Advances in Cancer Research Volume 72

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Advances in

CANCER RESEARCH Volume 72

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Advances in

CANCER RESEARCH Volume 72

Edited by

G e o r g e F. Vande Woude A B L-Basic Reseorch Progrmi National Cancer Institrite Frederick Cancer Research and Development Center I-rederick, Marylrind

G e o r g e Klein Microbiology and Ttrmor Biology Center Karolinska Institutet Stockholtn, Swedeti

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Contents

Contributors to Volume 72 ix

FOUNDATIONS IN CANCER RESEARCH Foulds’ Dangerous Idea Revisited: The Multistep Development of Tumors 40 Years Later George Klein

I. Foulds’ Rules 2 11. Oncogenetics 3 111. Other Destabilizing Mutations Caused by Loss of DNA

Repair Functions 11 IV. Molecular Biology of Multistep Carcinogenesis 14 V. Strong Selective Pressures Favor Multiple Escapes 1 7 V1. Conclusion 19 References 20

FOUNDATIONS IN CANCER RESEARCH Cancer Cells Exhibit a Mutator Phenotype Lawrence A. Loeb 1. Introduction 26 11. Historical Perspective 28 111. Requirements for a Mutator Phenotype 3 2 IV. Origins of Mutations 36 V. Candidate Target Mutator Genes 38 VI. Microsatellite Instability 4 3 VII. Theoretical and Practical Considerations 4 7 VIII. Summary and Perspectives 51 References 5 2

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Increasing Complexity of R a s Signal Transduction: Involvement of Rho Family Proteins Roya Khosravi-Far, Sharon Campbell, Kent L. Rossman, and Channing J. Der 1. Introduction 57 11. Ras Is a Point of Convergence for Diverse Extracellular Signal-Stimulated Pathways 60 111. Ras Activation of Raf-Independent Pathways Contributes to

Ras Transformation 65 1V. Ras Mediates Its Actions through Interaction with Multiple Effectors 6 9 V. Ras Activation of a GTPase Cascade: An Involvement of R h o Family Proteins in Transformation 78 VI. Rho Family Proteins Mediate Their Actions through Interaction with Multiple Effectors 85 VII. A Search for the Missing Link between Ras and Rho Family Proteins 95 VIII. Increasing Complexity of Ras Signal Transduction: A Boon or Bust for Drug Discovery and the Development of Anti-Ras Drugs for Cancer Treatment? 96 IX. Future Directions 97 References 9 9

B-Myb: A Key Regulator of the Cell Cycle Mark K. Saville and Roger I. Watson 1. Introduction 109 11. B-Myb Structure and Functional Domains 11 I 111. Transcriptional Regulation o f B-rnyb 115 IV. Modification of B-Myb Protein in the Cell Cycle 123 V. The Requirement for B-Myb in Cell Proliferation 1 2 7 VI. B-Myb Function 132 References 137

Alterations in DNA Methylation: A Fundamental Aspect of Neoplasia Stephen B. Baylin, James G. Herman, Jeremy R. Graff, Paula M.Vertino, and Jean-Pierre lssa

I. Introduction 142 11. Brief History of DNA Methylation in Eukaryotes 142 111. The Normal Roles for Cytosine Methylation in Higher Order Eukaryotes 144 IV. Abnormalities of DNA Methylation in Neoplasia 150 V. Mechanisms Underlying the DNA Methylation Changes in Neoplastic Cells 167 VI. An Overview of Tumor Progression That Incorporates the Roles of Altered DNA Methylation 186 VII. Clinical Implications of Altered DNA Methylation in Cancer 189 References 190

Contents

Ara-C: Cellular and Molecular Pharmacology S t e v e n Grant I. 11. 111. IV. V. VI. VII. VIII.

Introduction 198 Structure 200 Metabolism 200 Mechanisms of Cytotoxicity 205 Mechanism of Resistance 210 Signaling Pathways and Oncogene Interactions 213 Modulation of ara-C Associated Toxicity 220 Conclusions 225 References 225

Index 235

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Contributors

Nuinhers in parentheses indicate the pages on iubrch the authors’ contrihictions begin.

Stephen B. Baylin, The Johns Hopkins Comprehensive Cancer Center, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231 (141) Sharon Campbell, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 (57) Channing J. Der, Department of Pharmacology, Lineberger Comprehensive Cancer Center, Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599 (57) Jeremy R. Graff, The Johns Hopkins Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231 (141 ) Steven Grant, Departments of Medicine, Pharmacology, and Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 (197) James G. Herman, The Johns Hopkins Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231 (141) Jean-Pierre Issa, The Johns Hopkins Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231 (141) Roya Khosravi-Far, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (57) George Klein, Microbiology and Tumor Biology Center (MTC),Karolinska Institutet, S-17177 Stockholm, Sweden (1) Lawrence A. Loeb, The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195-7705 (25) Kent L. Rossman, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 ( 5 7 ) Mark K. Saville, Department of Medical Microbiology and Ludwig Institute for Cancer Research, Imperial College School of Medicine at St. Mary’s, Norfolk Place, London W2 lPG, United Kingdom (109)

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Contributors

Paula M. Vertino, The Johns Hopkins Comprehensivc Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21 23 1 (141) Roger J. Watson, Department of Medical Microbiology and Ludwig Institute for Cancer Research, Imperial College School of Medicine at St. Mary’s, Norfolk Place, London W2 1 PG, United Kingdom (109)

FOUNDATIONS IN CANCER RESEARCH

Foulds’ Dangerous Idea Revisited: The Multistep Development of Tumors 40 Years Later George Klein Microbiology and Tumor Bio/ogy Center Karolrnska Instrtiifet Stockholm. Suneden

I. Foulds’ Rules 11. Oncogenetics

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IV. V. VI.

