a). Virology 84, 348-358. Bryant, M. L., Roy-Burman, P., Gardner, M. B., and Pal, B. K. (1978b). Virology 88,389393. Bryant, M. L., Scott, J. L., Pal, B. K., Estes, J. D., and Gardner, M. B. (1981). Am. J . Pathol. 104,272-282. Buffet, R., Grace, J., and Mirand, E. (1969). Cancer Res. 29, 588-595. Cardiff, R. D., and Gardner, M. B. (1983). In “Understanding Breast Cancer: Clinical and Laboratory Concepts” (M. Rich, J. Hager, and P. Furmanski, Eds.), pp. 31 7-333. Marcel Dekker, New York. Cohen, J. C., and Varrnus, H. E. (1979). Nature 278, 418-423. Cohen, J. C., Traina, V. L., Breznik, T., and Gardner, M. (1982).J. Virol. 44,882-885. Czub, M., Czub, S., McAtee, F. J., and Portis, J. (1991).J . Virol. 65, 2539-2544. Dandekar, S., Rossitto, P., Picket, S., Mockli, G., Bradshaw, H., Cardiff, R., and Gardner, M. (1987). J . Virol. 61, 308-314. Faulkin, L. J., Mitchell, D. J,, Young, L. J. T., Morris, D. W., Malone, R. W., Cardiff’, R. D., and Gardner, M. B. (1984). J . Natl. Cancer Inst. 73, 971-982. Ferrick, D., Cho, K., Geminell-Hori, L., and Morris, D. W. (1992). Int. Immunol. 4,805-810. Fine, D. L., Arthur, L. O., and Gardner, M. B. (1978). J . Natl. Cancer Inst. 61, 485-491. Gardner, M. B. (1966). Arch. Environ. Health 12, 305-313. Gardner, M. B., Loosli, C. G., Hanes, B., Blackmore, W., and Teebken, D. (1970). Arch. Enviran. Health 20, 3 10-3 17. Gardner, M. B., Officer, J. E., Rongey, R. W., Estes, J. D., Turner, H. C., and Huebner, R. J. ( 197 1 a). Nature 232, 6 17-620. Gardner, M. B., Henderson, B. E., Estes, J. D., Rongey, R. W., <:asagrande, J., Pike, M., and Huebner, R. J. (197 I b). J . Natl. Cancer hut. 52, 979-981. Gardner, M. €3.. Heiderson, B. E., Rongey, R. W., Estes, J. D., and Huebner, R. J. (1973a). J. Natl. Cancer Inst. 50, 7 19-734. Gardner, M. B., Henderson, B. E., Estes, J. D., Menck, H., Parker, J. C . , and Huebner, R. J. (1973b).J. Natl. Cancer Inst. 50, 1571-1579. Gardner, M. B., Henderson, B. E., Officer, J. E., Rongey, R. W., Parker, J. C., Oliver, C;., Estes, J. D., and Huebner, R. J. (1973c).J. Natl. Cancer I T L S ~51, . 1243-1254. Gardner, M. B., Officer, J. E., Parker, J. Estes, J. D., and Rongey, R. W. (1974). Infficc. Immun. 10, 966-969. Gardner, M. B., Henderson, B. E., Estes, J. D., Rongey, R. W., Casagrande, J., Pike, M., and Huebner, R. J. (1976a). Cancer Res. 36, 574-581. Gardner, M. B., Klement, V., Rongey, R. W., McConahey, P., Estes, .I. D., and Huebner, R. J. (1976b). J. Natl. Cancer Inst. 57, 585-590. Gardner, M. B., Klement, V., Henderson, B. E., Meier, H., Estes, J. D., and Huebner, R. J. ( 1 9 7 6 ~ )Nature . 259, 143- 145. Gardner, M. B., Klement, V., Estes, J. D., Gilden, R. V., Toni, R., and Huebner, R. J. (1077). 1. Natl. Cancer Inst. 58, 1855-1857. Gardner, M. B. (1978). In “Current Topics in Immunology and Microbiology,” pp. 2 I5259. Springer-Verlag, Berlin.

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Kozak, C. A., Gromet, N. J., Ikeda, H., and Bucklei-,C . E. (1984).Proc. Natl. Acad. Sci. USA 81, 834-837. Kozak, C. A,, a n d ONeill, R. R. (1987).J. Virol. 61, 3082-3088. Lai, M. M. C., Shimizu, C. S., Rasheed, S., Pal, B. K., and Gardner, M. B. (1982).J. Virol. 41, 605-6 14. Law, L. W., and Moloney, J. B. (1961). Proc. Sac. Ex$. Biol. Med. 108, 715-723. Lilly, F., and Pincus, T. (1973). Adz). Cancer Kes. 17, 231-277. Linijoco, T. I., Dickie, P., Ikeda, H., and Silver, J. (1993).J. Virol. 67, 4163-4168. Lynch, W. P., Czuh, S., McAtee, F. J., Hayes, S. F., and Portis, J. (1991).Neuron 7,365-379. Lynch, W. P., and Protis, J. L. (1993).J. Virol. 67, 2601-2610. Medstrand, P., and Blomberg, J. (1993).J . Virol. 67, 6778-6787. Miller, A. D. (1992). Nature 357, 455-460. Miller, D. G., Edwards, R. H., and Miller, A. D. (1994).Proc. Nutl. Acad. Sci. USA 91,78-82. Morris, D. W., Young, L. J. ‘T., Gardner, M. B., and Cardiff, R. D. (1986).J. Virol. 58, 247252. Nusse, R., and Varnius, H. E. (1982). Cell 31, 99-109. O’Brien, S. J., Bernian, E . J . , Estes, J. D., and Gardner, M. B. (1983).,/. Virol. 47, 649-651. Odaka, T., Ikeda, H., Yoshikura, H., Moriwaki, K., and Suzuki, S. (1981). J . Null. Cuncer Inst. 67, 1123-1 127. Officer, J. E., Tecson, N., Estes, J. D., Fontanilla, E., Rongey, R. W., and Gardner, M. B. (1973). Science 181, 945-947. Oie, H. K., Gazdar, A. F., Lalley, P. A,, Russell, E. K., Minna, J . D., DeLarco, J., Todaro, G . J . , and Francke, U. (1978). Nature 274, 60-62. O’Neill, R. R., Hartley, J. W., Repaske, R., and Kozak, C. A. (1987).J. Virol. 61,2225-2231. Pal, B. K., Wright, M., Officer, J. E., Gardner, M. B., and Roy-Burman, P. (1973). Virology 56, 389-3547. Pal, B. K., McAllister, R. M., Gardner, M. B., and Roy-Burman, P. (1975).J.Virol. 16, 123131. Paquette, Y., Kay, D. G., Rassart, E., Robitaille, Y., and Jolicoeur, P. (1990).,J. Virol. 64, 3742-3752. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A,, Minna, J. D., and <;allo, K. C . (1980). Proc. Natl. Acad. Sci. U S A 77, 7415-7419. Portis, J. L., McAtee, F. J., Hayes, S. F. (1987).J. Virol. 61, 1037-1044. Rasheed, S., Gardner, M. B., and Chan, E. (1976).J. Virol. 19, 13-18. Rasheed, S., Toth, E., and Gardner, M. B. (1977). InttnJirology 8, 323-335. Rasheed, S., Charrnan, H. P., and Gardner, M. B. (1978). Viroloo 89, 605-609. Rasheed, S., Pal, B. K., and Gardner, M. B. (1982). Int. J . Cancer 29, 345-350. Rasheed, S., Gardner, M. B., and Lai, M. M. C. (1983). Virology 130, 439-451. Rasheed, S., and Gardner, M. B. (1983).I n t . ,/. Cancer 31, 491-496. Kassart, E., Nelbach, L., and Jolicoeur, P. (1986).J. Virol. 60, 910-919. Rettig, R. A. (1977). In “Cancer Crusade: T h e Story of the National Cancer Act of 197 1.” Princeton Univ. Press, Princeton, N.J. Rice, M. C., Gardner, M. B., and O’Brien, S. J. (1980). Biochem. G t n t t . 8, 915-928. Rohbins, D. S., and Hoffman, P. M. (1991).J. Neurozmrnunol. 31, 9-17. Robinson, H. L., Astrin, S. M., Senior, A. M., and Salazar, F. H. (l98l).J. Vzrol. 40, 745751. Rongey, R. W., Hlavackova, A., Lara, S., Estes, J,, and Gardner, M. B. (1973).J.Natl. Cuncer I w ~ . 50, 1581-1589. Rongey, R. W., Abtin, A. H., Estes, J. D., and Gardner, M. B. (1975).J. Natl. C u n c e ~ ~ n s54, l. 1149-1 156.

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SOL SPIEGELMAN Gunther S. Stent Department of Cell and Molecular Biology, University of California, Berkeley, Berkeley, California 94720

Sol Spiegelman was an archetypical example of a relentlessly driven scientist now rare and unlikely to arise under the relaxed ambiance of postmodernism. He was what Oswald Spengler called a “Faustian Man,” who viewed himself as being locked in an endless strife with the world, reaching for the infinite and never finding satisfaction. His career, which began just before the rise of molecular biology in the early 1940s and ended not long after the molecularbiologization of cancer research in the late 1970s, fascinated me ever since I met him 47 years ago, when I was a graduate student in physical chemistry at the University of Illinois. Over the years, I grew so obsessed with his tragic story of failed ambition that I was always thinking of turning it into a roman a clef, a project at which I may still try my hand. At first sight, Spiegelman seemed to have made a brilliant scientific career: He developed one of the experimental techniques without which present-day molecular biology would be unthinkable. By the time of his death in 1983, he had gained international fame and honors, prizes, honorary degrees, elections to prestigious academies, and, finally, in 1969, the directorship of the Cancer Research Center at Columbia University. Yet, all the while, he knew that for the people whose respect he craved most-the leaders of the nascent molecular biology of the 1950s and 1960s and of the molecularbiologized cancer research of the 1970s-he remained largely a figure of derision. Spiegelman’s failure to achieve his paramount professional aspiration -not to speak of the irony of superficially informed outsiders plying him with public honors while knowledgeable insiders perceived him as a semimountebank-was not simply attributable to his lacking the astral 203 ADVANCES I N C:AN(:EK RESEARCH. VOL.. 6.5

