Advances in Cancer Research Volume 54

ADVANCESINCANCERRESEARCH VOLUME 54

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ADVANCES IN CANCER RESEARCH Edited by

GEORGE F. VANDE WOUDE NCI-Frederick Cancer Research Facility Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 54

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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CONTENTS

CONTRIBUTORS TO VOLUME 51.........................................................................

xi

The Role of DNA Methylation in Cancer

PETERA . JONES AND JONATHAN D . BUCKLEY I. I1. I11. IV. V. VI . VII . VIII . IX. X.

Introduction ................................................................................................. CpG Islands ................................................................................................. Transduction of the Methylation Signal .................................................... DNA Methylation in Tumor Cells ............................................................. Methylation in Uncultured Tumor Tissue ................................................ Effects of Chemical Carcinogens on DNA Methylation........................... Role of DNA Methylation in Tumor Diversification ................................ DNA Methylation during Oogenesis and Spermatogenesis .................... DNA Methylation and Genomic Imprinting ............................................. Conclusion ................................................................................................... References., ..................................................................................................

1 2 4 7 9 11 13 14 16 19 19

Genetic and Epigenetic Losses of Heterozygosity in Cancer Predisposition and Progression

HEIDIJ . SCRABLE.CARMEN SAPIENZA.AND WEBSTERK. CAVENEE I. I1. 111. IV. V. VI .

Introduction ................................................................................................. Genetics and Predisposition ....................................................................... Loss of Heterozygosity and Tumor Progression ....................................... Loss of Heterozygosity in Mixed Cancer................................................... Epigenetic Inactivation of Alleles in Human Cancer............................... Conclusions .................................................................................................. References .................................................................................................... V

25 29

37 42 49

58 59

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CONTENTS

Genetic and Molecular Studies of Cellular Immortalization

JAMES R. SMITHAND OLIVIA M. PEREIKA-SMITH I. 11.

Introduction ..................................................................... Short-Tenn Analysis of Cell Fusion Products ...........................................

\‘.

Discussion .......I......................._........................................

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

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

64 73 76

The Function of RAS Genes in Saccharomyces cerevisiae JAJiES

R. BROACHAND ROBERTJ. DESCHENES

I. Introduction ......................................... 11. Sfodel for Ras Protein Function in Yeast ................................................... 111. Yeast Ras Proteins .........._... I\’, Components of the Ras-cAMP Pathway .............. .... . ...... ...... ........... V. Targets of the cL4MP-DependentProtein Kinase \’I, To What Signals Do R A S Genes Respond?............................................... VII. LVhat Is Ras Doing References...........................

79 82 86 101 111 122 128 132

Retroviral Integration in Murine Myeloid Tumors to Identify Evi-7, a Novel Locus Encoding Zinc-Finger Protein

N. G. COPELAND ASD N. A. JENKINS Introduction ..................... RI Slouse Strains ......................................................................................... Identification o f a New Common \ ’ i d Integration Site, Eci-f , in AKXD Myeloid Tuiiiors .................................................................... 11’. Relationship of Eci-l to Other Zinc Finger Proteins ................................ V. Activation of Transcription ofEl;i-I by Viral Integration in Fim-3 ......... 1’1. Additional Zinc-Finger Proteins Implicated in Neoplastic Disease .......

I, 11. 111.

................................... ............ ........................ ................................... I

141 143 145 151 154 154 154 155

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Metastatic Inefficiency

LEONARD WEISS I. I1. 111 IV V VI VII .

. . . .

Introduction ................................................................................................. Metastatic Inefficiency-Documentation .................................................. The Metastatic Process ............................................................................... Metastatic Inefficiency-Random and Nonrandom Events ..................... The Molecular Biology of Metastatic Inefficiency .................................... Consequences of Metastatic Inefficiency .................................................. Conclusions .................................................................................................. References ....................................................................................................

159 160 161 178 187 199 202 203

Growth Regulatory Factors for Normal. Premalignant. and Malignant Human Cells in Vitro

MEENHARD HERLYN.ROLAND KATH. NOELWILLIAMS. ISTVAN VALYI.NAGY. AND ULRICH RODECK I. I1. I11.

IV . V. VI .

Introduction ................................................................................................. Growth of Normal Human Cells in Vitro .................................................. Human Melanocytic Cells as a Model for Studies on Tumor Progression ................................................................................................... Growth Factor Independence of Human Tumor Cells from Metastatic Lesions ......................................................................................................... Autocrine Growth Stimulation of Human Tumor Cells and Strategies for Growth Inhibition .................................................................................. Summary ...................................................................................................... References ....................................................................................................

213 214 216 224 226 231 232

The Lymphopoietic Microenvironment in Bone Marrow

PAULW. KINCADE I. I1. I11. IV. V. VI .

Introduction ................................................................................................. Evolution of Experimental Approaches ..................................................... Long-Term Bone Marrow Cultures............................................................ Differentiation Steps and Lineages ........................................................... Lymphocytes in Long-Term Cultures ....................................................... Initiation of Long-Term Cultures ...............................................................

235 237 240 241 242 245

...

CONTENTS

Vlll

VII. VIII. IX. X. XI. XI1 XIII. XIV.

Essential Cells of the Microenvironment.................................................. Recognition and Adhesion between Cells Interleukin 7 ...

I

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

xv. References.......

,..................

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

246 250 252 254 256 257 259 261 261 264

Structure and Function of the B-Lymphocyte Epstein-Barr Virus/CSd Receptor

GLENR. NEMEROW, MARGARET D. MOORE,AND NEILR. COOPER I. Introduction ..... .................... 273 11.

111. IV. V. VI. VII.

Identification a Structure of CR2 ................ Structure and Function of the CR2 Ligands .............................................. Cellular and Tissue Distribution o Functional Pr Summary and Future Prospects ...................................... ........................... References.. ....

274 276 284 287 29 1 294 295

The Opportunistic Tumors of Immune Deficiency

HARRYL. IOACHIM Introduction ......................... ............ States of Immune Deficienc .. ... ,....,. 111. Opportunistic Infections ... IV. Opportunistic Tumors ................................. ................................................ V. Distinctive Features of Op VI. Spontaneous Regression of Opportunistic Tumors ................................... VII. Stromal Reaction of Tumors ........................................................ VIII. d Immune Surveillance of Tumors ...................... References. ....... ...................................................... ,........................... I. 11.

.I I..

30 1 302 303 304 31 1 311 312 3 13 315

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A Note on Concomitant Immunity in Host-Parasite Relationships: A Successfully Transplanted Concept from Tumor Immunology

GRAHAM F . MITCHELL I . Introduction ................................................................................................. I1 . Some Examples of Concomitant Immunity in Parasitized Mice ............. I11. Concluding Comments ............................................................................... References ....................................................................................................

319 320 327 330

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INDEX

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CONTRIBUTORS TO VOLUME 54 Numbers in parentheses indicate the pages on which the author’s contributions begin.

JAMES R . BROACH,Department of Biology, Princeton University, Princeton, New Jersey 08544 (79) JONATHAN D. BUCKLEY,Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern Calqornia, Los Angeles, Calqornia 90033 ( 1 ) WEBSTERK. CAVENEE, Ludwig Znstitute for Cancer Research, Royal Victoria Hospital and McGill University of Medicine, Montreal, Quebec H3A 1A1, Canada (25) NEILR . COOPER, Research Institute of Scripps Clinic, Department of Immunology, La Jolla, California 92037 (273) N. G. COPELAND, Mammalian Genetics Laboratory, BRI-Basic Research Program, NCZ-Frederick Cancer Research Facility, Frederick, Maryland 21 701 (141) ROBERTJ . DESCHENES, Department of Biology, Princeton University, Princeton, New Jersey 08544 (79) MEENHARDHERLYN,The Wistar lnstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213) HARRYL. IOACHIM, Department of Pathology, Lenox Hill Hospital, New York, New York, 10021, and College of Physicians and Surgeons, Columbia University, New York, New York 10032 (301) N . A. JENKINS, Mammalian Genetics Laboratory, BRZ-Basic Research Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21 701 (141) PETERA. JONES, Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033 (1) ROLANDKATH, The Wistar Institute ofAnatomy and Biology, Philadelphia, Pennsylvania 19104 (213) PAULW. KINCADE,Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 (235) GRAHAM F. MITCHELL,The Walter and Eliza Hall Znstitute of Medical Research, Melbourne, Victoria 3050, Australia (319) MARGARETD. MOORE,Research Institute of Scripps Clinic, Department of Immunology, La Jolla, California 92037 (273) xi

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CONTRIBUTORS TO VOLUME 54

GLENR. NEMEROW, Research Znstitute of Scripps Clinic, Department of Immunology, LaJolla, Calijornia 92037 (273) ULRICHRODECK, The Wistar Znstitute of Anatomy and Biology, P k l a delphia, Pennsylvania 19104 (213) CALMENSAPIENZA, Ludwig Znstitute for Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal, Quebec H3A 1A1, Canada (25) HEIDIJ . SCRABLE, Ludwig Znstitute f o r Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal, Quebec H3A LAI, Canada (25) JAMES R. SMITH,Roy M . and Phyllis Gough Huffington Center on Aging, and Departments of Virology and Epidemiology, of Cell Biology, and of Medicine, Baylor College of Medicine, Houston, Texas 77030 (63) O L I V LM~. PEREIRA-SMITH, Roy M . and Phyllis Gough Huffington Center on Aging, and Departments of Virology and Epidemiology, of Cell Biology, and of Medicine, Baylor College of Medicine, Houston, Texas 77030 (63) ISTVANVALYI-NAGY, The Wistar Znstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213) LEONARDWEISS, Department of Experimental Pathology, Roswell Park Memorial Znstitute, Buffalo, New York 14263 (159) NOELWILLIA-MS, The Wistar Znstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213)