A. Oncogenes B. Tumor Suppressor Genes C. DNA Repair Genes D. Genes That Influence Programmed Cell Death by Apoptosis Other Destabilizing Mutations Caused by Loss of DNA Repair Functions Molecular Biology of Multistep Carcinogenesis Strong Selective Pressures Favor Multiple Escapes Conclusion References

In his recent book on evolution, Darwin 2 Dangerous Idea, Dennett (1995) alludes to natural selection, an idea that has acted as a “corrosive acid,” dissolving established societal, ideologic, and religious structures. I have always regarded Leslie Foulds as the micro-Darwin of the neoplastic microevolution. Dennett’s title provoked the association that Foulds’ (1958)analysis of tumor progression, a term first used by Rous to describe the process whereby “tumors changed from bad to worse” (Rous and Beard, 1935), might have been corrosive as well, albeit on a much smaller scale, as befits a limited evolutionary process that is, moreover, pathological and a dead end in itself. Just as Darwinian natural selection has made creationistic myths obsolete in a single stroke, the concept that tumors develop by multiple, stepwise changes has dealt a fatal blow to the idea that has dominated cancer research during the first half of the century: that a single key event is responsible for tumor development. Biochemists, immunologists, cytogeneticists, and virologists kept searching, each with the methodology characteristic of their disciplines, for the decisive event that had to be discovered before the riddle of Advancer in CANCER RESEARCH 00hS-23OX/YX $25.00

Copyrighr 0 IYYX hy Academic Press. All righrs of reprorluurion In any trirm reserved.

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cancer could be solved. The Warburg hypothesis of a common metabolic disturbance, the mirage of a common cancer antigen, the universal cancer virus, and general chromosomal imbalance are some examples of the all-encompassing theories that were advocated with particular fervor. The multistep theory of cancer development originated from two main sources. Mathematical analysis of the age-incidence curves indicated that most of the major human tumors arose after five to seven mutation-like changes (Armitage and Doll, 1954; Farber and Cameron, 1980). Horizontal studies o n the natural history of tumor development at the tissue level, including Foulds’ own observations (Foulds, 1969; 1975), have defined some of these steps in histopathologic and biologic terms. The idea that tumors evolve after a series of mutations that provide the cell with a selective advantage within the gradual process of its emancipation from growth control has attained a dominating position in cancer research, just as Darwinian natural selection is accepted by all biologists (Klein and Klein, 1985a; Nowell, 1976).This concept is less generally recognized by the medical profession and only to a minor extent, if a t all, by nonbiologists. Actually, it makes good sense. It is generally accepted that cell division and the processes leading to it are regulated by a complex set of receptors as well as by signal transduction and transactivation pathways. The advent of knockout mice brought the unexpected news that several known genes that participate in this regulation can be deleted without fatal harm to the organism. Looking back a t the natural history of cancer as described by Foulds (1958) from o u r present knowledge concerning the regulation of the cell cycle by multiple signals, the loss of the controlled state as it occurs during neoplastic development must proceed through multiple steps.

1. FOULDS’ RULES The core of Foulds’ ideas is factorial analysis. In 1958, Foulds stressed that the neoplastic phenotype is composed of several unit characteristics, capable of independent reassortment and liable to independent progression. Progression was defined as a series of stepwise changes in several unit characteristics. This implied that progression could proceed along alternative pathways and, therefore, that each tumor of a given type had a certain degree of biologic individuality. During the past two decades, Foulds’ postulates have been provided with molecular meaning. There is only one major departure from the original text. Darwin developed his theory of natural selection without any knowledge of

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genes. A century later, Foulds formulated the rules of tumor progression but rejected the notion that mutations, in the strict sense of the word (i.e., changes a t the DNA level), could be responsible for its steps. H e accepted (Foulds, 1969) the conclusions of our early work (Klein & Klein, 1957; 1958),which postulated that the selection of ascites convertible variants isolated from solid tumors and of H-2 haplotype loss variants from H-2 heterozygous mouse tumors was due to variation and selection. He interpreted his own observations on the focal appearance of behaviorally different tumor cell variants in a similar way. Foulds did not believe, however, that phenotypic changes in a diploid cell could be caused by mutations because most of them would be recessive and remain “silent,” owing to the presence of a normal allele. H e chose to speak about neoplastic development, with a strong emphasis on the second word. This also became the title of his major monography (Foulds 1969; 1975). He attributed the steps of progression to epigenetic changes, akin to the phenotypic switches of differentiation. He was proved largely, if not completely, wrong on this point. Epigenetic, di’ ferentiation-related changes may occur and can be induced in tumors, but they are superimposed on a chain of consecutive changes at the DNA level. Tumor progression itself can be viewed as a combination of genetic and epigenetic changes, with mutations and DNA methylation, respectively, as paradigmatic mechanisms. There is only one known tumor that may have arisen by a purely epigenetic mechanism: the mouse teratoma, investigated by Mintz two decades ago (Mintz and Fleischman, 1981). The diploid tumor she used for these studies grew progressively and killed 100% of its grafted syngeneic hosts during an observation period of 8 years. Nevertheless, it could be fully normalized by implantation into an early blastocyst, whereupon it gave rise to all normal tissues of the mouse. Unfortunately, this important experiment has not been followed up a t the molecular level.