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brilliance of such three-star founders of molecular biology as Linus Pauling, Max Delbriick, Jacques Monod, or Francis Crick. He could have easily made it into the highly respected one- o r two-star category. But an overarching ambition, which energized a reckless perpetual motion, seemed to have prevented him from putting his far-from-negligible talents to the most effective use. He lacked not intelligence or imagination but the Sitzpeisch (i.e., the combination of persistence and patience) that all but authentic geniuses need to create great works. Even very good scientists, just as very good composers, hardly ever get it right on the first try. And when, as it usually does, their initial try fails, they build on its ruins, by modifying their starting concepts in the light of what went wrong. But Spiegelman was in too much of a hurry to try to turn a loss into profit. After each failure, he would start afresh, with an entirely new idea, and, often, with a different working material. Consequently, his episodic oeuvre lacked conceptual growth. Mozart, so it is said, was different. He didn’t need SitzJEeisch. He just put his perfect compositions on paper as the music entered his head and never needed to change a note. Francis Crick is the only Mozart-like genius in molecular biology I know; he (almost) always got it right the first time. I first met Spiegelman in May 1946 through Martha Baylor, who was in charge of the Illinois Chemistry Department’s Early American RCA electron microscope. Martha was a postdoctoral research biologist, married to Teddy Baylor (of the Texas Baylors who endowed Baylor University), then serving as a meteorologist-sergeant in the Army Air Corps, stationed at Chanute Field near Champaign-Urbana. Martha’s microscope was housed across the hall from the lab in which I worked as a graduate research assistant for the War Production Board’s Synthetic Rubber Research Program. There were few women around the Noyes Chemistry Laboratory in those days, and the attractive, charmingly smiling, freckle-faced, strawberry-blonde Martha in her white lab coat had not escaped my notice. My best friend and lab mate, Bill Treumann, who knew Martha and Teddy, introduced me to her. Although (or maybe, because) she was a married, older woman in her late twenties with a Ph.D., I developed a forlorn crush on her. Martha ran a sort of salon for physicists, chemists, and biologists in her cozy apartment, whose floors and walls were covered by Persian rugs. I had just turned 22 when she invited Bill and me to a party she was giving for a visiting seminar speaker, providing me with my first taste of urbane academic high life: wine-drinking, canape-munching graduate students, post-doctorates and junior faculty making clever

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conversation-a far cry from, and a tremendous improvement over, the callow fraternity and sorority mixers, dances and exchange dinners which had hitherto been the high points of my campus socializing. Martha’s guest of honor was Sol Spiegelman, then a 32-year-old Assistant Professor of Bacteriology at Washington University Medical School. Born in New York City in 1914, he had graduated from CCNY in 1939, and received his Ph.D. in cellular physiology from Washington University, St. Louis in 1944. Bill and I hadn’t troubled to attend his seminar, whose title was “Physiology and Genetic Significance of Enzymatic Adaptation,” because, being parochial physical chemists, we neither knew, nor cared to find out, what enzymatic adaptation was all about. Spiegelman turned out to be a short, aggressive man, who fixated his interlocutors with a penetrating stare, and talked staccato sentences in a tone suggesting that he was bringing enlightenment to Illinois. Provoked by his mannerisms, Bill and I started an argument with him about the applicability of thermodynamics to living systems. We didn’t know anything about living systems, and he didn’t seem to have a firm grasp on thermodynamics, which was our specialty. But we argued all the same, and in the end, we felt (no doubt, mistakenly) that we had shown him that he wasn’t as smart as he thought. Since Martha had given us to understand that Spiegelman was considered an up-and-coming star in his field, we asked her for some references to his publications, so we could get some idea what the work of a hot-shot biologist was like. She lent us a reprint of an article of his in the Annals of the Missouri Botanical Garden, on which, so she said, the seminar we missed had been based. We found to our surprise that the first two pages of his article presented a cumbersome derivation of equations describing a system of chemical reactions obeying zero- and first-order kinetics, which Illinois undergraduates were routinely asked to work out on the Chem Majors’ physical chemistry course final examination. Since my professors had left me with the impression that all the important discoveries in physical chemistry had been made by the 192Os, and that the art of research consisted of discovering an as yet unsolved problem, it occurred to me that biology might be a good field for me to get into if it took so little to have a paper published. Bill and I had considerable trouble making out the rest of Spiegelman’s article. When I looked at it again a few years later, driven by curiosity about this seminal experience that had set off my switch from physical chemistry to biology, I discovered that Spiegelman had been explaining enzyme adaptation in yeast in terms of a stabilization of intrinsically unstable enzymes by contact with their substrates. This idea

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had first been put forward by John Yudkin in 1938, and, although it turned out to be wrong, it opened the modern area of the study of enzyme adaptation. Transcending Yudkin, Spiegelman thought that his kinetic and genetic analyses showed that, once stabilized, the enzymes become self-duplicating, even in the absence of the genes that have the ability to form them in the first place. Martha told me that if, as our argument with Spiegelman suggested, I was interested in the connection between physics and biology, I should read the recently published What is Life? In this little book, the famous Austrian physicist, Erwin Schrodinger, announced that, thanks to a theory by the young German physicist Max Delbruck, a new era was dawning for the study of heredity. When I left Illinois a few weeks later for a nine-month tour of duty in Occupied Germany as a scientific consultant for the US. Department of Commerce, Schrodinger’s book was the only piece of scientific literature I took along. I was fascinated by What Is Life?, especially by Schroodinger’s assertion that it emerged from Delbruck’s general picture of the hereditary substance that living matter, while not eluding the presently known laws of physics, is likely to involve hitherto unknown “other laws of physics.” Schrodinger thought that there was no alternative to Delbruck’s molecular explanation of the gene, and that if it should turn out that the Delbruck picture failed, we would have to give up further attempts to understand the hereditary mechanism. T h e fabulous prospect that I might participate in turning u p “other laws of physics” so captivated me that I resolved to abandon physical chemistry and start studying genes once I had gotten my Ph.D. Delbruck, the young German physicist, had probably been drafted into the Wehrmacht and died on the Eastern Front, but perhaps there were people in the United States working along these lines whom I could join. While I was still stationed in Germany, I ran across an article in the January 20, 1947 issue of Time entitled “Tempest in the Cells,” which featured Spiegelman’s portrait, smiling, smoking a pipe. Time quoted the man I had met at Martha’s a year ago as having said that by starting to find out how genes d o their work, he had called into question a most sacrosanct doctrine in biology. Orthodox Mendelian genetics, as taught in every biology textbook, maintains that genes are the sole arbiters of heredity, but this view is incorrect because some cells, such as protozoa, yeasts, and cancer tissue, can change their chemical properties, thus defying their hereditary genes. Time reported that by labeling some of the protein molecules of the nucleus with radioactive phosphorus, Spiegelman and his colleague, Martin Kamen, had been able to provide direct support for Spiegel-

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man’s theory that the genes were sending out “partial replicas” of themselves-he called them “plasmagenes”-which enter the cytoplasm and multiply there independently. According to Spiegelman, it is not the genes that govern the cell’s global chemical properties, but the balance of plasmagenes, which can change under abnormal conditions that allow the favored plasmagenes to multiply, as rabbits once multiplied explosively in Australia after they had been introduced by Europeans. Time announced that Spiegelman had made an important breakthrough in the search for the physical nature of the gene, identifying it as a phosphorus-containing protein. (I didn’t know then, and Spiegelman didn’t seem to know, either, that Oswald Avery and his colleagues had shown three years earlier that the genes are made of DNA, rather than phosphorus-containing proteins.) This news story reinforced my resolve to switch into gene research after getting my Ph.D. If Spiegelman could get his picture into Time, why couldn’t I ? Not long after I got back to Illinois from Germany, I learned that, contrary to my lugubrious premonition, the young German physicistprotagonist of What Is Lqe? had not fallen on the Eastern Front, after all. Max Delbruck was alive and still wrestling with the problem of the physical nature of the gene-in America, to boot! So I applied for an NRC postdoctoral fellowship to help Delbruck find other laws of physics. I spent the summer of 1948 at Cold Spring Harbor, getting initiated into phage research, and then joined Delbruck as his first postdoctorate at Caltech in the fall. Spiegelman’s name came up occasionally in the seminars given at Cold Spring Harbor by resident researchers and drop-in visitors. More often than not, the references to him were critical or dismissive. Delbriick too gave a seminar, in which he presented his contribution to a recent conference in Paris, at which plasmagenes were a major topic of discussion. As I now learned, it was Tracy Sonneborn who had first put forward the plasmagene concept to account for his findings that the surface of Paramecium can change from one hereditarily stable structure to another without undergoing any gene mutation in the cell nucleus. Spiegelman had merely extended Sonneborn’s idea to the phenomenon of enzyme adaptation in yeast, after he had abandoned Yudkin’s hypothesis of the substrate-induced stabilization of unstable enzymes. In his talk, Delbruck pointed out that while he had nothing against plasmagenes, Sonnenborn’s findings could be explained also in an entirely different way, without invoking any genetic mechanisms-nuclear or cytoplasmic-at all: Suppose that there are two possible chemical pathways in the cell, one leading from substance a l to a3 via a2 and the