THE ROLE OF DNA METHYLATION IN CANCER Peter A. Jones and Jonathan D. Buckley Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033

I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction CpG Islands Transduction of the Methylation Signal DNA Methylation in Tumor Cells Methylation in Uncultured Tumor Tissue Effects of Chemical Carcinogens on DNA Methylation Role of DNA Methylation in Tumor Diversification DNA Methylation during Oogenesis and Spermatogenesis DNA Methylation and Genomic Imprinting Conclusions References

I. Introduction

Interest in a potential role for DNA methylation in the control of eukaryotic gene expression was first stimulated by two papers published in 1975 by Holliday and Pugh (1975) and Riggs (1975). Since that time considerable evidence has accumulated that 5methylcytosine is implicated in gene control. There have been well over 100 studies that have demonstrated an inverse relationship between methylation and expression, and numerous experiments have shown that methylation can inactivate genes. This evidence has led to the suggestion that hypomethylation may be a necessary but not sufficient condition for gene activity and has been summarized in numerous reviews published over the last few years. The reader is referred to these for a detailed description of the biochemistry of DNA methylation and its role in gene control (Doerfler, 1983; Adams and Burdon, 1985; Razin and Szyf, 1984; Cedar, 1988).All of the available evidence supports the idea that methylation plays a role in cellular memory and participates with other gene control mechanisms to stabilize transcriptionally inactive states. DNA methylation is clearly altered in many cancer cells, and the relevance of these alterations to malignancy has also been the subject of many review articles (e.g., Holliday, 1979; Riggs and Jones, 1983; Jones 1985). Some of the reasons for suspecting that the epigenetic 1 ADVANCES IN CANCER RESEARCH, VOL 54

Copyright 0 1990 by Academic Press, Inc All riehts of reoroduction in any form reserved

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PETER A. JOXES AND JONATHAN D. BUCKLEY

control exerted by 5-methylcytosine might be of relevance to carcinogenesis and tumor development were summarized in Advances in Cancer Research by Riggs and Jones (1983).This review will therefore concentrate on developments since that paper was published that have strengthened the hypothesis that aberrations in methylation are a key factor in the development and expression of the malignant state.

II. CpG Islands CpG methylation sites are markedly underrepresented in vertebrates (Josse et al., 1961) and are not scattered randomly throughout the genome but rather are clusterd in specific regions called CpG islands or HTF regions (Tykocinski and Max, 1984; Bird, 1986; Gardiner-Garden and Frommer, 1987). The enrichment for CpG within these islands is due to a lack of the overall suppression of the dinucleotide in eukaryotic genomes and also to their relative G + C richness (65% G + C compared to 40% for bulk DNA) (Bird, 1986). CpG islands have been found to be associated with the 5' ends of many tissue-specific and housekeeping genes as well as with the 3' ends of some tissue-specific genes. Bird et al. (1985)have estimated that there are -30,000 islands per haploid genome in the mouse. They suggested that the islands may be associated with genes and serve to identify sequences that ought to be constantly available in the nucleus for transcription. In support of this hypothesis, Lavia et al. (1987) used two randomly isolated islands as probes and detected multiple transcripts of RNA from several mouse tissues. Cloned cDNAs for the major transcripts of one island were isolated and used to construct a transcritipnal map. The authors found that the island contained the origin of a pair of divergent transcripts that were probably mRNA molecules. The results therefore support the view that CpG islands often mark genes and suggest that bidirectional transcription may be a common feature of island promoters. Many studies have shown that the CpG islands are unmethylated in all tissues tested. Bird et a2. (1987)analyzed both CpG frequency and methylation across part of the human a-globin locus. The CpG frequency was not reduced and none of the tested CpG sites were methylated in the DNA from erythroid or nonerythroid tissues, although flanking CpG sites were methyalted. This observation therefore confirms the results of a large number of other studies, which have shown that CpG islands are not methylated in the germ-line or adult tissues (Bird, 1986; Gardiner-Garden and Frommer, 1987). These kinds of

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experiments have added weight to the hypothesis of Bird (1986) that methylation of the CpG islands does not control gene expression of tissue-specific or housekeeping genes in normal development. Rather, the islands are protected from methylation and remain unmethylated, which has ensured their survival in the face of the strong tendency for 5-methylcytosine to deaminate to thymine. The clear exception to the general rule that CpG islands are not methylated is seen with genes located on inactive X chromosomes in female mammals. Numerous studies have shown that methylation of these areas is associated with transcriptional inactivity. Examples are hypoxanthine phosphoribosyltransferase (HPRT), glucose-6phosphate dehydrogenase (GGPD), and phosphoglycerate kinase (PGK), in which the islands are methylated on inactive X chromsomes but unmethylated on the active chromosome (Wolf and Migeon, 1985; Toniolo et al., 1988; Keith et al., 1986). Experiments with the mouse H P R T gene have also demonstrated that sites within the 5' region of the gene are completely unmethylated when carried on the active X and extensively methylated when carried on the inactivated chromosome. The same sites become demethylated in H P R T genes reactivated either spontaneously or after 5-azacytidine (5-Aza-CR) treatment (Lock et al., 1986). There is therefore considerable evidence that a methylationassociated mechanism participates in the silencing of genes on the inactive X chromosome. However, methylation does not appear to be the initial step by which the gene inactivation event occurs. X inactivation takes place early during embryogenesis and results in the inactivation, at random, of one of the initially active chromosomes. Lock et al. (1987) have demonstrated that methylation of the H P R T gene on the inactive X chromosome occurs after the inactivation event. Therefore, 5-methylcytosine in the CpG island is thought to stabilize the transcriptionally inactive state. If Bird's hypothesis (1986) is correct, then the methylation of CpG islands should never occur in cells that are part of the germ line. Indeed the germ line may be sequestered from the rest of the embryo during embryogenesis and escape X inactivation (Monk et al., 1987). DNA methylation therefore appears to be a mechanism whereby cells can control the expression of genes with similar promoter regions in the presence of ubiquitous transcription factors. As far as dosage compensation is concerned with the X chromosome, the methylation of the CpG island in somatic cells helps to ensure that only one chromosome is active, thus achieving functional hemizygosity for X-linked

4

PETER A. JONES AND JONATHAN D. BUCKLEY

genes. This has been the only clear demonstration of methylation of CpG islands to date; however, several indirect experiments have suggested that methylation may control genes located on autosomes. The metallothionein gene family is present in a broad range of eukaryotic species and is expressed in many different cell types and tissues. All vertebrates synthesize at least two metallothionein isoforms (MT1 and MT2), which have different amino acid sequences. The situation in humans is complicated by the presence of multiple M T l isoforms (Schmidt and Hamer, 1986).The single gene for MT2 is ubiquitously expressed in all human cells in response to cadmium; however, the two genes encoding MT1 isoforms are expressed in a highly specific reciprocal fashion that correlates with the embryonic germ layer origin of the cells. Cells derived from mesoderm and endoderm express predominantly the M T l e gene, whereas cells derived from ectoderm, intermediate mesoderm, or lateral mesoderm express predominantly the M T l f gene in response to a heavy-metal stimulus. Schmidt and Hamer (1986) showed that the ability of cells to express the different isoforms could be altered by prior treatment with S-AzaCR, suggesting that the reciprocity of gene expression in these cells was controlled by DNA methylation. Similar results were obtained for the M T I b gene by Heguy et al. (1986). These authors found that the S’-flankingregion of the gene was highly methylated in HeLa cells that did not express M T l b but unmethylated in hepatoma cells that do express the gene. The HeLa cells could be induced to express M T l b after S-Aza-CR treatment, suggesting a strong influence of methylation in inducing cis-acting suppression. Thus, methylation could play a key role in preferential silencing of a particular subset of genes in the presence of similar promoter regions and common transcription factors.