11. ONCOGENETICS Four worlds of genes are presently known that can contribute to tumor progression by constitutive activation, mutation, or deletion. They are, in order of their discovery, oncogenes, tumor suppressor genes, DNA repair genes, and genes that influence programmed cell death by apoptosis. There is a certain functional overlapping among these categories, as pointed out later. Some genes have multiple functions that may assign them to more than one category. It nevertheless seems practical to discuss the current state of the art under these four headings. Also, the tumorigenic process can be influenced by genes that belong to other functional categories. They may act

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by influencing vascularization, telomerase activity, invasion and metastasis, and immune escape. They are not the subject of this review.

A. Oncogenes The oncogenes catalogued and reviewed by Hesketh (1994)are highly conserved household genes. They are involved in signaling functions that can trigger or drive the cell cycle. Their illegitimate activation by structural o r regulatory changes may stimulate the cell to continuous proliferation. Some act by producing a growth factor in a cell that is normally programmed to respond to it, not to produce it. Others encode growth factor receptors o r signal-transducing proteins, which may mutate to emit a faulty signal, like a buzzer that has gotten stuck. Still others are transcription factors that have been illegitimately activated by mutation, retroviral insertion, chromosomal translocation, or gene amplification, with the stimulated DNA replication as a direct or indirect consequence. Activated oncogenes stimulate cell division in a dominant fashion. This meets one of Foulds’ objections, that cancer-related mutations would necessarily be recessive in somatic cells. Dominance does not mean, however, that an activated oncogene can transform any target cell. On the contrary, the transformation-sensitive target cell spectrum of each oncogene shows highly idiosyncratic features. Part of this is differentiation related. When the retrovirally transduced oncogenes were first discovered, it became apparent that each of them had its characteristic lineage- and/or differentiation-dependent “window” of transformable target cells (see Klein, 1982). Particularly informative experiments were performed with temperature-sensitive mutants of vsrc and v-erb B (Boettiger et al., 1977; Weintraub et al., 1982). These oncogenes could transform target cells of several lineages into undifferentiated neoplastic cells at the permissive temperature. Even a brief stay of the transformed cells at the nonpermissive temperature released the differentiation block imposed by the oncogene, however. Each cell type remained faithful to its lineage or, in Ephrussi’s term, its “epigenotype.” The oncogenes mutated to the terminal form of the respective lineage and stopped dividing. Subsequent reexpression of the oncoprotein at the permissive temperature could no longer transform the cells. This illustrates how the release of maturation arrest by a purely epigenetic switch can revert malignant behavior. The oncogene field has other idiosyncratic features. It is not easy to explain why some proteins within a certain signal-transducing chain or within a given receptor or transcription factor family can turn into an oncogene, whereas others within the same chain or family that might have been expected to have the same potential do not appear on the list of known oncogenes. It is also peculiar that some oncogene activation events-such as the

Foulds' Dangerous Idea Revisited

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constitutive switching on of c-myc by juxtaposition to an immunoglobulin locus in Burkitt lymphoma and rodent plasmacytomas (Klein and Klein, 1985b) or the creation of a new fusion protein by the bcrlabl translocation in chronic myelogenous leukemia (Konopka and Witte, 1985)-occur in all or most tumors of a given phenotype, whereas others are only found in a minority. B- and T-cell-derived lymphomas and leukemias are different in this respect in both mice and humans. All tumors of a given B-cell phenotype may show the same oncogene activation event, as exemplified by the Iglmyc and the Iglbcl-2 translocations in Burkitt lymphoma and in follicular lymphoma, respectively. T-cell leukemias and lymphomas of a given phenotype show a broad variety of different oncogene activation events (Rabbitts, 1994). In the human T-cell leukemias, oncogenes are mainly activated by chromosomal translocation, in contrast to murine T-cell tumors, in which activation is usually due to retroviral insertion (Jonkers and Berns, 1996). Still, the same broad variation prevails in both human and murine T cells. This difference between B and T cells may relate to the broader activability of T cells. Depending on the oncogene that has been juxtaposed to an an immunoglobulin locus by chromosomal translocation, the derived B-cell tumors differ with regard to their grade of malignancy. Activated myc is known to drive cell proliferation. It therefore makes sense that Burkitt lymphoma is a highly malignant tumor. It is also easy to rationalize that the Iglbcl-2 translocation-carrying follicular lymphomas are low grade because bcl-2 protects against apoptosis but does not provide a continuous drive to cell division (Korsmeyer, 1992). It is less easy to understand why bcl-I, now identified as cyclin D1, generates low-grade lymphomas after translocation to an ZgH locus (Weisenburger, 1991). This suggests that the activation of this single cyclin is not sufficient to provide a strong driving force for proliferation. Activation of c-myc by translocation into one of the physiologically rearranging immunoglobulin loci is thus a regular and probably rate-limiting (bottleneck)event in the development of Burkitt lymphoma and rodent plasmacytoma. In the course of solid tumor progression, c-myc or, alternatively, N-myc o r L-myc may be activated by gene amplification. This occurs only in a fraction of all tumors of a given type. In small cell lung carcinoma and neuroblastoma, myc amplification was found to be associated with the appearance of a more malignant variant (Little et al., 1983). There are many other examples of oncogene amplification. The prognosis-related amplification of c-erb B2, or m u , in breast carcinoma is particularly noteworthy among them (Slamon et al., 1987). Neither the mechanism of gene amplification nor its contribution to the malignant phenotype has been satisfactorily explained. In a particularly wellstudied model, the degree of dehydrofolate reductase amplification could be related to the level of methotrexate resistance (Biedler et al., 1983).Upon the release of the selective pressure by the drug, the amplified gene copies were