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other from substance b, to b, via b,, and that a2 inhibits the conversion of b, to b,, and b, inhibits the conversion of a , to a,. Then the cell can flip-flop between two mutually exclusive, alternative stable states, in one of which only as, and in the other of which only b,, is formed. As is usually the case, whenever two reasonable, internally coherent, alternative hypotheses are put forward to explain some biologic phenomenon, both will eventually be found to be applicable in one system or another; and so it turned out for Sonneborn’s plasmagenes and Delbruck’s chemical flip-flop scheme. Some types of cells do contain plasmagene-like entities endowed with genetic continuity in their cytoplasm, but many others undergo stable phenotypic changes, including those that Spiegelman had investigated, by flip-flop switching. 1 saw Spiegelman again in the spring of 1949, when Delbruck brought him into my lab in Pasadena and asked me to tell him what I was doing. Naturally, he didn’t remember me from our meeting at Martha’s party, and he didn’t seem terribly interested in my not exactly world-shaking phage experiments. Afterward, Delbruck told me that Spiegelman was looking for a job and that he had dropped in to ask whether there was a chance of his getting one at Caltech. Delbruck didn’t encourage him, and in the following year, Spiegelman was appointed Professor of Microbiology at my own Alma Mater, Illinois, where he stayed for nearly 20 years, until his final move to Columbia in 1969. My next encounter with him was in the spring of 1952 in Paris at the Pasteur Institute where, in the laboratory of Andre Lwoff, I was about to complete the last postdoctoral NRC fellowship of my Wanderjahre. He was still studying enzyme adaptation-soon to be rechristened “enzyme induction” in a manifesto cosigned by its half-dozen leading students, including Spiegelman-but in the meanwhile he had switched from yeast to bacteria as his experimental material. In making this change, he was no doubt motivated by the declining fortunes of the plasmagene idea as an explanation for enzyme adaptation and by the success of Jacques Monod’s recent studies at Pasteur of the inducible P-galactosidase of Escherichia coli, which bid fair laying the experimental and conceptual foundations for the analysis of the control of gene expression. Unfortunately for Spiegelman, this brought him into direct competition with Monod, another Faustian Man for whom he was no match. Monod was not only brilliant but had plenty of Sitzpeisch. I attended the seminar that Spiegelman gave at Pasteur on his work on enzyme induction, of which Francois Jacob gave an account in his autobiography, The Statue Within. Earlier in his story, Jacob caricatured Spiegelman as a “short, stocky . . . featherweight boxer,” but later lampooned his seminar as a corridu, with Spiegelman as toro, the “imperturb-

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able” Lwoff as presidente, the “elegant” Monod as matador, and with Roger Stanier (the Canadian “debonair giant”), Melvin Cohn (the “uninhibited young American”), and Martin Pollock (the Englishman with the “handsome, insolently aristocratic” face) as banderilleros, while the crowd of aficionados of enzyme induction shouts “ole‘!” at each pass. Nine years later, Jacob and Monod provided the basic framework for our present understanding of the regulation of gene expression in terms of their operon theory. In devising their theory, they had drawn mainly on their own work on the genetics and physiology of P-galactosidase induction, but they took into account also a broad panorama of relevant findings made by others, few of which, however, had been contributed by Spiegelman. A critical feature of the operon was Jacob and Monod’s postulation of a metabolically unstable messenger RNA onto which the DNA nucleotide base sequence of the gene to be expressed is transcribed for later translation into a polypeptide with the specific amino acid sequence encoded in the gene being expressed. It was by providing the experimental means for identifying and characterizing the putative messenger RNA that Spiegelman made the first of his two major, enduring scientific contributions: He forged one of the main tools-the specific RNA-DNA molecular hybridization technique-that would lead to the hegemony of Imperial Molecular Biology over the late 20th-century life sciences. T h e most important use that Spiegelman himself made of his RNADNA hybridization method was to demonstrate in 1963 that, in accord with what Jacob and Monod had assumed all along on first principles, only one of the two complementary DNA strands in the domain of any gene is actually transcribed into messenger RNA. Two years later, he developed a quantitative assay of the amount of DNA-hybridizable RNA present in an extract. By means of this procedure, he showed that the number of nucleolus organizer genes known to be carried by different mutant strains of Drosophila is equal to the number of DNA sectors per genome capable of forming hybrids with ribosomal RNA. According to the Science Citation Index, the paper in which he presented this quantitative method would be cited 1718 times in the next 17 years. T h e second of Spiegelman’s two major scientific contributions of lasting value soon followed his development of the DNA-RNA hybridization method, when he joined the molecular-biologic Gold Rush to uncover the replication mechanism of phages that carry RNA rather than DNA as their genetic material. The first RNA phage had then been recently discovered in New York City sewage by T. Loeb and N. D. Zinder, and indirect experiments had soon shown that replication of its RNA is

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catalyzed by an enzyme-RNA replicase-which is different from the bacterial RNA polymerase, which catalyzes the transcription of the host cell DNA into messenger RNA. Because of the intrinsic instability of the RNA replicase protein, no one had succeeded in purifying the active enzyme, until Spiegelman managed to do so by starting with an independently isolated RNA phage, which closely resembled Loeb and Zinder’s original strain, except that its replicase was much more stable. Spiegelman was then able to prepare reaction mixtures, containing only the purified RNA replicase and the four ribonucleoside triphosphates, in which RNA genomes extracted from intact phage particles, or virions, would replicate autocatalytically to yield infective progeny genomes. Upon prolonged maintenance of this in vitro replication process, the progeny RNA genomes became eventually reduced to less than half their original chain length. Spiegelman recognized that this decreased polynucleotide chain length represented a natural selection of fasterreplicating, shorter genomes from which all nucleotide sequences other than those necessary for the replicase-catalyzed in vitro replication process had been spontaneously deleted. This intriguing phenomenon seemed to be of little interest to Anglo-American students of evolution, but it made a big impact in Germany, where an institute devoted mainly to following up Spiegelman’s discovery of molecular Darwinism in the test tube was recently opened. Spiegelman left Illinois in 1969 to become Director of the Cancer Research Center at Columbia University in his native New York City. He was convinced that RNA tumor viruses were a major cause o f human cancer, and he intended to set u p research projects at Columbia aimed at cancer prevention and cure based on the insights into RNA replication provided by the study of RNA phages. For instance, he planned to stop the progress of human cancers by devising a “replicating magic bullet”an RNA molecule with no function other than self-replication and yet so avid for the tumor virus RNA replicase that, when injected into a cancerous cell, it would block all of the viral enzyme present and thus halt the replication of tumor virus RNA. Within a year, Howard Temin had shown, however, that, unlike the RNA phages, the classical avian RNA tumor virus discovered by Peyton Rous in 191 1 replicates indirectly, via a DNA intermediate whose formation on the RNA template provided by the infecting virion is catalyzed by a reverse trunscriptase. Spiegelman, therefore, abandoned his original strategic plan and, in the remaining 14 years of his life, focused his research efforts at Columbia on the detection and functional inhibition of reverse transcriptase in human cancer tissue. All the while, he rejected as highly improbable the rival theory of carcinogenesis, according

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to which tumors arise by the spontaneous or carcinogen-induced activation of normally dormant cancer-causing genes, or oncogenes. Despite the dozen or more papers on cancer published each year by Spiegelman and his many collaborators at Columbia, his reputation among the leaders of his new field of molecular oncology fared no better than it had earlier among the founders of “classical” molecular biology. In his autobiographical memoir, The Atheist and the Holy City (MIT Press, 1990), the Swedish tumor biologist, George Klein, quoted a vignette about Spiegelman by an unnamed colleague: “Usually you find Sol standing on the top of a mountain peak. He is waving with both arms and shouting so loudly that it can be heard throughout the valley: ‘Come up quickly, have a look at what I can see from here!’ Crowds of people begin rushing u p the mountainside. When they arrive, breathless, and stand beside Sol, they see nothing of what he claims to see. But nobody denies that the view is highly interesting.”

Spiegelman’s scientific life embodied the elements of true tragedy. On the one hand, the ideas he kept putting forward, which he wanted the coryphees of his discipline to acclaim as great and original, turned out to be neither original nor correct, beginning with his plasmagene theory of enzyme adaptation and ending with his advocacy of the (now generally discounted) paramount role of reverse transcriptase in human carcinogenesis and his rejection of the (now generally accepted) oncogene theory. On the other hand, the one contribution of extraordinary, everlasting importance he did make, n2:mely the development of the specific RNADNA molecular hybridization method, was not the kind of mindbending discovery for which Spiegelman wanted to be known. It was left to others to make use of this technique for the gene cloning procedures that revolutionized biologic research in the 1980s. T h e irony of the final denouement of his struggles-public honor and acclaim in the face of his failure to reach the high standards of achievement he had set for himself-was not lost on Spiegelman, and in 1983, age 67, the ebullient, feather-weight boxer died as an embittered scientific misanthrope. T h e tragedy of Spiegelman’s career may be hard to fathom for scientists not yet into their fifties, because Faustian Man driven by the Will to Power-in Spiegelman’s case not only the will to gain mastery over Nature but also the will to gain acknowledgment by those who were themselves masters that he was one of them-has largely disappeared from the contemporary scene. Likewise, the dominant role-model, three-star scientific personalities of unimpeachable integrity, of which Niels Bohr, Linus Pauling, and Max Delbruck were exemplars, have disappeared.

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Once upon a time, they set the standards of excellence, and their approval was worth more affectively to their colleagues than prizes and public fame. Had Spiegelman, with his rich intellectual endowment, been born 35 years later, he would now probably be, like many of the leaders of the second generation of molecular biologists, a multimillionaire partner in a major biotechnology company, secure in the feeling of his success and free of the pressure to win approval of no longer extant rhadamanthine arbiters of scientific quality. I regret Spiegelman’s passing, because he was an integral part of a bygone scientific scene which-warts and all-I preferred to what has now replaced it.

GROWTH DYSREGULATlON IN CANCER CELLS Arthur B. Pardee’ Department of Biological Chemistry and Molecular Pharmacology, Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 021 15

I. 11. 111. 1V. V. VI. VII. VIII. IX

X. XI. XII. XIII. XIV.

Introduction College and Graduate Training Cancer Research in 1950 Regulatory Processes Regulations in Higher Organisms Cancer and the Cell Surface T h e Restriction Point Hypothesis A Short-Lived Regulatory Protein Transcription from the Promoter Region of the Mouse Thymidine Kinase (TK) Gene Yi Complexes Cyclin E Is a Candidate R-Point Protein Cyclin Changes in Breast Cells Breast Cancer in Viva Future Problems References

1. Introduction This article recalls nearly a half century of my memories about the research area in which I work-mechanisms of growth regulation that are lost in cancer-as I saw the development from a primitive state in 1949 to 1994. This presentation is chronological although my path was not straight. Yet one discovery sometimes surprisingly led to another. According to the editors’ proposal, this is a personal overview and not a review in depth, and so it refers only to papers from my laboratory. Many relevant related articles can be found in these references. Regulation is at the heart of biology. T h e general question that has excited my imagination and focused my research is regulation of biologic processes in health and disease. My aim is to identify and characterize basic mechanisms, and how their derangements are responsible for defective growth of cancer cells. As expected, the answers are not simple and much is still to be learned. ’Dedicated to my mother, Elizabeth Beck Pardee, 1898-1942.