I l l . Transduction of the Methylation Signal Although we have known for >10 years that methylation and gene expression are inversely correlated, very little is understood of the mechanisms by which the methylation signal is transduced. Early experiments by Vardimon et al. (1982) showed that the expression of a cloned adenovirus gene was substantially inhibited by in vitro modification. Dramatic downregulation of expression of a variety of genes after methylation was also seen in other systems (e.g., Busslinger et al., 1983; Kruczek and Doerfler, 1983). For example, the transcription of the adenovirus 2 E2a gene is reduced below detectable levels by the methylation of three HpaZZ sites within its upstream region (Kruczek

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and Doerfler, 1983; Knebel et al., 1987). In vitro methylation of the cloned Moloney sarcoma virus (McGeady et al., 1983) or the Ha-ras genes (Borrello et aZ., 1987, 1988) limits their transforming activity after transfection. The in vitro methylation of specific sequences in the human P-globin gene has been shown to influence strongly its expression in either fibroblasts or erythroleukemia cells (Yisraeliet al., 1988). Thus, as mentioned earlier, the methylation of CpG sequences within the promoter region of genes can substantially and rapidly alter their expression. Studies on the regulation of eukaryotic gene expression have demonstrated that transcription results from the binding of multiple factors to the promoter region (Wolfee and Brown, 1988). Conceivably, methylation may block the binding of a particular transcription factor to its target sequence, thus preventing the activity of the gene in question. Several studies have shown that some but not all methylation signals are transduced in this way. We used synthetic oligonucleotides (Harrington et al., 1988) to investigate the effect of cytosine methylation on the binding of the transcription factor Spl (Dynan and Tijan, 1983)to its target sequence known as the GC box. The GC box sequence appears frequently in the promoters of genes containing CpG islands and might be an important sequence in which methylation could block factor binding. We failed to detect any effect of 5-methylcytosine in the internal CpG sequence of the GC box on Spl binding using DNase I footprinting or gel retardation analysis. These results did not address the possibility that the activity of Spl was influenced by the presence of a methyl group in its recognition sequence. These findings were confirmed and extended by Holler et al. (1988), who showed that Spl could bind and activate transcription frop a methylated GC box. Hoeveler and Doerfler (1987) found that although methylation of specific CCGG sequences within the E2a promoter of adenovirus DNA-blocked transcription, DNaseI protection analysis showed that the binding of factors to methylated promoter sequences was not influenced. Ben-Hattar and Jiricny (1988) have confirmed and extended the results found with Spl and investigated the effect ofmethylation of a single CpG sequence within the GC box on the activity of the thymidine kinase (TK)gene. The presence of a single 5-methylcytosine in the recognition sequence in the GC box blocked TK transcription but did not affect the affinity of the Spl protein for its respective recognition sequences. In contrast to these findings, cytosine methylation can block the binding of some transcription factors to target sequences. Experiments by Becker et aZ. (1987) have shown that the methylation

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PETER A. JONES AND JONATHAN D. BUCKLEY

of two cytosine residues could prevent the binding of an unknown transcription factor to the promoter region of the tyrosine aminotransferase gene. Watt and Molloy (1988) found that methylation blocked binding of a HeLa cell-transcriptional factor required for optimal expression of the adenovirus major late promoter. Thus, while the binding of some factors may not be affected directly by methylation, the binding of others may be strongly influenced by the presence of the modified base within the recognition sequence. Given the fact that methylation has such strong effects on the transcriptional activity of genes but does not necessarily block factor binding to upstream promoter regions, how then might the methylation signal be recognized in these cases? Some experiments have suggested that methylation may act by a more indirect mechanism to target DNA to inactive chromatin configurations. For example, Keshet et al. (1986)showed that the presence of cytosine methylation could direct transfected DNA to a DNase I-insensitive configuration after integration into chromatin. Detailed experiments by Buschhausen et al. (1987)also suggested that the inethylation signal may be transduced by a mechanism requiring a native chromatin configuration. These authors showed that the inhibitory effect of methylation on the herpes TK gene required that the promoter region become assembled into a chromatin configuration before the effects of methylation on transcription were seen. Naked methylated TK genes were expressed early after injection into TK- cells. However, if the methylated template was combined with histones before injection into the cells, the effect on transcription was immediate. The effects of methylation on gene expression are therefore likely to be complex and to require the presence of a chromatin structure in some cases for the appropriate recognition of the signal. One other mechanism by which methylation signals may be transduced is by the binding of negatively acting factors to methylated regions of DNA. Huang et al. (1984) and Wang et al. (1986a,b) have isolated proteins from human placenta that preferentially bind to certain methylated DNA sequences. Conceivably these sequencespecific and 5-methylcytosine-specific binding proteins might act to silence genes by binding to the promoter regions, thus precluding the access of more specific factors to their target sequences. Alternatively, they may stabilize inactive chromatin conformations. The mechanisms by which methylation signals are recognized therefore remain obscure. The binding of some, but not all, transcription factors may be influenced directly b y DNA modification, but methylation certainly does not directly block the binding of all transcription factors. The most likely explanation in the latter cases is that

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the signal is transduced by more indirect mechanisms such as altered chromatin conformation, which precludes and prevents the activities of the more specific factors. IV. DNA Methylation in Tumor Cells

Several publications since 1983 have expanded the data base that alterations in DNA 5-methylcytosine levels and patterns are very common in cultured tumor cell lines and primary tumors (Tables I and 11). Kuo et al. (1984)examined the methylation of HpaII and Hhal sites in DNA isolated from normal rat livers and from transplantable hepatomas. They found that Hhal sites in the a-fetoprotein gene were less methylated in hepatoma cell DNA than in liver DNA and found a generally decreased methylation of CCGG sequences in rat hepatoma DNA than in normal rat livers. Specific hypomethylation at a site within the c-myc gene was also seen in three of five human tumor cell lines by Cheah et al. (1984). Studies in OUI laboratory have shown that decreased methylation is not a universal feature of all human tumor cell lines, and substantial methylation decreases were not present in all explants of human pediatric tumors (Flatau et al., 1983). Methylation was decreased substantially in a fibrosarcoma, and in some neuroblastomas and rhabdomyosarcomas. However, Wilms’ tumor cell lines or medulloblastoma cell lines had methylation levels that were very similar to those obtained in human fibroblasts. Interestingly, four low-passage retinoblastoma cell strains had levels of methylation of -3.9%, which is a relatively high level of modification for human cells and tissues (Gama-Sosa et al., 1983). We also failed to detect consistently decreased methylation levels in a series of other human tumor cell lines using probes for the Ha-rus, TK, and al-collagen genes (Chandler et al., 1986). However, interpretation of experiments using cultured cell lines is complicated by the problem in obtaining the appropriate normal control cells. Also, there is substantial evidence that methylation levels and patterns can change in culture (Shmookler-Reis and Goldstein, 1982; Wilson and Jones, 1983), which also complicates interpretation of the results. Decreased 5-methylcytosine levels have been observed during metastasis in nude mouse models (Table I). Liteplo and Kerbel (1987) determined the total levels of methylation in a series of related highly metastatic cell lines isolated from a poorly metastatic human melanoma tumor cell line MeWo. The authors interpreted the observations to support the hypothesis that alterations in cytosine methylation may play a part in the generation of tumor cell heterogeneity.

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DNA AM^^^^^^^^^^

IN

TABLE I CULTURED CELLS AND TRANSPLANTABLE TUMORS

Tumor type

Assay method

Transplantable rat hepatomas Human tumor cell lines Human melanoma cells

Gene probe with a-fetoprotein c-myc Probe

Human pediatric tumor explants Human tumor cell lines Human lung and other tumor lines

HPLC

HPLC

Gene probes Calcitonin and other chromosome 11 probes

Methylation level

Reference

Decreased

Kuo et al. (1984)

Decreased in three of five Decreased in metastatic nodules Variable

Cheah et al. (1984)

Variable but heterogeneous Increased

Liteplo and Kerbel (1987) Flatau et al. (1983) Chandler et ~ l . (1986) Baylin et al. (1986); DeBustros et al. (1988)

In contrast to these observations, which have generally found decreased DNA methylation in cell lines associated with transformation and malignancy, Baylin and his colleagues (1986,1987; DeBustros et al., 1988) observed substantial hypermethylation within specific regions of human chromosomes in tumor cells. These authors examined small-cell lung carcinoma and lymphoma for methylation of the calcinonin gene located on chromosome l l p . The unusual hypermethylation patterns were found less frequently in other tumor cell types, suggesting that there was a correlation between abnormal calcitonin gene methylation and differentiation events. These studies have subsequently been extended for other markers on chromosome l l p (DeBustros et aZ., 1988). It appears that there is a hot spot for abnormal methylation on the short arm of chromosome 11, an area known to harbor several putative tumor suppressor genes. Baylin has postulated that the increased regional DNA methylation within these tumors may participate in, or mark, chromosomal changes associated with gene inactivation events central to the development of tumors during multistep carcinogenic changes. In view of the current interests in tumor suppressor genes (Klein, 1987), the concept that increased methylation may be associated with the silencing of suppressor genes within tumors is particularly attractive and provocative.