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lost. Extrapolating this to the case of oncogene amplification, Bishop (1983) suggested that the persistence of amplified oncogenes in growing tumor cells must mean that they provide the cell with a selective advantage. Amplification of an androgen receptor gene in hormone-independent variants of prostatic carcinoma is consistent with this concept because increased receptor expression facilitates the capture of the ligand, even if it is present only in a low concentration (Kallioniemi and Visakorpi, 1996).

B. Tumor Suppressor Genes The earliest indications suggesting the existence of genes that may antagonize tumor development came from several sources. Fusion of normal with malignant cells showed, unexpectedly, a regular suppression of tumorigenicity, as long as the chromosome complement derived from the normal parent was maintained (reviewed by Klein, 1976). Reappearance of tumorigenicity was associated with the loss of specific chromosomes from the normal parent. Another early finding was the isolation of more or less “normalized” revertants from tumor cell cultures under saturation conditions in which continued DNA synthesis of the tumor cells was made suicidal by highly radioactive thymidine or a combination of 5-bromodeoxyuridine and blue light. Although several types of revertant cells could be isolated and characterized, the interesting ones were those that maintained the original transforming oncogene in an unchanged form. For example, the viral Ki-ras gene was still active after helper rescue from the phenotypic revertants. The transformed phenotype was suppressed by one of several downstream-acting genes, exemplified by Krev-1 (Noda, 1993; Yanagihara et al., 1990). The molecular approach has shown that tumor development and progression are accompanied by numerous genetic losses. Studies o n colon, breast, prostate, kidney, lung, and brain tumors have created the inipression that gene inactivation may be more frequent than oncogene activation (Collins and James, 1993; Fearon and Vogelstein, 1990; Kallioniemi and Visakorpi, 1996; Kok et al., 1996; Kovacs et al., 1988; Lee, 1995; Yokota and Sugimura, 1993). The most meaningful functional information came from the study of two suppressor genes, RB and p53. It can hardly be a coincidence that the transforming proteins of three different DNA tumor viruses (SV40, adenovirus, and papillomavirus) have R B and p53 as their cellular targets and that they use essential domains of their transforming proteins to cancel their function (Selivanova and Wiman, 1995; Weinberg, 1995). This surprising convergent evolution among viruses of different origins may have been driven by the common requirement of resting cells to enter the cell cycle, to establish latent viral persistence. RB and p53 play important and different roles as cell cycle checkpoint con-

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trols. RB has a pivotal position a t the R (restriction) point that controls the transition from C1 to S phase (Weinberg, 1995). The nonphosphorylated form of the protein stops the cycle in middle to late G1 phase. This block to cell cycle progression serves not only as a control post in proliferating cells but also as a prerequisite for the exit of cells from the cycling compartment by differentiation or by senescence. The position of the RB protein within the complex chain of events that must take place to allow the cells to transgress the cycle is well known. Triggering of the cycle by appropriate ligand-receptor interactions and subsequent signaling elicit the activation of cyclin-cyclin-dependent kinase (CDK) complexes (Hall and Peters, 1996; Hunter and Pines, 1994; Sherr, 1993). This leads, after several steps, to the inactivation of RB by phosphorylation. Phosphorylated RB releases a bound transcription factor, E2F, that transactivates numerous genes and represses some. This is obviously a gross oversimplification of a much more complex situation with many additional participants, including a family of RB-like molecules (p107, p130, and others) and several different E2F-like factors. Only RB, however, has been identified as a tumor suppressor gene, and none of the E2F family members have been identified as potential oncogenes in vivo. Somewhat surprisingly, E2F1 knockout mice were found to develop a variety of tumors, perhaps owing to the loss of the suppressor function of E2F (Yamasaki et al., 1996). The loss of RB plays a direct causative role in retinoblastoma and is also essential for the development of osteosarcomas in RB families. It can also contribute to the progression of other tumors, including sarcomas and prostatic and bladder carcinomas (Knudson, 1993). A central theme of the oncogene field repeats itself: the same gene may act in a regular bottleneck capacity in causing tumors within a certain cell lineage; whereas in tumors derived from other cell types, it contributes to some late event that occurs only in part of the tumors during their progression and that may render them more invasive, metastatic, or less responsible to local growth-regulating signals. The earliest molecular analyses of familial retinoblastomas have shown that Foulds' objection to the mutational basis of tumor development and progression is not valid for recessive mutations either. Foulds could not see how a recessive mutation that affects only one of two alleles could be expressed in a diploid cell. It turned out, however, that one RB allele is mutated in the germ line, as Knudson originally postulated (Knudson, 1971), whereas the second is cancelled by chromosome loss, mutation, deletion, or mitotic crossing over during somatic development (Cavenee et al., 1983). In about 7 0 % of the tumors, the normal allele is eliminated through the loss of the whole chromosome by nondisjunction, with or without the duplication of the remaining chromosome. Different cytogenetic mechanisms operate in different individual tumors, even if they arise in the same patient, but the loss of the normal allele is their invariable common denominator.