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A most important characteristic of cancer cells is their growth at the wrong places and times. “Cancer is a wound that does not heal” (quoted from Dr. Harold Dvorak). A wound generates new cells until it has healed. But in cancer, cell growth does not stop. Mutations of a few oncogenes and tumor suppressor genes create disharmonies in biochemical expression that upset cellular balances and the control of cell growth by altering various parts of the growth regulatory mechanism. What goes wrong so that cells continue to grow in wrong times and places? We now have evidence to support a reasonable hypothesis of growth regulation which I am going to discuss, including some of our most recent investigations. I hope these will provide a basis for further clarification, on which to build treatment modalities. To summarize, much evidence now supports our early hypothesis that a major control of normal animal cell proliferation is exerted at the restriction point (R-point) located in the late G1 phase of the cell cycle. This control mechanism limits the sudden onset of DNA synthesis and its related biochemical processes (Pardee, 1974). The R-point process is deregulated in cancer cells (Pardee, 1987). Cyclins, cyclin dependent kinases (cdks), retinoblastoma (pRB), and other proteins have recently been implicated in the R-point process. Deranged expression of cyclins, particularly of cyclin E, is a major contributor to defective growth control of cancer (Dou et al., 1993; Keyomarsi and Pardee, 1993).

II. College and Graduate Training

My bachelors degree at the University of California, Berkeley in 1942 was in chemistry and physical sciences. This training in thinking quantitatively about molecules and kinetics was a fine preparation for biochemical research. Regarding biochemistry, “tierchimie is schmierchimie” was the viewpoint of‘ my chemistry professors. Nevertheless, I did the unheard of by taking a biochemistry course in 194 1. T h e laboratory exercises involved blood and urine analyses. Structures of small molecules were described, but metabolism was still generally primitive. Pathways of glycolysis and the Krebs cycle were worked out but little was known about most biosynthetic pathways. Structures of macromolecules were a mystery; no protein had been sequenced and nucleic acids were seriously believed to be tetranucleotides. Genetic concepts regarding cancer were absent. My graduate work from 1942-1947, with an intervening period of war work, was done in Linus Pauling’s group at CalTech. He was trying to bring chemistry to immunology, through studying antigen-antibody reactions in vitro. However, little connection was made between chemis-

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try and other problems of biology. I took a plant biology course, and learned that one cannot store plant extracts overnight at room temperature, as one does chemical reactions. They become moldy! Ill. Cancer Research in 1950 In 1947 I decided to start cancer research. To set the stage, a main idea at mid-century was that the disease is caused by a defective metabolic process, a concept consistent with biochemical research at that time. Otto Warburg had demonstrated that glycolysis and respiration are uncoupled in tumors; this was one of the few positive clues. Tumors were found to progress through a series of steps, going from bad to worse, and so one expected to find several changes from normal tissues. Nonetheless, Jesse Greenstein and others compared numerous enzymatic reactions in normal and cancer tissues but could not find a definitive difference. Treatment depended primarily on surgery; antineoplastic drugs were not used until a few years later. Hormones were involved in some tumors, which led to Huggins’ hormone therapy for prostate cancer. I had to make a choice between studying enzymology and hormones, and decided to work with Van Potter at Wisconsin on enzymology. Fortunately I got one of the first Merck fellowships; $3000/year was then easy for a young couple to live on! Dr. Potter was among the first to study metabolism using ground-up tissues, made with the Potter homogenizer. He focused on respiration and the Krebs cycle. Conditions were primitive by today’s standards. For example, biochemical supply companies did not exist, so we made our own ATP, NAD, cytochrome C, etc. Of course, no kits were available. I got a good foundation in cancer research and biochemistry, and published several papers on my studies. With Charlie Heidelberger we showed that acetate is used by the Krebs cycle, representing an early application of labeled compounds (Pardee et al., 1950). Investigating control of Krebs cycle events (Pardee and Potter, 1948) sowed the seeds for discovering feedback inhibition (see later). But, I did not discover anything revealing about cancer after two years. I concluded that the subject was not ready for a biochemical breakthrough, and that more basic knowledge was needed. So I studied bacterial metabolic regulation for the next 20 years, and discovered several processes that later applied to cancer. IV. Regulatory Processes Before the mid- 1950s, biochemistry was mainly dedicated to discovering metabolic pathways, a great achievement now summarized in the

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familiar metabolic maps. However, they provide a static picture. What was the density of travel along these metabolic roads and byways? By what mechanisms is this flow regulated? The activities of enzymes were mainly ascribed to substrate concentrations, according to the MichaelisMenten equation, and to the action of a few naturally existing inhibitors and activators. The main mechanisms of metabolic regulation were discovered in the mid-1950s: gene induction and repression, noncovalent feedback inhibition of enzymes, leading to allostery, and covalent protein modification such as phosphorylation. Many of these were found with the bacterium Escherzchza coli. The general principle of regulated cell signaling is that there are three parts: the extracellular signal, an intermediary signal transducing system, and the target. This intermediate part is a regulator which looks two ways, receiving the signal and transferring its message to modify the proper molecular functions. In research I did at the Pasteur Institute with Jacques Monod and Francois Jacob in the late 1950s we asked the question: What is the genetic-biochemical mechanism by which a simple bacterium makes an enzyme only when it is needed for growth? Lactose must be hydrolyzed by P-galactosidase before E . coli can use it as a source of carbon and energy. Remarkably, the bacteria make this enzyme only when lactose is the best available carbon source, and not otherwise. T h e three components for regulating this enzyme’s production are the external signal lactose, the gene coding for the enzyme which is the target, and the signal transducer. We discovered that signal transduction is carried o u t by a novel kind of protein which we named the repressor (Pardee et al., 1959). This repressor attaches to a special regulatory region of P-galactosidase DNA, and thereby blocks the gene’s transcription. This binding is released when lactose combines with the repressor, and the gene initiates production of the enzyme within minutes. Thus, not only does this system produce the enzyme when it is useful, but it stops production when lactose is used up so that the signal disappears. Biologic processes are regulated at many different levels, of which gene activation is just one. Another is inhibition of activity of an enzyme after it has been made. Its catalytic activity can be turned off and on by signaling molecules. One such mode of regulation, feedback inhibition, senses when a sequence of reactions overproduces a product, and adjusts the overall process accordingly by changing the rate of the beginning step. Feedback of biochemical sequences was described in both Edwin Umbarger’s laboratory and mine in the mid-1950s. Working on the pathway for synthesis of pyrimidines, which requires more than six steps, Richard Yates and I found that a final product inhibits the first

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enzyme in the entire sequence. If this end product is overproduced, it shuts down the whole pathway at its start, providing a very tight control and avoiding wasteful overproduction of all the products (Yates and Pardee, 1956). This explains how bacteria growing under variable conditions produce the molecules they need in just the right amounts to make a new cell. What is remarkable about this system is that inhibitory end product and substrate molecules d o not look at all alike. However, inhibitors and substrates usually have very similar structures because they compete for the same enzyme binding site. John Gerhart and I solved this problem of apparent nonspecificity. T h e enzyme has two interacting protein parts, one catalytic and the other regulatory. Substrate molecules bind specifically to the catalytic part, and end product inhibitor binds to the regulatory part (Gerhart and Pardee, 1956). Thus, this pattern of linked regulatory and catalytic components is the same at the very different biochemical levels of gene and enzyme. One of Jacques Monod’s greatest insights was to recognize the central role of such dual “allosteric” sites in a wide variety of regulated biologic processes. Louise Prestidge and I at this time also found links between syntheses of the major macromolecules. Bacterial nucleic acid synthesis depends on a constant supply of amino acids for continual protein synthesis (Pardee, 1956), later named stringent control. Conversely, protein synthesis requires continual nucleic acid synthesis (Pardee and Prestidge, 1956), a forerunner of the discovery that mRNA synthesis is required to make proteins. Other areas of regulation on which my students and I worked during the 1950s and 1960s include a demonstration of inducibility of active transport systems (Pardee, 1957), identification and purification of the first protein involved in a transport process (Pardee, 1968), and demonstrations of bacterial cell cycle events including periodic syntheses of enzymes and macromolecules (Abbo and Pardee, 1960; Masters and Pardee, 1965; Gudas and Pardee, 1974; Smith and Pardee, 1970). V. Regulations in Higher Organisms

A great principle is that the same basic mechanisms apply to all cells, even when established with a simple bacterium. Jacques Monod said “What is true for E . coli is true for the elephant.” But not vice versa: elephant cells use these same principles but their control machinery is vastly more complex than that of a bacterium. This similarity of biochemical regulatory mechanisms is remarkable because the role of regulation in multicellular organisms is to coordinate 1012 cells; defective control in a single cell can produce cancer. In contrast, the function of

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regulation for bacteria is to permit as rapid growth as possible. Bacteria are asocial cells; they grow like uncontrolled cancers! VI. Cancer and the Cell Surface

By 1960, some major steps in the development of current ideas were discoveries of DNA structure, mRNA, systems for synthesis of RNA and protein, all of molecular biology, signal transduction pathways, recognition of instability of RNA and of proteins (turnover), and ability to culture mammalian cells. In addition, there had been major progress in techniques of cancer research: recognition that mutations and genetic changes are at the roots of the disease, cancer viruses, ability to culture cells in vitro, and the first successful chemotherapy, with folate analogues. The molecular basis of regulation had become an active subject for investigation (Pardee, 1959). What brought me back to cancer research was an invitation to a meeting in Peru and Colombia in 1964, kindly arranged by Van Potter. I wrote a paper for it, suggesting that changes at the cell surface are responsible for cancer (Pardee, 1963), which was far ahead of its time because growth factor receptors had hardly been discovered. After this meeting I started to work on the surfaces of normal and cancer cells. These studies led to discoveries by David Foster, Dennis Cunningham, and me on the alterations of transport in cancer cells (Foster and Pardee, 1969; Cunningham and Pardee, 1969), of phospholipid metabolism in membranes (Cunningham, 1972), and to Max Burger and Allen Goldberg’s investigations of altered lectin binding (Burger and Goldberg, 1966). VII. The Restriction Point Hypothesis I decided in 197 1 that it was time to tackle a major problem that I was not confident I could solve. T h e question is what niechanisms regulate the growth of normal cells that are upset in cancer cells. What is the molecular basis and biochemistry behind the poorly controlled growth of cancer cells? To gain a background in this subject, I took a sabbatical with Michael Stoker at the Imperial Cancer Research Fund in London. Relatively little was known about the kinetics of cell growth at that time. Howard and Pelc described the four phases of the cell cycle. After a cell divides and forms two cells, each daughter cell starts around the cycle to make DNA in S phase and then goes through its mitosis and division. Mammalian cells in an adult are not growing most o f t h e time; their intermittent growth replaces lost cells. T h e growth of a tissue de-