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V. Methylation in Uncultured Tumor Tissue

In contrast to the situation with cultured tumor cell lines, where there is always the question of potential artifacts being induced by culture conditions and the difficulty in obtaining appropriate normal tissue for comparison, there have been substantial advances made in our knowledge of methylation changes in uncultured tumors (Table 11).Generally decreased levels of DNA methylation were observed in a careful study of a large number of human tumors by Gama-Sosa et al. (1983) using high-performance liquid chromatography (HPLC) techniques. The authors proposed that metastatic neoplasms have significantly lower genomic 5-methylcytosine levels than most benign neoplasms or normal tissues. Similar observations have been published by Bedford and van Helden (1987), who reported decreased levels of methylation in metastatic prostatic carcinoma cells but not in less malignant tumors. A thorough study completed by Feinberg et al. TABLE I1 DNA METHYLATION IN UNCULTURED TUMORS Tumor type Diverse primary and secondary tumors Prostrate carcinoma Colon adenomas and carinomas Colon and lung carcinoma

Rat stomach tumors Human lymphoid and myeloid malignancy

Assay method

Matched control

Methylation change

Reference Gama-Sosa et al. (1983)

HPLC

No

General decrease

HPLC

Yes

HPLC

Yes

Specific genes

Yes

HPLC

Yes

Specific gene (pepsinogen) Specific gene (calcitonin)

Yes

Bedford and van Decrease in metastatic Helden (1987) carcinoma, not in nonmetastatic 8-10% decrease in Feinberg et al. all examined (1988) Feinberg and Decrease in Vogelstein benign and malignant (1983a,b); tumors Goelz et al. (1985) Decrease Ichinose et al. (1988) Heterogeneous Ichinose et al. (1988) pattern Baylin et al. Increased (1987)

Yes

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PETER A. JONES AND JONATHAN D . BUCKLEY

(1988) using human colonic cells has shown an unequivocal hypomethylation of tumor DNA. These studies were aided by the availability of normal tissue in close proximity to the tumors, guaranteeing that the appropriate controls were available. They showed an average reduction in genomic 5-methylcytosine content of 8-10% in all colon adenomas and adenocarcinomas. In contrast to the earlier reports, there was no significant difference between benign and malignant tumors, suggesting that the alteration in methylation occurs very early in the genesis of these particular tumors. The studies by Feinberg et al. (1988) complemented earlier work on changes within specific loci in tumors (Feinberg and Vogelstein, 1983a,b; Goelz et al., 1985). The earlier observations had demonstrated alterations in methylation levels of specific genes including growth hormone, y-globin, a-chorionic gonadotropin, and ycrystallin in all 23 neoplastic growths examined (Goelz et al., 1985). The data suggested that hypomethylation was a consistent biochemical characteristic of human colonic tumors and, since it was also seen in the adenomas, the methylation defect preceded the acquisition of malignancy. Goelz et al. (1985) also made the suggestion that the hypomethylation might inhibit chromosome condensation and lead to chromosome mispairing and nondisjunction. There is evidence that drug-induced hypomethylation can lead to decondensation and chromosome abnormalities (Schmid et al., 1983;Bianchi et al., 1988).Thus, methylation defects may be important in promoting allelic deletions, which are thought to be important in the genesis of tumors (Vogelstein et al., 1988). A study by Ichinose et a2. (1988)demonstrated alterations in methylation of the tissue-specific gene pepsinogen within stomach neoplasms (MNNG). induced in rats by N-methyl-N‘-nitro-N-nitrosoguanidine The methylation patterns of the genes were different from those of the normal tissues, but there was not a simple correlation between methylation and expression of the genes. However, the observation that the patterns were altered is consistent with the other data discussed earlier, which have shown that methylation is changed within tumor cells. Baylin et al. (1987) have also examined the methylation of the calcitonin gene in human lymphoid and acute myeloid malignancies. I n support of their observations with cultured cells (see earlier), these studies showed that there was an increase in the numbers of CCGG sites methylated in the 5’ region of the calcitonin gene in 90% of patients with non-Hodgkin’s lymphoid neoplasms and in 95% of tumor cell DKAs extracted from patients with acute nonlymphocytic leukemia.

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In general the evidence suggesting that methylation changes are associated with the transformed state has accumulated since the early 1980s. The studies with uncultured tissues have been particularly important in establishing a relationship between these two parameters.

VI. Effects of Chemical Carcinogens on DNA Methylation Since the pioneering studies of Drahovsky and Morris (1972) and Drahovsky and Wacker (1975), many studies have demonstrated that chemical carcinogens can interfere with the DNA methylation system (Riggs and Jones, 1983). Several later studies have extended these findings. For example, carcinogens can induce decreases in genomic 5-methylcytosine in normal bronchial epithelial cells (Wilson et al., 1987). Not all cells respond to carcinogens by heritable decreases in DNA methylation. Krawisz and Lieberman (1984) could find no substantial 5-methylcytosine decreases in Raji cells, S49 cells, or human diploid fibroblasts after treatment wich chemical carcinogens or ultraviolet (UV) radiation. However, these latter studies may not have been sensitive enough to detect biologically important methylation changes. There are two corollaries to the hypothesis that chemical carcinogens act in part by inhibiting DNA methylation: (1) Chemical carcinogens should be able to activate nonexpressed genes in suitable selectable systems in a similar manner to that demonstrated for 5-Aza-CR. (2)Agents that inhibit methylation such as 5-Aza-CR should be carcinogenic. Many experiments since 1984 have addressed these issues. MacArthur et al. (1985) showed the treatment of a cadmiumsensitive metallothionein-negative mouse cell line with two directacting carcinogens N-ethylnitrosourea or N-acetoxy-2acetyIaminofluorine, or with UV irradiation, induced a substantial increase in phenotypically stable cadmium-resistant variants. The increased cadmium resistance of the cells was demonstrated to be due to the activation of metalloethionein genes by the carcinogens. Previous studies from this group (Lieberman et al., 1983) had demonstrated that UV radiation could induce MTI gene activation, and that this was associated with extensive DNA demethylation. Thus, it is possible the mechanism of activation of metallothionein inducibility was due to a demethylation event induced by the carcinogen. Further studies by Barr et al. (1986) showed that an inactive but functionally intact hamster TK gene could be activated by MNNG in Chinese hamster cells. The authors proposed that the chemicaI carcinogen activated the TK

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gene by focal demethylation resulting in low TK activity, whereas demethylation throughout the genome resulted in much higher levels of TK activity. Ivarie and Morris (1986) have also shown that MNNG and 5-Aza-CR can induce a 50- to 100-fold increase in the reversion frequency of a mutant HeLa cell line harboring an HPRT gene silenced by methylation. Thus, chemical carcinogens such as MNNG have epigenetic affects in addition to their well-known ability to be mutational agents. Some DNA-alkylating agents can promote enzymatic methylation at CpG sites in certain cases. Ethyl methane sulfonate (EMS) can silence prolactin gene expression in GH3 cells (Ivarie and Morris, 1982), and direct experiments have shown that methylation of poly dC-dG with EMS stimulates the methyl-accepting ability of the DNA by rat DNA methyltransferase (Farrance and Ivarie, 1985).Carcinogens can therefore have multiple effects on DNA methylation, acting either to decrease or to increase methylation depending on the specific lesion induced in DNA. Macnab et al. (1988) have demonstrated that some viruses such as herpes simplex virus can induce the hypomethylation of host cell DNA synthesized after infection. The inhibition of host cell DNA methylation may therefore be an important step in the transformation of cells by herpesviruses. The second corollary of the hypothesis that DNA methylation and carcinogenesis are linked predicts that agents that inhibit DNA methylation should be capable of transforming cells. Our early experiments with 5-Aza-CR (Benedict et al., 1977) showed that the drug was capable of transforming the 10T1/2 cell line in addition to inducing changes in differentiation within the cells (Constantinides et al., 1977). These experiments have been confirmed by Rainier and Feinberg (1988). Studies by Harrison et aZ. (1983)and Gadi et al. (1984) showed that some differentiated cell types induced by 5-Aza-CR were also tumorigenic. Walker and Nettesheim (1986) demonstrated that 5-AzaCR induced the transformation of rat tracheal epithelial cells in primary culture. Carr et al. (1984) showed that the drug could induce tumors in male Fisher rats, and Dendra et al. (1985) reported that the drug could potentiate the initiation of carcinogenesis in rats being fed diets containing various liver carcinogens. Finally, Samid et al. (1987) have shown that 5-Aza-CR can reinduce the transformation of TUS transfected cells that have been reverted to a nontumorigenic phenotype by interferon treatment. This evidence clearly shows that 5-Aza-CR can induce the transformation of cells and also that carcinogens that can inhibit DNA methyl-

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ation are capable of activating the expression of some genes known to be silenced by methylation. Overall the data strongly support the notion that methylation and carcinogenesis are linked. VII. Role of DNA Methylation in Tumor Diversification

Interpretation of experiments relating methylation changes to tumor progression are complicated because it is not certain whether the alterations observed are a cause or a result of the transformed phenotype. Fresh information that methylation plays a role in tumor progression and control of cellular behavior during metastasis has been obtained by a series of experiments using 5-Aza-CR to change DNA methylation patterns and to test the effects of the changes on tumor diversification and metastasis. Frost and Kerbel (1983)and Frost et al. (1984)were the first to show that 5-Aza-CR could strongly influence the tumorigenicity of cells by altering their immunogenicity. These initial findings were expanded by Kerbel et al. (1984),who suggested that the changes in tumorigenic potential were induced by altered methylation patterns. The same kinds of changes could also be seen at similar frequencies by treatment of the cells with agents known to be mutagenic, such as MNNG and EMS. Thus some of the effects of these carcinogenic and mutagenic agents on cellular behavior may be due to epigenetic effects on DNA methylation. Liteplo and Kerbel (1987) also showed that 5-Aza-CR could induce TK activity in a spontaneously enzyme-deficient murine tumor line when its metastatic potential was also altered by drug treatment. This provided additional evidence that methylation might be implicated in the control of the mtastatic phenotype. A series of studies from Olsson’s lab (Olsson and Forchhammer, 1984; Olsson et al., 1985a,b) showed marked effects of methylation changes on tumor cell behavior. They demonstrated that clones of murine Lewis lung carcinoma cells selected for nonmetastatic potential could be converted to a metastatic phenotype by brief exposure to 5-Aza-CR. The ability of human tumor cell lines to grow in semisolid medium, a property often associated with tumorigenicity, was also shown to be markedly responsive to 5-Aza-CR treatment. Olsson et al. (1985b) also tested the response of cloned lines of human squamouscell lung carcinoma and small-cell lung carcinomas to 5-Aza-CR7phorbol esters, and retinoic acid. 5-Aza-CR brought about a shortening of doubling time and increased cloning efficiencies, whereas the other two agents appeared to act in opposite directions. WhiIe the effects of 5-Aza-CR on cellular phenotype were quite variable, the experiments