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Progression through the cell cycle can be inhibited by several other proteins as well. Some have been identified as tumor suppressors, but others have not. Some of the more prominent examples deserve special mention. DNA damage is a potent inducer of p53. This switches on p21 (also known as WAF-l), a protein that can inhibit all cyclin-CDK complexes (Xiong et a/., 1993; elDeiry et al., 1992). Although p53 is an important tumor suppressor gene, p21 is less prominent in that capacity but is occasionally found mutated in prostatic cancers (Gao et d., 1995). A transforming growth factor p-inducible protein, p27, can inhibit one or more cyclin-CDK complexes as well but is not known as a tumor suppressor gene either (Toyoshima and Hunter, 1994). In contrast, p16 and its close relative p l 5 , which specifically inhibit cyclin Dl-CDK4 complexes, were identified as suppressor genes because they have mutated or became methylated in many different tumors. The differential significance of the various cyclin- and CDK-inhibitor proteins in relation to tumor development has not been fully explained (Hall and Peters, 1996).

C. DNA Repair Genes More than half of all tumors carry mutant p53, the most frequently mutated gene in human tumors (Selivanova and Wiman, 1995). The tumorassociated p53 mutants have lost the ability to bind to specific p53 binding sites on double-stranded DNA by the middle domain of the protein molecule, or have only a strongly reduced specific DNA binding. Most are missense point mutations, suggesting a positive selection for mutant p.53, in contrast to RB, which is usually inactivated by nonsense mutation. The high frequency of p53 mutations is consistent with the broad involvement of this protein in the control of the cell cycle and apoptosis, its response to DNA damage and other inducers, and its ability to influence tumor development in several different ways. Certain p53 mutants have a direct transforming effect in vitro, even on p53 knockout fibroblasts, and can thus be regarded as oncogenes (Dittmer et al., 1993; Hann and Lane, 1995; Milner, 1994). Other mutants have no direct transforming activity but have lost their suppressor function. They no longer bind specifically to DNA and cannot arrest the cycle. Some of these mutants act as dominant negatives, owing to their ability to heterodimerize with wild-type p53 and thereby inhibit its function (Dittmer et al., 1993; Zambetti and Levine, 1993). p53 has been called the “guardian of the genome” (Kastan et af., 1991; Lane, 1992). Normal p53 is a short-lived protein that is expressed a t a low level. Damaged DNA or other p53 inducers, such as adenoviral E1A (Debbas and White, 1993; Lowe and Ruley, 1993) or elevated c-myc (Hermeking and Eick, 1994), raise the p53 level by stabilizing of the protein. The accumulated p53 arrests the cycle by the transcriptional transactivation of p21,

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as mentioned earlier. DNA repair enzymes gain tine to work during this growth arrest and to correct some of the damage. If they are successful, the level of p53 declines, and cell division can proceed. RB is essential to normal development; p53 knockout mice, however, develop relatively normally but are highly tumor prone as adults (Donehower et al., 1992).Germline p53 mutations in humans are associated with the LiFraumeni multicancer syndrome (Malkin, 1993; Strong et al., 1992). Although the tumor spectra in men and mice are different, tumor incidence is believed to increase in the absence of the guardian function because the lack of the p53-mediated growth arrest response to DNA damage permits the survival of many genetic variants that would have been eliminated otherwise. Mutated p53 can thus also be considered a destabilizing gene. Other destabilizing genes are discussed in a separate section later. As already mentioned, high levels of p53 can induce growth arrest and elicit apoptosis. Different pathways are involved in these two reactions. Wildtype p53 plays an important role in a t least one major apoptotic pathway that is nonfunctional in mutant p53-carrying and p53-negative cells (Evan et al., 1995). Thus, p53, which is basically regarded as a tumor suppressor gene, can influence the probability of programmed cell death, as can a variety of activated oncogenes, including c-myc.

D. Genes That Influence Programmed Cell Death by Apoptosis More specialized genes regulate the probability of programmed cell death by apoptosis. These genes can also act as important modifiers of tumor development. The potential to elicit a nucleolytic breakdown of DNA is part of the normal developmental and functional repertoire of all organisms. It is essential for morphogenetic remodeling in lower forms. Its genetic control has been thoroughly analyzed in the nematode Caenorhabditis elegans, in which the full set of apoptosis-preventing and apoptosis-triggering genes has been identified (Horvitz et al., 1994). In vertebrates, a vitally important apoptotic function is responsible for the removal of lymphocytes that have not been called on by the cognate antigen within a limited period after they have rearranged their TCR or immunoglobulin genes. Prevention of their apoptotic destruction may favor neoplasia. The most important known apoptosis-modifying gene in mammalians, bcl-2, has been discovered as a result of its illegitimate rearrangement by chromosomal translocation in human follicular lymphoma (Tsujimoto et al., 1984). Most follicular lymphomas carry a reciprocal translocation between the IgH cluster on chromosome 14 and a gene on chromosome 18. In a minority of cases, the light-chain genes are involved on