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pends on the net fraction of cells that are proliferating. If all cells were growing and doubled every day, and no cells died, each of us would be larger than the volume of the earth in a couple of months! It is thus important to understand the mechanism that switches cells between the resting and growing states according to the need for new cells. Renato Baserga was among the few scientists who performed pioneering studies of metabolism of cells in culture. My conception of the R-point (Pardee, 1974) was the critical turning point for me. To consider cell growth control, I started with the cell cycle. The hypothesis is that a cell has to make a decision prior to entering S phase as to whether to go on and synthesize DNA and to produce two cells, o r to enter into the quiescent state. This decision is important for cell proliferation, and is determined by external conditions such as interactions with the growth factor IGF-1 (Yang and Pardee, 1986). One cell can communicate with others through circulating protein growth factors. These proteins announce their arrival at the outer membrane of a target cell by binding to specific receptors, thereby “ringing the doorbell” to tell a chain of second messenger proteins within that cell to carry on the message. A subset of regulatory genes is the target for the extracellular factors that determine whether a cell grows. This communicating mechanism is altered by mutation in some cancer cells, leading to defective growth control, and can make a cell grow when and where it should not. T h e first important control determines emergence from quiescence, and the second late in G1 acts at the R-point to control onset of DNA synthesis. Once a cell has gotten beyond the R-point, the rest of the cycle is not tightly controlled. It’s easier for a cancer cell than for a normal cell to get beyond this R-point, the point of no return. Lee Hartwell conceived of the same kind of regulation for yeast at this same time, based on his experiments with cell cycle defective mutants, and named it START. VIII. A Short-Lived Regulatory Protein

We proposed that the G1 regulatory machinery is unstable (Rossow et al., 1979). This instability would provide the basis for a good mechanism to control cell division. A protein that must accumulate but is constantly breaking down provides a much sharper control than a stable protein which finally accumulates to the critical amount. It’s like one’s bank account: the balance is very precisely determined by input versus expenditure. This plus/minus o r push/pull kind of regulatory mechanism is very effective, an example of the YinIYang concept of balanced opposing

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forces which is widely found in nature. Balance is very important for controlled growth, and is easily upset in cancer cells. The idea is that DNA synthesis starts when an unstable protein accumulates and reaches a critical level during GI phase. If one adds an inhibitor of protein synthesis so that no new protein is made, and also any previously synthesized unstable proteins are degraded and disappear. Thus, the amount of such proteins will decrease while total protein synthesis is inhibited. After the inhibitor is removed, synthesis resumes and eventually the labile protein reaches the critical level. Extra time is required, however, for resynthesizing the protein, whose duration is related to the length of inhibition. We calculated that the R-point protein in 3T3 cells has a half-life of about 2.5 hours. Strikingly, when Judith Campisi did this same experiment with benzo[alpyrene-transformed tumorigenic 3T3 cells they moved forward to S as soon as the inhibitor was removed (Campisi et al., 1982), without apparent delay. The R-point protein is either stable in these tumor cells, or there is so much of it that its decay does not limit cell progression. This hypothesis led us to seek such a labile G1 protein by using twodimensional gel electrophoresis. Many experiments by Bob Croy using this technique revealed only one protein, of molecular weight 68 kDa, that satisfied all criteria for a R-point protein: made in G1, labile in the untransformed cells, stable in the transformed cells (Croy and Pardee, 1983). Thus, a protein with such unusual properties does exist. However, this search was stopped because there was so little p68 protein that we were unable to determine its properties o r make an antibody. IX. Transcription from the Promoter Region of the Mouse Thyrnidine Kinase (TK) Gene

It was therefore time to try a different approach to the problem of regulation of the GI/S transition. A number of molecular events occur concomitantly when DNA synthesis starts, such as production of histones and of several enzymes that are under the same general control by growth factors and inhibitors as is DNA synthesis. Our novel idea was to use one of these events as a marker of the transition between GI and the DNA synthetic parts of the cell cycle and to determine its molecular biology. The great advantage of studying such a single event is its relative simplicity. One can immediately proceed to molecular genetics, whereas the overall synthesis of DNA is very complicated indeed. We investigated the cell cycle regulation of T K , and asked how that regulation is affected in tumor cells (Coppock and Pardee, 1985). This approach led to our

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analysis of the molecular events that regulate the entry of cells into S phase. Jean Gudas, Glenn Knight, and I selected mouse T K as our marker because a good deal was known about it. Its activity appears at the same time as DNA synthesis in synchronized cells. Its mRNA increases 50-fold a couple of hours before the protein, at about the R-point, so we studied this earlier event (Gudas et al., 1988). The upstream mouse T K region was sequenced by Jean Gudas and Judy Fridovich-Keil. A minimal region at the 5'-end of the gene is sufficient to direct its efficient G 1/S-phase specific regulated expression (Pardee et al., 1959). This 5' region regulates induction of the S-phase events. T h e point of greatest interest is that this region has three nucleotide consensus sequence sites to which proteins bind: MT1, MT2, and M T 3 (Gudas et al., 1992). Recently we have shown by mutational analysis that these three sites have different functions in the G1 to S progression (Fridovich-Keil et al., 1993). MT1 is essential for T K transcription because its mutation in a construct abolishes expression of a downstream reporter gene. Mutation of the MT2 sequence, which binds E2F, causes constitutively expressed transcription, indicating a repressing function of E2F. MT3 mutations had less effect, but may increase the basal level of T K expression in GO cells. X. Yi Complexes

Qing-Ping Dou identified the protein complexes called Yi that bind to the M T sequences in the murine T K promoter and regulate this enzyme's production (Dou et al., 1991, 1992, 1994). After normal cells emerge from quiescence, a new complex named Yi2 appears shortly before DNA synthesis starts, just what we would expect if it is related to the R point. When a protein fraction from nuclear extracts that does not form Yi2 complex is incubated with ATP, the complex appears, which suggests that an ATP-dependent kinase in these cells can activate assembly of Yi2. An SP1 binding site located in MT1 is constitutive (i.e., not cell cycle dependent), and serves as a control to show that not every binding is changing. In 3T3 cells the Yi2 complex only appeared in S-phase but not in G1 or GO, however, David Bradley found a very different picture in BPA3l transformed cells; this complex was seen during G1 phase (Bradley et al., 1990). Furthermore, expression of a reporter gene (P-globin) under control of the T K promoter was constitutive in these transformed cells, suggesting that their cell cycle related transcription is deranged. In spite

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of this, T K mRNA did not appear until the end of G1. T h e basis for this constitutive transcription but absence of mRNA is loss of the T K premRNA by post-transcriptional processes (Bradley et al., submitted for publication). This result is consistent with an important physiologic role of transcription from T K promoter-like motifs. MT2 and its complexes have similar kinetic and mobility properties to those of the several E2F complexes discovered by Joseph Nevins’ laboratory from studies with adenovirus-infected cells. Dr. Dou identified GO/G1 and S complexes that bind to the very similar MT2 and E2F motifs and contain a murine E2F protein (Dou et al., 1994). These complexes also contain p107 protein. Another complex appears which contains cyclins E and cdk2 as the cells approach S. In S phase the E2F-S complex also contains cyclin A but not cyclin E. Some of the proteins in these complexes have been identified, including the retinoblastoma protein and its related p107, a DNA binding E2F protein, cyclins A and E, and cdc2 and cdk2 kinases. These complexes can be converted from one to the other, consistent with change from a negatively G1-complex to a positively acting S-complex regulating start of DNA synthesis. MT3 binds quite different complexes than does MT2. One is an immediate-early (GO/Gl) complex we named TKE, and also a Yil complex in G1. Genes coding for two proteins that bind to M T 3 were isolated by Anne Crozat and Gyongyi Molnar, using expression cloning (Crozat et al., 1994). One is the Egr-1 early response gene. T h e other is novel and codes for a protein of about 90 kDa whose function is under investigation. XI. Cyclin E Is a Candidate R-Point Protein

Cyclins are prime cell cycle regulators central to the control of major checkpoints in eukaryotic cells. Originally discovered in eggs of sea urchins and clams, cyclins are now found in mammalian cells and yeasts as well. There are quite a few members of this protein family. These proteins are named cyclins because they increase at specific times during each cycle of cell division. Starting from a resting state and through a cell cycle, different cyclins appear sequentially. T h e first are the D cyclins, Cyclin E appears later in G1, followed by A and then in G2 by B. Cyclins, which were discovered by Tim Hunt and Joan Ruderman, regulate the cdk kinases (first discovered by Paul Nurse) that put phosphates onto gene regulating proteins such as pRb. This process thereby modifies gene functions and G 1 progression. Antibodies against cdk2 and cdc2 show that activities related to these kinases appear at the times when kinases increase in the cycle. Specific complexes containing each of