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suggested strongly that alterations in DNA methylation had profound influence on the behavior of tumor cells as measured by the expression of several different parameters. The role of DIVA methylation in the expression of the metastatic phenotype in B16 melanoma cells was investigated by Trainer et al. (1985). Both 5-Aza-CR and 5-fluoro-2-deoxycytidine caused dosedependent increases in the abilities of B16 cells to form experimental lung metastases. Measurements by HPLC on 5-methylcytosine levels after treatment showed that these were decreased by the drug. The metastatic capacity of human tumor cell lines in nude mice has also been found to be strongly influenced by 5-Aza-CR (Ormerod et al., 1986). The drug induced a 40-fold increase in the number of lung tumor nodules compared to control cell lines. The cells populating the lung tumor nodules retained increased metastatic capacity through several cycles of growth in uitro followed by reinjection into nude mice. Examination of the methylation levels showed that 5-Aza-CR had induced a significant decrease in methylation directly after treatment but that this extensive hypomethylation was not maintained in the cell lines derived from the lung nodule lines. These studies have shown in general that 5-Aza-CR can markedly increase the rate of tumor progression and diversification. However, in certain cases 5-Aza-CR can retard the progression of cells to a more malignant phenotype. Babiss et al. (1985) showed that the rate of progression of adenovirus 5-transformed rat embryo cells was retarded by 5-Aza-CR treatment. Several experiments have shown that other drugs such as hydroxyurea that do not cause DNA hypomethylation and may in fact cause hypermethylation (Nyce et al., 1986) can also substantially alter the metastatic activity of cells (Frost et al., 1987; McMillan et af., 1986; Alvarez et al., 1988).Thus not all of the effects of 5-Aza-CR on tumor metastasis may necessarily be due to changes in DNA methylation. Clearly the drug might act in some cases to change the phenotype in cells by mechanisms more related to its inherent toxicity. However, it remains true that 5-Aza-CR is the most potent agent for altering tumor progression and metastasis in these defined systems. The final proof that 5-Aza-CR acts through a DNA methylation-linked mechanism will require the isolation of the putative genes activated by drug treatment. VIII. DNA Methylation during Oogenesis and Spermatogenesis

Several studies have demonstrated substantial differences in the methylation of DNA sequences in sperm and oocytes. These differences in methylation may be important in controlling the preferential

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expression of genes from either haploid genome during embryogenesis. Groudine and Conklin (1985) examined the chromatin structure of constitutively expressed genes, tissue-specific genes, and inactive genes in chicken sperm chromatin. They found specific sites of hypomethylation in sperm DNA within constitutively expressed genes, but not within globin genes or inactive genes. This corresponded to the location of altered chromatin structure (hypersensitive sites) in somatic tissue and spermatogonial cells. The authors also found that considerable de novo methylation occurred during spermatogenesis, so that regions within and around the genes became methylated but hypersensitive sites did not. This de novo methylation occurred between the spermatogonial stage and the first meiotic prophase. The undermethylated regions were postulated to play important roles in the activation of the paternal genome during embryogenesis. Monk et al. (1987) examined globin methylation during mouse development. The egg genome was strikingly hypomethylated whereas the sperm genome was methylated at a level similar to that found in mature tissues. Monk et al. (1987)also found a loss of genomic methylation during preimplantation development, with the embryonic and extraembryonic lineages becoming progressively and independently methylated to different extents. Sanford et al. (1987) have examined the methylation levels of dispersed, repeated, and low-copy-number gene sequences during gametogenesis and early embryogenesis in the mouse. These sequences were extensively hypomethylated in diplotene oocytes and highly methylated in DNA from sperm. Thus the results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavagestage embryos from inner cell masses were methylated at intermediate levels consistent with the idea that the different methylation levels present in sperm and eggs were carried through to the early embryonic stages of development. These findings are in line with the idea that DNA methylation may play a role in genomic imprinting in mammalian development. Since earlier studies by Chapman et al. (1984) and Rossant et al. (1986) had shown that these same sequences were highly methylated in the embryonic portion of the conceptus by 7.5 days after conception, the initial differences between the maternal and paternal genomes may be obliterated at later stages of development. However, not all sequences that are differentially methylated by passage through the paternal or maternal lines behave in this way, since some differences persist into adult stages of development (see later).

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IV. DNA Methylation and Genomic Imprinting The maternal and paternal genetic complements of mammalian embryos are not functionally equivalent during development (Surani et al., 1987). After fertilization the two haploid gene sets cooperate to direct the development of a complete embryo, and there is substantial evidence that memory of the gametic origin of the two haploid gene sets is maintained during early development. This process, called imprinting, refers to the marking of certain genes for differential utilization by their passage through the maternal or paternal germ lines. Experiments with mice have clearly shown that imprinting is confined to specific chromosomal regions, and it has been proposed that the phenomenon exists in mammals to prevent the formation of homozygotic fetuses (Surani et al., 1987). Imprinting must be established before or during gametogenesis, must persist during early cell division, must be stably inherited during DNA replication, and should potentially be reversible during new gametogenesis (Monk, 1988). As pointed out in several reviews, DNA methylation is a particularly attractive molecular mechanism for the propagation of imprinting (Monk, 1987,1988). Indeed several papers have clearly shown that the methylation status and sometimes the expression of transgenes in mice can be dependent on whether they have been inherited from the mother or father mouse. Reik et al. (1987) used random DNA insertions into transgenic mice to probe the genome for modified regions that might be subjected to methylation changes during transmission from male or female mice in different strains. One of the seven loci studied showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. Importantly, the methylation pattern was faithfully reversed upon each germ-line transmission to the opposite sex. Similar results were obtained b y Sapienza et al. (19871, who also showed that the methylation patterns of exogenous DNA sequences in transgenic mice could be changed by switching their gamete of origin in successive generations. Swain et al.(1987)extended these experiments to study the methylation and expression of an autosomal transgene constructed by fusion of the Rous sarcoma virus long terminal repeat (LTR) and the myc gene. If the transgene was inherited from the male parent it was expressed in the heart and in no other tissue. On the other hand, if the gene was inherited from the female parent, it was never expressed in any tissue tested. The pattern of expression correlated precisely with the imprinted methylation state evident in all tissues. Thus, the meth-

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ylation of the gene acquired by its passage through the female parent was eliminated during gametogenesis in the male, which resulted in the subsequent expression of the gene in the heart. While these experiments do not show that the regions studied are necessarily the ones normally imprinted, and therefore relevant to mammalian development, they nevertheless show conclusively that the methylation status of a gene can be altered by its passage through the male or female germ line. Given the observation that methylation can lead to the suppression of genetic information, it may well be that early mammalian embryos are functionally hemizygous for some genes located on autosomes. This may be a mechanism to ensure that parthenogenic development could not occur. All of these results are consistent with DNA methylation playing a role in marking the paternal and maternal gene sets during early development. However, definitive proof that methylation is involved in impriting will require the isolation of a gene differentially expressed during early embryogenesis depending on its gametic origin. Nevertheless, the fact that there is evidence for differential gene utilization during development, and that methylation differences are substantial, strongly suggests that these differences may have relevance to the development of certain kinds of cancer. Many of the tumors that arise in early childhood are thought to have developmental etiology, and imprinting could conceivably play a role in the development of certain kinds of human cancer. It is not certain that imprinting exists in humans. However, it would appear that both maternal and paternal chromosomes are necessary for normal human embryogenesis, since hydatidiform moles, which contain only paternal genes, do not develop normally (Lawler et ul., 1982). This cannot be due simply to a requirement for heterozygosity, since some hydatidiform moles derive from a double fertilization by two separate sperm and are heterozygous (Lawler et al., 1982). Results from our laboratory on the methylation patterns of individual alleIes of the rus gene have also suggested that individual alleles of genes may be differentially methylated in humans (Chandler et ul., 1987). These experiments showed unequivocally that the two rus alleles present in human cells can bear different methylation patterns. If individual alleles can be differentially methylated, then it might be anticipated that this could alter the inheritance of certain kinds of human cancer. For example, if cells are functionally hemizygous for certain autosomal genes, then the frequency of occurrence of deleterious mutations at these loci would be many orders of magnitude higher than that anticipated for a diploid cell. The differential methyl-