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chromosome 2 or 22. The juxtaposed sequence from chromosome 18 has been identified by cloning the 14;18 translocation breakpoint and designated as bcl-2. It was found that bcl-2 elevates the apoptotic threshold by heterodimerizing with another protein, bax (Oltvai et al., 1993), which promotes apoptosis in the homodimeric form. Transgenic mice that carry immunoglobulin enhancer-bcl-2 constructs have chronic lymphocytosis (McDonnell et al., 1989; Strasser et al., 1991). Moreover, their B cells survive for abnormally prolonged periods in vitro. Some develop slow-growing, low malignant lymphomas late in life. Their human counterpart, follicular lymphoma, is also low grade. Occasionally, a second translocation that juxtaposes myc to an immunoglobulin locus may occur in the human lymphoma (Karsan et al., 1993; Gauwerky et al., 1988). This invariably leads to high-grade lymphoma. The same is found in crosses between bcl-2 and myc transgenic mice. It is easy to rationalize the fact that constitutive activation of a gene that protects from apoptosis but provides no drive for continuous cell division only gives rise to low-grade lymphoma, perhaps triggered by the normal microenvironment that is predestined to stimulate legitimately activated lymphocytes. It is also understandable that myc, a proliferation-stimulating transcription factor, provides an impetus for high-grade lymphoma development. It is more difficult to rationalize the fact that the gene cloned from 11;14 translocations in human low-grade lymphoma, bcl-2, now identified as cyclin D1, induces low-grade rather than high-grade tumors, as mentioned previously (Harris et al., 1995). This may imply that a single cyclin may not be sufficient, in contrast to myc, to push the cell through the whole cycle, without the aid of other factors. This is also consistent with the fact that no other cyclins were found to act as oncogenes, with the possible exception of cyclin E, which is highly expressed in some tumors at an advanced stage of progression (Dou et al., 1996). Other oncogenes that may protect cells from apoptosis include the tyrosine kinase gene v-abl, which acts through a bcl-2-independent pathway (Cotter, 1995; Fernandes et al., 1996). Proteins encoded by DNA tumor viruses (e.g., the Epstein-Barr virus-encoded membrane protein LMPl ), protect their B-cell hosts from apoptosis by elevating cellular bcl-2 expression (Henderson et al., 1991; Okan et al., 1995). As mentioned earlier, p53 is only required for certain apoptotic pathways, such as for radiation-induced apoptosis in lymphocytes. Loss of p53 elevates the apoptotic threshold of lymphocytes exposed to radiation and DNA-damaging agents such as mitomycin C and etoposide, but not to dexamethasoneinduced apoptosis. High levels of bcl-2 expression were observed to block some apoptotic pathways, but not others (Strasser et al., 1994). Protein products of the bcl-2 gene family, which includes bax, bad, and several other genes, interact to form various homodimers and heterodimers that can ei-

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ther accelerate or inhibit programmed cell death (White, 1996). Like other p53 target genes, bax is a unique p53-regulated gene. Its induction by genotoxic stress requires not only that p53 is functional but also that the cells are apoptosis “proficient” (Liebermann et al., 1995). Protection against apoptosis by p53 mutation is also important in another oncogene-related context. Activated myc can drive cells not only to proliferate but also to apoptose, depending on the cell type and the environmental conditions (Evan et al., 1992). This may be related to o r even dependent on the fact that activated c-myc can induce p53. The same domains of c-myc are required for transformation as for apoptosis. Mutations in p53 may protect cells driven by activated myc genes (e.g., Burkitt lymphoma cells) from apoptosis. Counterbalancing effects of this type may play a major role in multistage carcinogenesis. Protection of Burkitt lymphoma cells from apoptosis may be important also from another point of view. Most Burkitt lymphoma cells express CD77 (BLA), identified as a cell surface glycolipid (Wiels et al., 1981). The same unusual marker also appears on the surfaces of normal germinal-center centroblasts and centrocytes (Gregory et al., 1987).These cells migrate from the mantle zone of the lymph nodes, rearrange their immunoglobulin genes on the way, and tend to undergo massive apoptosis when they arrive to the interior of the germinal center. This probably reflects the fate of the B cells that failed to meet an appropriate activating complex on the way. A ligand, verotoxin, which binds to CD77, was found to elicit apoptosis. This suggests that CD77 may be a receptor for a natural ligand that triggers apoptosis within the germinal center. With Barbro Ehlin-Henriksson, we recently found that all 9 tested Burkitt lymphoma cell lines that expressed BLA a t a high level carried only mutant but no wild-type p53, whereas only 9 of 14 low CD77 expressors had mutated p53 in single or double dose. Selection for p53 mutations may thus protect cells with the BL phenotype from myc-driven and possibly even physiologic apoptosis. Thus, p53 can play four different roles in tumor development and progression-as an oncogene, a tumor suppressor gene, a destabilizing gene, and an apoptosis-influencing gene-even though its action in the latter capacity may be more indirect and tied to the function of p53 in cell cycle regulation.