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these cyclins were obtained by imrnunoprecipitation with the antibodies against them. Their associated kinase activities (using H- 1 histone as an artificial substrate) were determined and showed that the antibody to cyclin E did not precipitate any activity from resting cells nor until 12 hours of growth when DNA synthesis had just started. Cyclin A appeared a little later than 12 hours. Cyclin B2 antibody precipitated a kinase activity much later, at around the time of mitosis (Keyomarsi et al., 1993). To determine if cyclin E behaves according to the three conditions that define the putative restriction point protein, Dr. Dou carried out the following tests (Dou et al., 1993). The first criterion for the B protein is its activation in late G1 in nontransformed cells. He determined when cyclin E and cyclin A proteins appear in Gl/S. An antiserum to human cyclin E recognized two main bands by western blotting; one of which, p52, probably represents the mouse cyclin E protein since its accumulation correlates well with CycE-H1K activity during the cell cycle (see later). A mouse p55 protein identified by an antiserum to human cyclin A was not present in GO cells, and increased in S phase, coincident with CycE-H 1K activity. Antibodies against Cdk2 and Cdc2 showed that the corresponding proteins were not strongly cell cycle regulated. These data suggest that accumulation of cyclin E and cyclin A proteins at G l / S regulate activities of their respective kinase activities. Dr. Dou also used antibodies to precipitate the corresponding cyclin and cdk proteins from synchronized cells, and measured H 1 kinase activities in the precipitates. These activities were low in GO, and increased at different times of the cell cycle. CycE-H 1K and cdk2-H 1K activities started to increase around the R point (before 12 hours). CycAH 1K and cdc2-H 1K gradually increased after 12 hours, parallel to the kinetics of DNA synthesis. These timing data are most consistent with cyclin E being the R-protein. Test 2 for an R-point candidate is that in nontransformed cells there should be a delay before the labile protein is restored to its former level after a period of inhibited protein synthesis. The R-protein should be unstable and so the extra time is required for its resynthesis. Anticyclin E immunodetected p52 protein extracted from nontransformed A3 1 cells was very low for 12 hours in G1 and during inhibition with cycloheximide (CHX) for 5 hours. It increased only several hours after inhibition was released. Cyclin A p55 protein was almost undetectable during inhibition, and gradually increased several hours after removing CHX. In contrast, cdk2 protein was relatively constitutive during this process. We expected CycE-H 1K and CycA-H 1K activities to behave similarly to their cyclins during this time. Indeed, after several hours of inhibition

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we observed two- to four-fold decreases in these H 1K kinases. Their activities started to recover only several hours after CHX was removed. Therefore, cyclin proteins are probably the labile components responsible for cycle dependent regulation of cdk kinases, and these results also are consistent with one of these cyclins being the R-point protein. Test 3 for the R-point protein is its fast recovery in transformed cells from CHX inhibition. We expected that cyclin-associated H 1K from BPA3l cells would show little delay in appearance, or else it would remain at a high level after the pulse of CHX. CycE-H 1K and CycA-H 1K at 12 hours were two- to three-fold higher in BPA3l than in A31 cells. CHX reduced these H l K activities in both cells, but it remained higher in BPA3 1 than in A3 1 cells. Both activities recovered to high levels much earlier in BPA3 1 cells than in A3 1 cells. From these cell cycle pulse-chase experiments, we conclude that accumulation of either cyclin E and cyclin A proteins and of their dependent kinases satisfies all of the three properties which define the R-point protein. Therefore these cyclins may be proliferation-regulating in the normal cells, a process that is deranged in tumor cells. XII. Cyclin Changes in Breast Cells

Our first approach in determining whether, and which, cyclins are altered in human cancer was to investigate their properties, production, and appearance in proliferating cells during the cell cycle. Khandan Keyomarsi found several types of deranged expression of cyclins in these proliferating breast tumor cell lines versus normal human breast cells (Keyomarsi and Pardee, 1993). Until very recently it was not possible to grow these cells under comparable conditions. Thanks to cell biologic studies by Ruth Sager we were able to grow them in the same medium so as to permit their meaningful comparison. Several changes that were seen in all o r most of the cancer cells include: (1) eightfold amplification of cyclin E gene in one tumor line, 64 fold overexpression of its mRNA, and altered expression of its protein; (2) deranged expression of cyclin E protein in all (10 of 10) tumor cell lines studied; (3) increased cyclin mRNA stability; (4) general overexpression of mitotic cyclins and of cdk RNAs and proteins in 9 of 10 tumor lines; and (5) deranged order of appearance of cyclins in synchronized tumor versus normal cells, in which mitotic cyclins appeared prior to G1 cyclins. These multiple changes in cyclin expression in human breast cancer cells suggest that cyclins may be a new class of growth dysregulating oncogenes. They may function redundantly in cancer, with overexpression andlor amplification of one cyclin replacing the cell cycle functions of others.

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When western immunologic blots of cyclins A and B, and also of the related kinase cdc2 from normal human breast tissue, were compared to those from three tumor specimens, the cyclins were expressed very differently. One also sees other dramatic differences in their mRNA expressions in the cultured normal and tumor cells. The cyclin E proteins that are recognized by antibodies are different. In normal cells there is one protein with a molecular weight of about 50,000 Da. In various tumors there are greater or smaller amounts of this protein, and others are present that have different molecular weights. Total cyclindependent H1 kinase activity is elevated in the tumor cells. Thus, in breast tumor specimens a dramatic alteration in cyclin protein E was very often observed. T h e other cyclins also changed, though less dramatically. Amounts of mRNAs of cyclins A and B, which are normally expressed in G2, are increased in many tumors. This is odd because one would expect only G1 cyclins to be changed since growth regulation is in G1. Cyclin changes in synchronized cells were also studied. A normal breast epithelial cell and a breast cancer cell did not show dramatic changes in cyclin E mRNA when compared for their time courses of cyclin expression. In the normal cells, G1 events are mostly related to D and E cyclins. In contrast, we saw a different picture in the tumor cell line. T h e most striking difference is that the A and B cyclins appeared late in the normal cells, but appeared early in the tumor cells. T h e B cyclins which normally are made in preparation for mitosis, now appear early in G1, even before DNA synthesis starts. This upsetting of the pattern of cyclin expression in this tumor cell line could be very important, because a G2 cyclin might be able to replace a G1 cyclin functionally, and give a faulty signal for initiating cell proliferation. Cancer is a progressive disease. We speculate that cyclin patterns change from bad to worse as cancers go on. The development of a mechanism for aberrant expression of cyclins in breast cancer is probably a multistep event. We speculate that cyclin mRNA is originally unstable, but becomes more stable in the early stages of breast cancer. Therefore, it is easier for a cell to accumulate it. At a later stage another change increases its production. Even later, some non-G1 cyclins appear at the wrong time in the cycle and trigger DNA replication. Finally, cyclins are grossly overproduced and thereby take over the system. The cyclin E gene is amplified in some cancer cells, which speeds the process even more. These experiments with cyclins and their dependent kinases are consistent with the cell biology in telling us that one can explain the ability of tumor cells to grow aberrantly by accumulating cyclin changes, probably

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at first of cyclins E and A, which thereby become easier to produce in tumor cells than in normal cells. XIII. Breast Cancer in Vivo

Khandan Keyomarsi has recently examined cyclins in cancer biopsy specimens (Keyomarsi et al., 1994). T h e normal mammary epithelial specimens used for comparison were obtained from nearby tissue. She found in nearly all cancer specimens that cyclin E in particular is grossly altered in activity, amount, and structure. In addition, irrespective of their tumorigenicity potentials or estrogen-receptor status, she observed with antibodies overexpression of one, two, or all three cyclin E-like proteins ranging in size from 35 to 50 kDa, whereas in the normal tissues only one major protein of about 50 kDa was found. These results using breast cancer tissues and normal adjacent nontumor biopsy material are very encouraging to indicate that altered expression of cyclin E can also occur in vivo. Since a changed “picture” of cyclin E protein expression is observed in tumors, this cyclin might be used diagnostically or prognostically in breast cancer. XIV. Future Problems

There is much to be done (Pardee, 1989). Some other problems suggested by our studies include finding the components of Yi complexes, working back to earlier events that turn on cyclins, the involvement of D cyclins, and the relation of these T K related processes to the initiation of DNA synthesis. What is the role of all these kinases? What proteins are their targets? What proteins are phosphorylated and what are the consequences? Finally, I want to stress the importance of investigating the multiple molecular levels of regulation for complex processes such as cell growth, and the derangements that occur in cancer cells (Pardee, 1994). Our major research emphasis today is on regulating transcription by binding of transactivating proteins to promoter motifs. Another major focus is on the multiple roles of protein phosphorylations in signal transduct ion pathways. Evidence is strong, however, that major controls exist at numerous other molecular levels as well. These include pre-mRNA processing, pre-mRNA degradation, mRNA degradation, control of translation, permanent protein modifications, protein degradation, reversible covalent protein alterations, noncovalent protein interactions with small molecules and with other proteins, and effects of relocations within cell compartments. These various controls are exhibited in many biologic processes.

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Some general thoughts have guided our ongoing research studies. First, growth control depends primarily on external factors, such as growth factor proteins. At least two of these factors act sequentially in the G1 part of the cell cycle, one specifically prior to the restriction point during preparation for DNA synthesis. When we try to understand growth-controlling mechanisms, we have to think about the ability to prepare for DNA synthesis and what is needed for that. This regulatory process is mediated by an unstable protein with a short half-life, which must accumulate in sufficient quantity if DNA synthesis is to be initiated. That’s where G1 cyclins come into the picture. The underlying genetic and biochemical mechanism involves cyclins, and also cdk kinases, and pRB. These mechanisms are just beginning to be understood. T h e significance of our research is threefold. First, these studies are very relevant to cancer because they deal with defective growth control at the molecular level. We focus on the most critical event, namely preparation of cells for the onset of DNA synthesis. Normal and transformed cells differ in their ability to regulate this decision. Second, this research leads to new insights into mechanism for regulating gene expression in normal cells, providing results of broad applicability. Third, use of human epithelial cells, enzymes, genes, and drugs involved in therapy or diagnosis make these studies clinically relevant. ACKNOWLEDGMENTS T h e author thanks his many colleagues whose names are in these references for their contributions to this research. T h e work was mostly supported by Grant GM 24571 from the National Institutes of Health.

REFERENCES Abbo, F. E., and Pardee, A. B. (1960). Biochim. Biophys. Acta 39,473-485. Bradley, D. W., Dou, Q.-P., Fridovich Keil, J., and Pardee, A. B. (1990).Proc. Natl. Acad. Sci. USA 87,93 10-93 14. Bradley, D. W., Fridovich-Keil, J. L., and Pardee, A. B. Submitted for publication. Burger, M. M., and Goldberg, A. R. (1966). Proc. Natl. Acad. Scz. USA 57, 359-366. Campisi, J., Medrano, E. E., Morreo, G., and Pardee, A. B. (1982).Proc. Natl. Acad. Scz. USA 79,436-440. Coppock, D. L., and Pdrdee, A. €3. (1985).J. Cell. Physiol. 124, 269-274. Croy, R. G., a n d Pardee, A. B. (1983). Proc. Natl. Acad. Sci. USA 80, 4699-4703. Crozat, A., Molnar, G., and Pardee, A. B. (1994).Mol. Cell. Biol.,in press. Cunningham, D. D. (1972).J.Biol. Chem. 247, 2464-2470. Cunningham, D. D., and Pardee, A. B. (1969). Proc. Natl. Acad. Sci. USA 64, 1049-1056. Dou, Q.-P., Fridovich-Keil, J., and Pardee, A. B. (1991).Proc. Natl. Acad. Sci. USA 88, 11571161.