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ation could also result in different mutation frequencies for the two alleles because of the known inherent mutagenic activity of 5methylcytosine (Radman and Wagner, 1986). Cytosine residues undergo spontaneous deamination to uracil, which-not being a normal component of DNA-is readily recognized and repaired by DNA excision repair enzymes. Methylated cytosine residues, on the other hand, deaminate at a higher frequency to produce thymine residues. Since the double-stranded DNA now has a TG pair mismatch, two repair mechanisms are possible: replace the T with C or G with A, with the latter giving rise to a permanent base substitution. Although there is some evidence that the cell differentially excises the T rather than the G in this situation (Brown and Jiricny, 1987), it has been suggested that the methylated cytosine residues are hot spots for mutation (Radman and Wagner, 1986). In fact the majority of restrictionfragment-length polymorphisms in the human genome are associated with C-to-T transitions (Barker et al., 1984). The hvo alleles of a gene in the same cell may therefore have different mutation rates if they bear different DNA modification patterns. Thus, if the genes have been imprinted by their passage through the paternal or maternal germ lines, there is the possibility that the gene inherited from each parent might have different mutation frequencies. There is in fact some evidence that such a situation might occur in Wilms’ tumor, which has been associated with homozygosity for chromosome l l p (Schroeder et al., 1987).The observation that homozygosity for specific chromosomes is found in many cancers (Hansen and Cavenee, 1987) has provided experimental evidence for a two-hit model for carcinogenesis, originally proposed by Knudson (1971).Sporadic tumors were postulated to result from two somatic events such as a mutation of one allele followed by loss of the normal allele, whereas familial tumors might be caused by inheritance of a mutant allele followed by loss of the wild-type gene. In most investigations on the allelic compositions of tumors, genetic markers within the tumors have been compared to normal tissue from the same individual without regard for the parental origin of the markers. However, strikingly unusual results have been achieved with Wilms’ tumor: in all of seven cases studied, the allele lost was that inherited from the mother (Schroder et al., 1987; Reeve et al., 1984). In five of these cases the tumor was unilateral and was not associated with any developmental anomaly, implying that each was due to a new somatic mutation on the paternally derived chromosome. This non-Mendelian behavior of alleles during the formation of this tumor suggests strongly that imprinting might be involved. Thus, the

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differential methylation patterns imposed on genes during their passage through paternal and maternal germ lines may well alter their susceptibility to spontaneously occurring mutations and thus alter the genetics of human cancer. X. Conclusions

The evidence that DNA methylation plays a fundamental role in carcinogenesis and tumor diversification has strengthened considerably. Carcinogens have been shown to inhibit methylation and to activate genes. Agents that inhibit methylation can transform cells and markedly increase the rate of tumor diversification. Many studies have shown that considerable alterations in methylation are evident in naturally occurring human and animal neoplasms at early stages of tumor development. Studies on methylation changes in early embryonic development have raised the interesting possibility that 5-methylcytosine could alter the genetics of develpment of childhood cancers. Overall the evidence is pervasive, but definitive proof will only be available when genes responsible for transformation and malignancy are isolated and characterized within these systems.

ACKNOWLEDGMENT This work was supported by grant R35 CA49758 from the National Cancer Institute.

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Wilson, V. L., Smith, R. A,, Longoria, J., Liotta, M. A., Harper, C. M., and Harris, C. C. (1987).Proc. Natl. Acad. Sci. U.S.A. 84,3298-3301. Wolf, S. F., and Migeon, B. R. (1985). Natzre (London)314,467-469. Wolfee, A. P., and Brown, D. D. (1988). Science 241,1626-1632. Yisraeli, J., Frank, D., Razin, A,, and Cedar, H. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 4368-4642.

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GENETIC AND EPIGENETIC LOSSES OF HETEROZYGOSITY IN CANCER PREDISPOSITION AND PROGRESSION Heidi J. Scrable, Carmen Sapienza, and Webster K. Cavenee Ludwig Institute for Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal,Quebec H3A 1Al Canada

I. Introduction 11. Genetics and Predisposition A. Cytogenetics of Retinoblastoma B. Molecular Genetics of Predisposition to Retinoblastoma 111. Loss of Heterozygosity and Tumor Progression IV. Loss of Heterozygosity in Mixed Cancer A. Association between Developmental Malformations and Tumors B. Tumors with Phenotypically Distinct Elements V. Epigenetic Inactivation of Alleles in Human Cancer A. Predisposition B. Progression VI. Conclusions References

I. Introduction There is an increasingly large body of evidence that supports the involvement of genetic lesions in the etiology of human cancer. The aggregation of tumors with occurrence incidences much greater than would be expected by chance in families such as that shown in Fig. 1is perhaps the single strongest formal corroboration of this notion. The occurrence of several types of cancer in individual members of a family is indicated in Fig. 1A. An attempt to discern a formal genetic definition of predisposition to the colon cancer trait is futile because its transmission is clouded by a spotty pattern of distribution. When the same family is categorized for a different phenotypic characteristic, stomach cancer, familial aggregation is also apparent; the formal description of a mutation giving rise to this disease is also unclear because transmission appears limited to the first few generations. Furthermore, the frequent occurrence of endometrial cancer in this family provides a phenotype that is not particularly informative given its necessarily sex-limited nature. Consolidation of the preceding phenotypic data makes it clear that cancer is prevalent in this family and that 25 ADVANCES IN CANCER RESEARCH, VOL. 54

Copyright 8 1990 by Academic Press,Inc.

A l l riEhts of reDroduction in any form reserved.

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A

I

T 7

B

FIG.1. Aggregation of cancers of different histogenesis in a family. (A) Individual members ofthe pedigree who developed cancers of the colon (diagonal stripes), stomach (crosshatching), or endometrium (dots). (B) Segregation of cancer in the family. In all cases, filled symbols represent affected individuals; circles represent females and squares represent males. This family was first ascertained by Warthin (1913).

cancers of each type have occurred uniquely in each affected individual. A phenotypic distillation accomplished by removing the qualifying tissue distribution designation and relying only on whether the family members have developed a tumor or not is shown in Fig. 1B. In this simplified display, cancer appears in the family with the formal genetic behavior of an autosomal dominant Mendelian trait. That is, disregarding the site of disease, approximately half the progeny of an affected parent have developed a tumor and no sex bias is obvious.

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This raises two paradoxes: (1) how a single mutation can give rise to any type of cancer given the complex nature of such a phenotype, and (2) how a single mutation can elicit diseases that are quite disparate with respect to the tissue affected. Regarding this latter point, it may be important to note that although this family has developed more than one tumor type, the distribution is actually quite limited in light of the large number of potential cancers that are not apparent. It may also be germane to note that familial aggregation of tumors of mixed histological types is often mirrored by the sequential occurrence in other individuals of second primary lesions after being affected first by cancer of different histogenesis. The final point is that in families such as that shown in Fig. 1, who appear to be transmitting heritable risk, the clinical description of the disease invariably shows focal lesions. This, of course, suggests that the mutation predisposes to, but is not itself sufficient to cause, the disease. On the surface this is entirely consistent with the notion that cancer represents the phenotypic manifestation of the accumulation of a critical load of genetic damage (Foulds, 1957; Nowell, 1976). This proposal is outlined in Fig. 2. The predictions it makes are of two types depending on the entry point into which the pathway is viewed. If this point is at the beginning, the circuitry can be viewed as an initiating genotoxic event with clonal outgrowth, perhaps due to incurred prolifEvents :

tNormaltInitiation+

Promotion-

ProgressionFTumor4

FIG.2. Pathway by which the increasing accumulation of genetic damage leads to malignant progression. N, Normal cell; I, initiated cell; Ti, Tz,TS, T4 refer to progressively more damaged cells in the promotion and progression stages of malignancy.Filled symbols indicate those cells that have undergone lethal mutations. This figure is redrawn from its original proposal by Nowell (1976),and all subsequent figures in this review utilize it in various permutations.

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eration advantages (N + I, Fig. 2). There is, however, a large body of experimental evidence indicating that this initial damage must be genetically fixed and then compounded. A cellular mechanism that causes biallelic fixation of the first event could cause the irreversible conversion to an expanded initiated clone (I 4TI, Fig. 2). Further damage to this clone would then permit (or perhaps even force) it to a stage of higher malignancy (TI + Tz, Fig. 2). Iteration of this process would then culminate in a fully malignant clone (Tz + T3 + Tq, Fig. 2). If the entry point is at the end of the pathway, then one is considering a cellular mass (T4)that is greatly removed from normality and that carries all of the genotoxic damage suffered in each of the previous stage transitions. This hypothesis, then, predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each ofthe events less the last one, and so on. Thus, the dissection of the pathway by which a normal cell becomes fully malignant may be viewed as the unraveling of a nested set of aberrations. Although this model does not specify the nature of the events that cause this malignancy progression, there is much reason to believe that many entail activating or inactivating mutations of genes encoding growth-related molecules. The intimate involvement of chromosomal aberrations in the neoplastic pathway has been postulated since the early part of this century. The major proponent of this idea was Boveri, who noticed that mitotic abnormalities often led to aberrant development of sea urchin embryos. These observations were concordant with the frequent occurrence of nuclear and mitotic abnormalities in carcinoma biopsy samples described during the same period by von Hansenmann. Since these pioneering studies, the technology necessary to elucidate chromosomal structure has become progressively more powerful. Malignant cells of many types have been subjected to cytogenetic analysis, and a massive number of deviations from the normal karyotype have been described, including chromosomal aneuploidy, translocation, deletion, and regional amplification (Helm and Mittelman, 1987).Moreover, many of these somatic alterations occur at high frequency in particular malignancies and so appear to be specific in that sense. Whether these chromosome aberrations are the cause or the result of the neoplastic process is generally unclear, but the studies described here make a strong primafacie case for the former, at least in some cancers. Furthermore, the power of molecular genetics in sorting through this array of cytogenetic abnormalities and in drawing mechanistic inferences regarding submicroscopic genetic and epigenetic defects in the process is illustrated by some of the studies reviewed here.