111. OTHER DESTABILIZING MUTATIONS CAUSED BY LOSS OF DNA REPAIR FUNCTIONS In 1909, Ehrlich wrote a widely quoted sentence, often regarded as the first formulation of the immune surveillance theory. He stated that the “complex

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fetal and postfetal development” must involve “many aberrations” and that these would allow tumors to develop in an “enormous frequency,” unless eliminated by the “defense forces of the organism.” Ehrlich meant immune defense. We know now, however, that the first line of defense is not immunity (except against virus-transformed cells), but DNA repair. We should have known it before. The rare recessive condition known as xeroderma pigmentosum affects a single enzyme system responsible for the excision of thymidine dimers from the DNA of keratinocytes damaged by ultraviolet light (see Lambert et al., 1995). Hundreds of carcinomas develop in all daylight-exposed areas of the skin. It has also been known for a long time that inherited chromosome fragility is associated with increased cancer frequencies in ataxia telangiectasia, Fanconi anemia, and Bloom syndrome (reviewed by Kidson, 1980). The multicancer syndrome of humans with inherited p53 mutations and of the p53 knockout mice, mentioned earlier, is a more recent example. The equipment of DNA repair enzymes is faultless in this case, but they are not given time to work properly, owing to the absence of the p53-dependent growth arrest. There was more to come. Yeast geneticists have often pointed to the possible analogies between cancer and the destabilizing mutations in yeast that affect some part of the DNA repair enzyme system (Hartwell and Kastan, 1994). Loeb has also suggested that the mutator phenotype is a prerequisite for multistage carcinogenesis (Loeb and Cheng, 1990). The fidelity of DNA replication, the mechanisms of DNA repair, and the mechanisms of chromosome segregation were suggested as the vulnerable points that may trigger or favor cancer development when damaged. More specifically, spindle errors produce aneuploidy, spindle pole errors may lead to polyploidy, and faulty replication can generate translocations, deletions, and amplification. Most recently, mismatch repair defects were identified as the causative factor in familial hereditary nonpolyposis colon cancer (HNPCC), also called Lynch syndrome (Peltomaki and de la Chapelle, 1996). At least five highly conserved mismatch repair enzymes, known from bacterial and yeast genetics, were alternatively mutated in different families with the syndrome. First, a genetic linkage between HNPCC and a site on chromosome 2p was discovered in some families. Tumors arising in the HNPCC families showed frequent microsatellite instability, characterized by slippage in repeat number. This was taken to indicate that mismatch repair defects were the cause of HNPCC. Later, the human homologue of the bacterial mismatch repair enzyme MutS was found to map to 2p, and mutations in the corresponding human gene MSH2 were shown to be responsible for HNPCC. A second HNPCC locus, MLH1, was discovered by linkage analysis on chromosome 3p. Three further loci have been identified as well. Importantly, sporadic tumors were also found to carry mutations in genes involved in hereditary HNPCC. Mutations in mismatch repair genes induce multiple replication errors

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(RER),leading to microsatellite instability, also called the RER-positive phenotype (Casares et al., 1995). Although most of the tumors from identified HNPCC patients were RER-positive, the frequency of RER-positive tumors largely exceeds that of HNPCC patients. Most of the known HNPCC families carry MSH2 or M L H l mutations that map to chromosome 2 and chromosome 3 , respectively. Most Finnish families have chromosome 3 mutations, indicating a founder effect, whereas chromosome 2 mutations are more common in the United States (Peltomaki and de la Chapelle, 1996). In families carrying germline mutations, the mutation in the second allele occurs during somatic development and is then associated with microsatellite instability. HNPCC families show not only an increased colon tumor incidence but also endometrial, stomach, gallbladder, pancreas, and urinary tract tumors. It is not yet clear whether all HNPCC patients carry a mismatch repair gene mutation or whether all carriers of such a mutation can be regarded as HNPCC patients. Among sporadic tumors, microsatellite instability was found in colon, stomach, and endometrial cancers. Although genetic instability is thus clearly an initiating or a contributory factor to many forms of tumor development, the idiosyncratic nature of the tumor spectra generated by the different repair gene mutations is difficult to explain. It suggests the probable existence of lineage- or differentiationdependent differences in repair gene function or, as in the case of xeroderma, in the mutagenic exposure. Genetically determined variations in the likelihood of different tissues to undergo neoplastic transformation were documented during the early history of experimental cancer research (Heston, 1959). Inbred strains of mice selected for high incidence of mammary carcinoma, lung adenoma, thymic lymphoma, and adrenal carcinoma were found to carry multiple genes that increased the probability of tumor development. These genes were shown to act at several different levels. Selective breeding of other mammalian species, which favors complex phenotypes, such as the race horse or dog breeds with complex behavioral patterns, leads to the fixation of many different genes that contribute to the ultimate phenotype. Each gene contributes to the multigenetically determined trait in an indirect way and is neither necessary nor sufficient by itself. Selective inbreeding of mice for high mammary tumor incidence has led to a similar result. Some of the known susceptibility genes promote mammary cancer development by influencing the hormonal environment. Others make the host permissive for the replication of a cancer-promoting retrovirus (MMTV). Similarly, selection for high leukemia incidence has fixed genes that favor the replication of leukemia-promoting viruses (MuLV)and other genes that impair the specific immune response of the host. Genes that influence retroviral replication or immune surveillance against retroviral proteins are not highly relevant for human carcinogenesis. A third