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Dou, Q. P., Levin, A. H., Wang, J., Helin, K., and Pardee, A. B. ( 1994).J. B i d . Chem., 269, 1306- 1313. Dou, Q.-P., Levin, A. H., Zhao, S., and Pardee, A. B. (1993). Cancer Re.,. 53, 1493-1497. Dou, Q.-P., Markell, P., and Pardee, A. B. (1992). Proc. Natl. Acad. Sci. USA 89,3256-3260. Foster, D. O., and Pardee, A. B. (1969).J. Biol. Chem. 244, 2675-2681. Fridovich-Keil, J. L., Markell, P. J., Gudas, J. G., and Pardee, A. B. (1993). Cell Growth Differn. 4, 679-687. Gerhart, J. C., and Pardee, A. 8. (1956).J. Biol. Chem. 237, 891-896. Gudas, J. M., Knight, G. B., and Pardee, A. B. (1988) Proc. Natl. Acad. Sci USA 85, 47054709. Gudas, J. M., Fridovich-Keil, J. L., Datta, M. W., Bryan, J., and Pardee, A. B. (1992). Gene 118,205-216. Gudas, L. J., and Pardee, A. B. (1974).J. Bacten’ol. 117, 1216-1223. Keyomarsi, K., O’Leary, N., Molnar, G., Lees, E., Fingert, H., and Pardee, A. B. (1994). Cancer Res., 54, 380-385. Keyomarsi, K., and Pardee, A. B. (1993). Proc. Natl. Acad. Sci. USA 90, 1 1 12-1 116. Masters, M., and Pardee, A. B. (1965). Proc. Natl. Acad. Sci. USA 54, 64-70. Pardee, A. 8. (1956). Proc. Natl. Acad. Sci. USA 40, 263-270. Pardee, A. B. (1957).J. Bactpn’ol. 73, 376-385. Pardee, A. T h e control of enzyme activity. (1959).In “The Enzymes, 2nd Ed.” (P. D. Boyer, H. Lardy, and K. Myrback, Eds.), Vol. 1, pp. 681-716. Academic Press, San Diego. Pardee, A. B. (1963). Natl. Cancer Inst. Monogr. No.14, 7-20. Pardee, A. B. (1968). Science 162, 632-637. Pardee, A. B. (1974). Proc. Natl. Acad. Sci. USA 71, 1286-1290. Pardee, A. B. (1987). Cancer Res. 47, 1488-1491. Pardee, A. B. (1989). Science 246, 603-608. Pardee, A. B. (1994).J. Cell. Biochem., 54, 375-378. Pardee, A. B., Heidelberger, C., and Potter, V. K. (1950).J. Biol. Chem. 186,625-635. Pardee, A. B., Jacob, F., and Monod, J. (1959).J . Mol. Biol. 1, 165-178. Pardee, A. B., and Potter, V. R. (1948).J. Biol. Chem. 176, 1085-1094. Pardee, A. B., and Prestidge, L. S. (1956).J. Bacterial. 71, 677-683. Rossow, P. W., Riddle, V. G. H., and Pardee, A. B. (1979). Proc. Natl. Acad. Sci. USA 76, 4446-4450. Smith, H. S., and Pardee, A. B. (1970).J. Bacterial. 101, 901-909. Yang, H. C., and Pardee, A. B. (1986).J. Cell. Physiol. 127, 410-416. Yates, K. A,, and Pardee, A. B. (1956).J. Biol. Chem. 221, 757-770.

INDEX

A Adducts benzo[a]pyrene dial-epoxide-DNA, determination, 82 bulky, carcinogenicity of tobacco and, 81-82 Adenovirus developmental stages, 148- 150 molecular biology, 156- 158 Aflatoxin B, carcinogenicity, reasons for, 87 f53 mutations and, in definition of relationship to hepatitis B virus, 3839 viral infectious hepatitis and, relationship, 88 Alkylating agents, cancer-chemotherapeutic, cytotoxic activity, BCNU incorporation in analysis, 79 Alleles, for genetic control of MuLV in LC mice F v - l * resistant, 188 Fv-4 resistant, 188-189 Air pollution, ambient, carcinogenicity studies, 169- 170 Amphotropic viruses development, from MuLV cancer resistant mice, 177 effect of Fv-4R on, 190 Aneuploidy, carcinogenesis and, role in cellular transformation, 2 1 Aralkylating agents, mutagenic action studies, 83 Aromatic amines electrophiles and, metabolic generation, 64-65

structural requirements, physical organic chemistry studies, 70-73 in vivo chemical modifications of DNA, 60-64

B Bacteriophages, replication mechanism, work of Sol Spiegelman on, 2 10-2 1 1 Base-substitution mutations from aralkylating agents, studies in Eschm'chia coli, 83 and oncogene activation, 96 Benzo[a]pyrene, as archetypal environmental carcinogen, 44 BK virus, simian virus 40 and, similarities, 155 Bladder cancer, aromatic amines and, 2526,60-64 Boveri, Theodor, somatic mutation theory and, 20 Breast cancer cyclin E in vzuo expression, 226 effect of radiation, 90 role of estrogens, 22 Breast cells, cyclin changes, 224-225 Breast-feeding, avoidance, for prevention of human type C retrovirus transmission, 196 Bulky adducts, carcinogenicity of tobacco and, 81-82

C Cancer, see also specific types of cancer age and, relationship, 30-31

229

230

INDEX

Cancer (continued) exogenous sources, radiation and soot, 23-25 smoking-induced, hereditary influences versus environmental factors, 2728 Carcinogenesis aromatic amines and nitrosamines, and in uzuo modifications of DNA, 6064 cell proliferation and cell death in, 18 chemical, exogenous sources, 23-28 effect of hormonal factors, 22 experimental, radiation source, 23-25 gene alterations in, identification, 35 humoralist concept, 21-22 Malthus’s views on, 18 p53 mutations and, 38 psychosomatic factor involvement, theories, 21-22 somatic mutation theory originator, 20 support for, and Tp53 mutation changes, 88-89 tobacco, historical origins, 26-27 tumor viruses involved, 39 two-stage, and polycyclic aromatic hydrogens, relationship, 46-47 Carcinogens chemical, detoxification, enzymes involved, 74-75 growth-inhibitory activity, in rats, 49 methylation, in analysis of molecular mechanisms of carcinogenicity, 78 Cell culture, development, historical origins, 143-144 Central nervous system, effects of MuLV spongiform polioencephalopathy, analysis by induction into wild mice, 184- 185 Chemical carcinogens detoxification, enzymes involved, 7475 exogenous sources, 23-28 growth-inhibitory activity in rats, 49-50 Chemotherapy, and mutations of oncogenes, 78-79 Chromatography, high performance liquid, Jee High performance liquid chromatography

Clark, William, studies by Beth Israel Hospital and Harvard Medical School, 131-137 biologic forms of primary cutaneous melanoma, at Massachusetts General Hospital, 121-123 melanocytes, at Tulane University, 117I20 with Pigmented Lesion Group, 126131 Temple University, 123-126 tumor biology, in Maine, 1 15- 117 Coal tar benzo[a]pyrene as carcinogenic agent in, 80 carcinogenicity, role of polycyclic aromatic hydrocarbons, 73 fluorescence pattern, 43 Colon cancer, role of oncogenes and tumor suppressor genes, 35 Cutaneous melanoma, determining susceptibility of phenotype, 130 Cyclins, and cell cycle regulation in eukaryotic cells, 222-223 Cytogenetics, salivary chromosomes, 14

D Darwin, Charles, views on origin of cancer, 18 Darwinism, and Mendelian inheritance, comparison, 19 Debrisoquin, metabolism, role in lung cancer, 74 Deoxyguanosine, role in carcinogenicity of nitrosamines, 65-66 Deoxyribonucleic acid, see DNA Dietary mycotoxins, as cause of liver cancer, 86-87 Diethylstilbestrol, growth-inhibitory potential in tumors, 51 Dimethylnitrosamine, see Nitrosamines DNA chemical alkylation biological consequences, 66 cytotoxic action, analysis with mustards, 58-59 effect on hydrolysis abilities, 55-56 effects of mustard gas, 5 5

23 1

INDEX

effects of in uivo carcinogens, 57-58 recombinant eukaryotic genes and, composition studies, 162 simian virus 40 DNA and, importance, 158 replication cycle, in analysis of physicochemical nature of genes, 19 transfection of tumor, in isolation of genes causing cancer, 34-35 ultraviolet absorption spectrum alterations, effect of mustard gas, 55 DNA tumor virus, developmental stages, 148-150 Drosophila melanogmter breeding tests, in gene phenotype determinations, 5 radiation carcinogenesis, confirmation studies, 24 Dysplastic nevus, precursor state, 130

E Ecotropic viruses, development from MuLV cancer resistant mice, 177-178 Electrophiles, metabolic generation, 6465 Epstein-Barr virus, role in human carcinogenesis, 39 Escherichia coli P-galactosidase production, components regulating, 216 metabolic regulation studies using, 2 16 Estrogens role in breast cancer, 22 synthetic, growth-inhibitory potential in tumors, 5 1 Estrone, role in breast cancer, 45 Eukaryotic cells cell cycle regulation, effect of cyclins, 222-223 transcriptional control and regulation, 162

F Fisher, R.A., role in study of population genetics, 6

G P-Galactosidase, production, con~ponents regulating, 216 Gene mutations Mendelian inheritance as explanation for, 19 study of, in 19th century, Mendelian inheritance versus Darwinism, 19 Genes DNA replication cycle, Watson-Crick model, 19 Fv-4, major features, tabular presentation, 189 nature of, 1930s studies regulating, 9 nucleic acid structure, 1930s views, 10 Genetics generality in rules of, 10-1 1 population historical origins, 6 importance of randomness and Mendelism, during 1930s, 19 techniques for study, in 1930s, 13-14 Guanine, ring-fission reactions, 56