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II. Genetics and Predisposition

As already discussed, the earliest event in the pathway toward tumorigenesis is termed the initiating event in experimental chemical carcinogenesis. In the human population, such ,initiations may be transmitted as inherited predisposition (Fig. 3). At least 50 different forms of human cancer have been observed to aggregate in families as well as to have corresponding sporadic forms (Mulvihill, 1977). Obviously, these individuals represent a valuable resource in attempts to define the targets of initial genotoxic damage. In many of these cases the aggregation occurs with a pattern consistent with the transmission of an autosomal dominant Mendelian trait reminiscent of the family in Fig. 1. This interpretation is, however, at odds with three lines of evidence. First, if a single mutation were sufficient, in and of itself, to elicit a tumor, then families segregating for autosomal dominant forms of cancer would be expected to have no normal tissue in the diseased organ. This expectation is in direct contrast to the clinical observation of discrete tumor foci amidst normal, functional tissue in such individuals. Second, elegant epidemiological analyses (Knudson, 1986) of sporadic and familial forms of several cancer types have indicated that the conversion of a normal cell to a tumor cell requires multiple events. Finally, there is a substantial body of evidence derived from somatic

FIG.3. Pathway by which increasing genetic damage leads to malignancy: genetic nomenclature. N, Normal; P, predisposed; H, homozygote; H1, H2, H3, homozygotes with increasing levels of progressional damage. Such a pathway must necessarily begin in a single somatic cell in sporadic cases but can initiate in any cell in heritable cases as proposed by Knudson (1971).

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cell hybrids that indicates dominance of the wild-type phenotype in the presence of tumorigenic mutations (Stanbridge, 1987; Klein, 1987). Thus, in the context of the malignancy progression models in Figs. 2 and 3, the transition of cells from one stage to the next could be viewed as an accumulation of genetic lesions.

A. CYTOCENETICS OF RETINOBLASTOMA One important line of evidence in support of a genetic origin for cancer is the frequent finding of constitutional chromosomal abnormalities in patients with specific types of tumors. Perhaps the best characterized of these are deletions involving the chromosome region 13q14, which are found in normal tissues of 3-5% of children with bilateral retinoblastoma, a tumor of embryonic neural retina (Francke, 1976). Several such cases have now been reported and, although these deletions were rare and the extent of deletion varied considerably, the smallest overlapping region of aberration was the band 13q14. Furthermore, analysis of tumor cells from patients with normal constitutional karyotypes indicated that -5% of cases had tumor-specific deletions of chromosome 13, each of which included the q14 band (Balaban et al., 1982; Benedict et al., 1983; Squire et al., 1985).Though striking, the significance of these observations was unclear for several reasons. First, only a small proportion of cases carried visible constitutional or somatic deletions. Second, in those cases in which a 13q14 deletion was apparent in all cells, not all retinal cells were neoplastic; clearly these alterations were insufficient to elicit disease. Finally, tumor (but not constitutional) karyotypes have shown several other chromosomal aberrations in addition to deletion of 13q14, such as triplication of lq23-lqter and an isochromosome 6p (Squire et al., 1985). These complications notwithstanding, deletion cases have been useful in defining the region of the genome likely to contain a locus involved in the genesis of retinoblastoma through inference from the physical map and also genetic-linkage mapping. In the latter, activity levels of esterase D, an enzyme of unknown physiological function, were shown to be reduced in patients with deletions of 13q14 as compared to their karyotypically normal family members (Sparkes et al., 1980).This enzymatic activity also displayed isozymic forms in the human population, so that the cosegregation of a specific allelic variant with retinoblastoma could be determined in families (Sparkes et al., 1983). In fact, no meiotic recombination events could be detected, strongly suggesting that the genetic locus influencing tumor develop-

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ment resided in the 13q14 region. This was particularly important given the aforementioned rarity of cytogenetically detectable deletions of chromsome band 13q14 and because the primary mutation has not been characterized in most familial-form retinoblastoma cases. The involvement of this region has been substantiated by the identification and cytogenetic analysis of a remarkable kindred (Strong et al., 1981). In this family, both affected and unaffected members were described who carried an interstitial 13q14 deletion. However, several of the unaffected members also carried a chromosome 3 that included an interstitial translocation of this 13q14 region. Thus, all family members who were constitutionally monosomic for the region developed disease, whereas all who were either disomic or trisomic were spared. These data strongly suggested that the deletional event predisposed to retinoblastoma, that this region did indeed contain a locus or loci involved with tumor development, and that the initial mutation was unlikely to be acting in a dominant genetic fashion at the level of the individual tumor cell. Nonetheless, in the context of the requirement for multiple events in tumorigenesis (Knudson, 1986), such deletions could act as the first “hit” and, when they are germinal, they could confer the risk of tumor formation in an autosomal dominant manner, depending only on the number of cells at risk for further damage.

B. MOLECULAR GENETICS OF PREDISPOSITION TO RETINOBLASTOMA A model has been proposed (Knudson, 1971; Hethcote and Knudson, 1978)encompassing the aforementioned cytogenetics and the observation that familial cases are generally multifocal and bilateral whereas sporadic cases typically manifest as unilateral, unifocal disease of later diagnosis. According to the model, as few as two stochastic mutational events are required for tumor formation. The hereditary cases would have inherited a germinal mutation that does not in itself cause the tumor but rather predisposes each retinal cell to a further transforming event. In this model, the nonhereditary cases would arise by a similar mechanism but both events would have to occur in the same somatic cell. Thus, the two forms of the disease could be viewed as resulting from the same two-step process at the level of the aberrant retinal cell, the difference being the inheritance or somatic occurrence of the first mutation. Several predictions can be made about the nature of the second tumorigenic event in this model. First, the autosomal dominant hereditary form of retinoblastoma, in the absence of a gross chromosomal deletion, should involve the same genetic locus as that invoIved in

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cases showing large deletions of chromosome 13. Thus, the first step in the pathway toward tumorigenesis in these cases should be a submicroscopic mutational event at the tumor ( T M R ) locus. Second, the same genetic change that has occurred as a germ-line mutation in hereditary retinoblastoma should occur as a somatic genetic alteration of the TMR locus in a retinal cell in nonhereditary retinoblastoma. Third, the second step in tumorigenesis in both heritable and nonhereditary retinoblastoma should involve somatic alteration of the normal allele at the TMR locus in such a way that the mutant allele is unmasked. Thus, the first mutation in this process, although it may be inherited as an autosomal dominant trait at the organismal level, is, in fact, a recessive defect in the individual retinal cell. The model that arises (Cavenee et al., 1983) from these considerations is shown in Fig. 4, which outlines specific chromosomal mechanisms that would allow phenotypic expression of a recessive germinal mutation of the T M R locus. In this model, the heritable form of the disease arises as a germinal mutation of the TMR locus and is inherited by an individual who therefore is an obligate heterozygote (t/+) at the TMR locus in each of his somatic and germ cells. A subsequent event in any of his retinal cells that results in homozygosity for the mutant allele (i.e., mutant at the TMR locus on both chromosome homologs) will ultimately result in a tumor clone. Chromosomal mechanisms that could accomplish this loss of constitutional heterozygosity include (a) mitotic nondisjunction with loss of the wild-Qpe chromsome, which would result in hemizygosity at all loci on the chromosome; (b) mitotic nondisjunction with duplication of the mutant chromosome, which results in homozygosity at all loci on the chromosome; or (c) mitotic recombination between the chromosomal homologs with a breakpoint between the TMR locus and the centromere, which would result in heterozygosity at loci in the proximal region and homozygosity throughout the rest of the chromosome including the T M R locus. Regional events such as gene conversion, deletion, or mutation must also be considered. Heritable and sporadic retinoblastoma could each arise through the appearance of homozygosity at the TMR locus, the difference being two somatic events in the sporadic case as compared to one germinal and one somatic event in the heritable case. The approach that has been taken to examine these hypotheses relies on the variability of DNA sequences among humans. Chromosome-specific, single-copy segments of the human genome, isolated in recombinant DNA form, can be used to recognize polymorphisms at the corresponding chromosomal locus. Sequence variation in restriction endonuclease recognition sites, giving rise to restriction-

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tmr

33

t

Chromosomes :

FIG.4. A model for chromosomal mechanisms that can accomplish the conversion of a normal (N) cell to a cell that is homozygous (H) for inactivation of a tumor (TMR)locus. Predisposition occurs either by inheritance or by somatic occurrence of a mutation that converts a wild-type (+) allele to an inactive allele (TMR).A tumor could then occur by elimination of the remaining wild-type allele by nondisjunction (A), nondisjunction/ duplication (B), mitotic recombination (C), or regional aberration (D), as shown by Cavenee et aE. (1983).

fragment-length polymorphisms (RFLP) of the locus defined by the probe, are revealed as distinct bands on an autoradiogram and represent alleles of the locus (one from the paternally derived and one from the maternally derived chromosomal homolog) and behave as Mendelian codominant alleles in family studies. These RFLP can be used as linkage markers in inherited disorders, including retinoblastoma. If a disease locus is located close to a polymorphic RFLP marker locus, they are likely to segregate together in a family. Therefore, the genotype of DNA markers can be used to infer the genotype at the retinoblastoma locus, and thus to predict if the offspring has inherited the

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predisposition. Chromosome segregation during tumorigenesis can also be determined in each patient by comparing the child’s constitutional and tumor genotypes at each of these marker loci. Recombinant DNA segments have been isolated from human chromosome 13 and used in several such cases to determine somatic changes in the germline genotypes in the manner illustrated in Fig. 4. Detailed analyses of many retinoblastomas (Cavenee et al., 1983, 1985; Dryja et al., 1984) have shown that such events are common and are detected in about three-fourths of all retinoblastoma tumors. These rearrangements fall into four different classes as illustrated in Fig. 4. For example, in 20 of 33 tumors, one constitutional allele was missing at all informative loci along the entire chromosome, and 19 of these tumors contained two intact chromosomes 13 as determined either b y cytogenetic analysis of the tumor cells or densitometric quantitations of the autoradiographic signal of the remaining alleles. Therefore, these losses of alleles must involve two separate events: a nondisjunction resulting in loss of one chromosomal complement and either a duplication of the remaining homolog or an abnormal mitotic segregation of the chromosomes resulting in isodisomy as illustrated by the Retin 409 case in Table I. In one case, data consistent with the sole loss of chromosome 13 were obtained. Evidence for mitotic recombination between the chromosome homologs was provided in 4 of the 33 tumors (one example is Retin 412, Table I). The constitutional genotype was maintained at all informative loci in 9 of the 33 tumors, and therefore, in these cases the mechanism of attainment of homozygosity could not be determined. These studies strongly suggest that the second component in tumor initiation consists of a specific chromosomal rearrangement resulting in physical loss of the balancing wild-type allele. This inference was corroborated by examining cases of heritable retinoblastoma and showing that the chromosome 13 homolog retained in these tumors was derived from the affected parent as would be predicted. Two examples are shown in Table I: Retin KSBH and Retin

462F. It is noteworthy that although the unmasking of predisposing mutations at the RBI locus occurs in mechanstically similar ways in sporadic and heritable retinoblastoma cases, only the latter carry the initial mutation in each of their cells. Heritable cases also seem to be at greatly increased risk for the development of second primary tumors, particularly osteogenic sarcomas (Kitchin and Ellsworth, 1974). This high propensity may not be merely fortuitous but may be genetically determined by the predisposing RBI mutation. This notion of a pathogenetic causality in the clinical association between these two rare

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TABLE I FOR LOCION CHROMOSOME 13q IN RETINOBLASTOMAS Loss OF HETEROZYCOSITY AND OSTEOSARCOMAS Alleles present at locus" Tumor type Retinoblastoma Sporadic Retin LA69 Retin 409 Retin 412 Heritable Retin KS2H Retin 462F Osteosarcoma Sporadic Osteo 03

Tissueb D13S1

D13S7

D13S4

D13S5

013853

Mechanism

N T N T N T

1,s 2 2,2 2,2,2 172 14

1,l 1 172 2,292 2,2 2,2

N T N T

2,2 2,2 1,2" 172

1,l 1,1 1,l 1,1

Mitotic recombination

1,2

Isodisomy

N 132 T 24 Osteo 06 N 192 T 1,s Osteo 09 N 22 T 2,2 Second primary to retinoblastoma Rbl-1 N 1,2 T 171 Rb108 N 19 T 17172

Chromosome loss Isotrisomy Mitotic recombination Isodisomy

191

1,l 1J 172 171 2,2 2,2,2 172 171

Isodisomy Isodisomy Translocation, isodisomy Translocation, isodisomy

a Alleles designated in bold type are combinations that were heterozygous in constitutional tissue. N, Normal; T, tumor. 'D13S6 was examined, not D13S1. Not determined.

'

tumor types was tested by determining the constitutional and osteosarcoma genotypes at RFLP loci on chromosome 13 (Hansen et al., 1985). The data (Table I) indicated that osteosarcomas arising in retinoblastoma patients had become specifically homozygous around the chromosomal region carrying the RBI locus. Furthermore, these same chromosomal mechanisms were observed in sporadic osteosarcomas, suggesting a genetic similarity in pathogenetic causality. These find-

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ings are of obvious relevance to the interpretation of human mixedcancer families such as the one in Fig. 1, as they suggest differential expression of a single pleiotropic mutation in the etiology of clinically associated cancers of different histological types. A likely explanation for the association between retinoblastoma and osteosarcoma is that both tumors arise subsequent to chromosomal mechanisms that unmask recessive mutations in either one common locus that is involved in nornial regulation of differentiation of both tissues, or in separate loci that are located closely within chromosome region 13q14. In either case, germ-line deletions of the retinoblastoma locus may also affect the osteosarcoma locus. Deletions are likely to be an important form of predisposing mutation at the RBI locus because a considerable fraction of bilateral retinoblastoma cases carry visible constitutional chromosome deletions, and submicroscopic deletions have been detected by reduction of esterase D activity and by molecular analyses using a cDNA for a gene that is, in all likelihood, the transcription product of the retinoblastoma locus (Friend et al., 1986; Lee et al., 1987; Fung et al., 1987). The information derived from these studies raises two points relevant to familial predisposition to cancer. Chromosomal mechanisms capable of unmasking predisposing recessive mutations occur in more than one tumor and, at least for chromosome 13, clinically associated tumors share this mechanism of pathogenesis. This latter point suggests that these loci have pleiotropic tissue specificity; however, this pleiotropy appears to be restricted to a small number of tissue types. A more extensive discussion of this point in relation to the model in Fig. 4 follows. The data in this section, however, suggest a general approach to identifying the chromosomal positions of loci the recessive alleles of which predispose to human cancer. The approach takes advantage of specific and frequent somatic chromosomal alterations in tumors and draws its power from the conjoint use of such information and familial genetic analysis. Clearly, one would anticipate that a segregating tumor trait will be genetically linked to the predisposing mutation that elicits it (Hansen and Cavenee, 1987). This single characteristic should provide a means of distinguishing predisposing from progressionally acquired genetic damage, since the latter would be unlikely to be genetically linked to the former in families. In any case, the relevance of this use of genetic analysis to define the first steps of malignant progression, which involve initial monoallelic genotoxic damage and its biallelic fixation (Fig. l),is established by these studies. The more general applicability of the approach has been established as well and is reviewed in Nordenskjold and Cavenee (1988).

LOSS OF HETEROZYGOSITY IN CANCER PREDISPOSITION

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111. Loss of Heterozygosity and Tumor Progression

The models shown in Figs. 2 and 3 predict that the ultimate stage of malignancy should encompass all previously occurring aberrations in addition to its own particular event. Clearly, the foregoing section suggests that the attainment of complete defectiveness at a “tumor locus” is one means of initiating the pathway of tumorigenesis. There is no a priori reason that similar mechanisms should be excluded from comprising at least some more distal events as well. In order to test this hypothesis we searched for a disease system characterized by the increasing acquisition of histologically-defined malignant criteria. This seemed a real consideration because other means of clinical progression could comprise, for example, a dominantly acquired increase in drug resistance, invasive capacity, metastatic potential, or growth factor responsiveness, as shown in Fig. 5. We chose the glial tumors for these first efforts at utilizing genotypic

E

Imr

Additional Events : Hemi/ Homozygosiiy- other locus

Chromosomes: +

IQ

i++tmr

it

10

Oncogene Activation Growth Factor Response Growth Foctor Receptor Amplification Drug Resistance Metastatic Potential

FIG.5. Additional genotoxic damage is required in the progression of homozygously defective (H) cells toward frank neoplasia. Potential events in the later steps of the pathway that could contribute, singly or in combination, are listed at right.

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analyses to place tumors into various stages of malignant progression. Gliomas, as a class, are the most common primary neoplasms of the central nervous system. Tumors of this type can be subclassified according to their cellular differentiation, displaying either astrocytic, oligodendrocytic, ependymal, or mixed composition, with astrocytic tumors occurring most frequently. Prognoses for individuals having astrocytoma vary according to the histopathologically-assessed malignancy grade of the tumor; however, all adult malignancy grades (grades II-IV) of astrocytoma respond poorly to radiation and/or chemotherapy and the 5-year survival rate for individuals with the most malignant form, glioblastonia (or GB, astrocytoma grade IV) is