H Haldane, J.B.S., role in study of population genetics, 6 HeLa cells, poliovirus replication and, limitations, 146 Hepatomas, proteins of, absence of bound dye, 51-52 High performance liquid chromatography, in analysis of chemical nature of polycyclic aromatic hydrocarbons, 80-8 1 Hormones carcinogens and, methylcholanthrene in study of, 45-46 role in carcinogenesis, 22 HPLC, see High performance liquid chromatography Human genetics, cytogenetic studies, in 1930s, 6-8 Human type C retrovirus, transmission via breast-feeding, 196 Humoralism, historical origins, 2 1-22

232

INDEX

I International Congress of Geneticists (1932) plant and animal breeding papers presented, 4 studies regarding genetics, 4-5 Ionizing radiation, carcinogenicity studies, 23-25

K Kidney cells, monkey adenovirus production, complications from simian virus 40, 150 for production of poliovirus stocks and vaccines, 147-148

L Leitch, work on experimental paraffin cancer, 40-4 1 Leukemias, pre-B cell, pathogenesis in MuLV-infected LC mice Liver cancer, dietary mycotoxins and, 8687 Lung cancer benzo[a]pyrene dial-epoxide-DNA adducts, determination in nontumorous parenchyma, 84 involvement of TP53 gene mutations, 84 pharmacogenetic aspects of initiation, 74 skin cancer and, comparison of causes, 85 Lymphomas, incidence rate in MuLVinfected LC mice, 180 Lymphotropic virus, human T-cell, role in human carcinogenesis, 39

Malthus, views on origins o f cancer, 18 Mammary tunior virus, type B, incidence levels in wild mice, 192

Melanocytes melanoma cells and, comparison, 128 study of, historical origins, 118 Melanocytic neoplasia, precursor state, stromal changes in lesions of, 134 Melanomas antigens, as reflection of tumor progression, 128 cutaneous, determining susceptibility of phenotype, 130 expression of multiple growth factor genes by, in vitro studies, 128 metastasis, attributes predicting, 1 16117 primary cutaneous, biologic forms, studies, 122-123 Melphalan, carcinogenicity, and mutation rate, 79-80 Mendelian inheritance, as acceptable explanation for gene mutations, in 1930s, 19 Mendelism Darwinism and, comparison, 19 symmetry and randonmess, influence on study of genetics in l930s, 1213

3-Methylcholanthrene, in study of relationship between hormones and carcinogenesis, 45-46 @-Methylguanine, repair, ability of human cells, 77 Methyl methanesulfonate, methylation of DNA using, 67 N-Methyl-N-nitrosourea, .see Nitrosoureas MMTV, see Type B mammary tumor virus Mouse, wild aged populations MuLV transmission, milk-borne method, 178 sporadic lymphomas, detection o f MuLV p30 antigen, 177 LC (Lake Casitas) amphotropic and ecotropic viruses, transmission method, 182 Fu-4 resistance allele, frequency levels, 191 genetic control of MuLV in use of Fu-lh resistance allele, 188 use of Fv-4 ecotropic resistance allele, 188- I89

233

INDEX

incidence rate of type B mammary tumor virus, 192 lymphomas, incidence rate, 180 MuLV features, tabular presentation, 180 nongenetic control of MuLV, use of selective breeding techniques, 187-188 pre-B cell leukemias, pathogenesis, 183-184 spongiform polioencephalomyelopathy presence, 179-181, 184-185 transmission methods, 194 RNA tumor viruses, nature and function, 171-172 trapping procedure, 173- 174 MT2 protein complexes, E2F complexes and, similarities, 222 Muller, H.J., 1 statements regarding purpose of genes, 10 views on role of mutations in carcinogenic process, 28-29 Multiple allele hypothesis, origins, 7 MuLV, see Murine leukemia virus Murine leukemia virus congenital transmission, in LC mice, 182 genetic control in LC mice use of Fv-lb resistance allele, 187- 188 use of Fv-4 ecotropic resistance allele, 188-189 and human type C retrovirus, similarities, 192-194 milk-borne transmission method, 178 nongenetic control in LC mice with selective breeding techniques, 187188 occurrence in wild mice, 174- 175 p30 antigen, detection in sporadic lymphomas of wild mice, 177 Murine mammary tumor virus, see Type B mammary tumor virus Mustard gas cytotoxic effects on DNA, 54-55 as inhibitor of carcinogenesis, 48-49 dermal, mode of action, 52 vesicant action, similarities with radiation, 29 Mutations, see Gene mutations

Mycotoxins, dietary, as cause of liver cancer, 86-87

N Neoplasia melanocytic parenchymal-stromal interactions, 136-137 precursor state, stromal changes in lesions, 134 role in mutation of oncogenes and tumor suppressor genes, 163 studies by William Clark, 123-125 Nitrosamines carcinogenic action, in vivo reactions causing, 65-69 effect on DNA, in vivo chemical modifications, 60-64 methylation, correlation with cancer initiation in target tissues, 62-63 structural requirements, studies using physical organic chemistry, 70-73 Nitrosoureas, tumor induction incidence, in rats, 76

0 Oncogenes activation using base-substitution mutations, 96 using mutation of cellular protooncogenes, 37 mutations, from cancer chemotherapy, 78-79

P p30 antigen, MuLV, detection in sporadic lymphomas of wild mice, 177 $53, aflatoxin B , and, in definition of relationship to hepatitis B virus, 38-39 Papillomavirus in conversion into carcinomas, effect of polycyclic aromatic hydrocarbons, 60 role in human carcinogenesis, 39

234

INDEX

Pardee, Arthur cancer research in 1950, 215 college and graduate training, 214 Polioencephalomyelopathy, spongiforni, incidence in MuLV-infected LC mice, 179-181, 184-185 Poliovirus methods of distribution, 144-145 pathologic characteristics, 145- 146 vaccine production, use of monkey kidney cells, 147- 148 Polycyclic aromatic hydrocarbons chemical nature, high performance liquid chromatographic studies, 8081 DNA and, location of chemical modification, 69 DNA as in uzuo target, 59-60 production methods, 41 relationship to sterols, 44-46 structural requirements, studies using physical organic chemistry, 70-73 tumor-initiating action, 94 two-stage carcinogenesis and, studies using mouse skin, 46-49 use as radiomimetic compounds, 50 Population genetics importance of randomness and Mendelism, during 193% 19 origins, 6 Pott, Percival, writings on carcinogenicity of coal and soot, 24-25 Primary cutaneous melanoma, biologic forms, studies, 122- I23 Protein complexes MT2, similarity to E2F complexes, 222 Yi, role in thymiciine kinase production, 22 1-222 Proto-oncogenes, activation of oncogenes using, 37

R Related protovirus hypothesis, viral oncogene hypothesis and, comparison, 171 Restriction point cell cycle and, 2 19 role in carcinogenesis, 2 14

Retinoblastoma, incidence in children, time-dependence, 34 Retroviruses cell-to-cell transmission, 170 human type C, murine leukemia virus and, similarities, 192-194 type C, in feral mice, characteristics, I73 RNA-DNA hybridization method, contribution of Sol Spiegelman, 209 RNA tumor viruses, seE Retroviruses Rous sarcoma virus, discovery of viral origin oncogenes using, 37

S Salivary glands, giant chromosomes, physical location of genes using, 2 Sex determinations, genotypes and, early discoveries in 1930s, 3 Shale oil, carcinogenicity studies, 25 Simian virus 40 acquisition by monkeys, 152- 154 BK virus and, similarities, 155 contamination during adenovirus production, 150-152 DNA chromosomes, structure, tabular presentation, 158-159 p53 detection using, 163 transcription maps and, studies using eukaryotic cells, 160 Skin, mouse, chemical carcinogenesis, twostage mechanism, 38 Skin cancer, and lung cancer, comparison of causes, 85 Smallpox, preventive practices for, development procedures, 142-143 Smog, see Air pollution, ambient soot carcinogenicity chemical basis for, 25 polycyclic aromatic hydrocarbons in determination, 24 fluorescence spectra, 42 Spiegelman, Sol biographical data, 203-205 contributions to studies of reverse transcriptase in human cancer, 2 1021 I

235

INDEX

enzyme studies, 206 RNA-DNA hybridization method and, 209 work on replication mechanism of phages, 210-211 Spongiform polioencephalomyelopathy, incidence in MuLV-infected LC mice, 179-181, 184-185 Squamous cell neoplasia, TP53 mutations, detection, 85 SV40, see Simian virus 40 Synthetic estrogens, growth-inhibitory potential in tumors, 5 1

T Tamoxifen, breast cancer and, treatment modality for, 54 Thymidine kinase gene cell cycle regulation in tumor cells, 220 role in production of Yi protein complex, 22 1-222 Tobacco, carcinogenicity, historical origins, 26-27 Transmission genetics, discoveries regarding, in 1930s, 2-3 Tumor cells, cell cycle regulation, thymidine kinase gene and, 220-221 Tumors, growth inhibitory potential of synthetic estrogens, 51 Tumor suppressor gene, p S 3 , role in human carcinogenesis, 36 Tumor viruses, see also specific viruses effect of immunosuppressive drugs, 39 Epstein-Barr, role in human carcinogenesis, 39 papillomavirus conversion into carcinomas, effect of polycyclic aromatic hydrocarbons, 60 role in human carcinogenesis, 39 transfection of DNA from, in isolation of cancerous genes, 34-35

Two-stage cdrcinogenesis, polycychc aromatic hydrocarbons and, relationship, 46-47 Type B mammary tumor virus, incidence levels in wild mice, 192 Type C retrovirus, see Human type C retrovirus; Murine leukemia virus

v Viral diseases historical origins, 14 1 poliovirus methods of distribution, 144-145 pathologic characteristics, 145- 146 vaccine production with monkey cells, 147-148 smallpox, preventive practices for, development procedures, 142-143 Viral Oncogene Hypothesis, carcinogenesis and, role of virogenes and oncogenes, 170 Virogenes Fu-qR, infection prevention in wild mice, blockage of exogenous retroviruses, 195 role in carcinogenesis, 170 Virus Cancer Program, RNA tumor viruses study, 169

W Watson-Crick model, and discoveries of physicochemical nature of genes, 19 Wright, Sewall, role in study of population genetics, 6

Y Yi protein complex, role in thymidine kinase production, 221-222

I S B N 0-12-00bbb5-3