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set of proven but little-known genes that were found to influence tumor development in the high tumor strains at the level of the target tissue may be more pertinent. The existence of such genes was shown by experiments based on the transplantation of the normal tissue-mammary gland, thymus, lung, o r adrenal-from strains with high or low incidence of the corresponding tumor, into common F1 hybrid recipients. F1 hybrids derived from the crossing of two homozygous strains accept transplants from both parental strains. Having been implanted into the same host, the target tissues of a low- and a high-cancer-prone strain were thus exposed to the same hormonal, viral, and other potential carcinogens. Tumors arose with a significantly higher frequency in the tissues of the high-cancer strain than in the tissues of the lowcancer strain in all four systems. How could genes selectively influence the propensity of a given cell type t o undergo neoplastic transformation in a given host environment? Tissuespecific repair, tissue-specific apoptosis, or the scavenger mechanism might be affected. These may or may not belong to the same category of phenomena as those responsible for the previously discussed idiosyncratic features of tumor incidence in patients with certain repair defects. Another tissue-specific cancer-prone case may be of interest in the same context. We have found that the high susceptibility of the BALB/c strain to plasmacytoma induction by pristane oil is determined, at least in part, at the level of the target cell. Tumors induced in reciprocal chimeras between the susceptible BALB/c and the resistant D B N 2 strain originated exclusively from BALB/c cells in both combinations (Silva et al., 1991 ). The special susceptibility of the BALB/c cell may be due to a high propensity to generate Iglmyc translocations, the common denominator of all mouse plasmacytomas. Resistance is dominant over susceptibility, and nearly all mouse strains, except BALB/c and NZB, are resistant. It is conceivable that BALB/c may be deficient in some mechanism that protects B cells against illegitimate nonhomologous translocations that may occur during physiologic Ig-locus rearrangement. Alternatively, or in addition, the postulated BALB/c defect may affect an apoptotic or scavenger function that can eliminate cells that have committed such an error.

IV. MOLECULAR BIOLOGY OF MULTISTEP CARCINOGENESIS How does the current molecular information fit with the progression rules of Foulds? And how is the responsibility for the emancipation of somatic cells from multiple host control divided among the four worlds of genes? Are there other worlds, in addition to these four? Several mutational steps that contribute to the development and progres-

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sion of colorectal, prostatic, and breast carcinomas and of malignant gliomas have been identified at the molecular level. The analyses of Vogelstein and his associates and others have defined the sequence of events in colorectal tumors (Fearon and Vogelstein, 1990). They include the mutation of the APC gene on 5q (in polyposis-related colon cancer), activation of K-ras, loss of DCC, a gene that encodes an adhesion protein, loss or mutation of p53, and further changes, reflected by aneuploidization. Some of these changes can be related to the well-defined progression steps from benign polyps through various stages of adenoma, into frank and eventually metastatic cancer. The horizontal study of individual patients has confirmed Foulds' dictum that progression can follow several alternative pathways. The development of astrocytomas into malignant glioblastoma involves a large number of losses, as shown by the loss of heterozygosis (LOH) technique and also by some oncogene activation events (James and Collins, 1992). Particularly noteworthy are the losses of the CDKN2 genes (encoding p16 and p l 5 ) that can inhibit the cyclin-CDK cascade. According to our understanding, p16 binding to CDK4 inhibits the formation of CDK4-cyclin D complexes. Low o r absent expression of p16 or CDK4 amplification favors the phosphorylation of RB, leading to its inactivation. Other changes during astrocytoma progression include the amplification of M D M 2 and of both EGF and PDGF receptors. These changes are akin to oncogene activation events, increasing the drive to cell proliferation. Documented losses involve both p53 and RB and numerous unidentified genes, as indicated by LOH. A particularly significant change, associated with transition to the highest grade of histopathologic malignancy, is a loss of heterozygosis on chromosome 10. Collins and others (Collins and James, 1993; Jiang et al., 1993; Lowe and Ruley, 1993; Strauss et al., 1995; Debbas and White, 1993; Hermeking and Eick, 1994) have pointed out that deregulation of the cyclin D-CDK-CKI-RB pathway may be an obligatory step in tumorigenesis in this and other systems. Examples include overexpression of cyclin D1 in tumors with an intact RB and p16, normal cyclin D1 expression in tumors with inactivated RB, and others. Importantly, overexpression of cyclin D1 has the same effect as loss of p l 6 , and both together have an effect similar to loss of RB. All these changes may converge in targeting the restriction point ( R ) of the cell cycle. Note that p16 and some other inhibitors may be inhibited by DNA methylation, an epigenetic change, rather than by mutation, in the course of tumor progression (Herman et al., 1995; Jones, 1996; Merlo et al., 1995). Three known susceptibility genes have been identified as being responsible for inherited breast cancer proneness: BRCAl, BRCAZ, and p53 (Bieche and Lidereau, 1995; Cannon-Albright and Skolnick 1996; Feunteun and Lenoir, 1996). The spectrum of tumors in the breast and in other organs is different in the syndromes associated with the germline mutations of these three genes and may also differ among different mutations of the same gene.

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BRCAl mutations are associated with both overian and female breast cancers, whereas BRCA2 mutations favor the development of only breast cancer, but in both females and males. Many other chromosome regions that may show LOH in breast tumors have been identified (Cox et al., 1994; Lee, 1995). The RB gene is mutated or deleted in about one third. RB loss appears to be associated with increased invasiveness. Amplification of the c-erb B2 oncogene is a prognostically unfavorable sign (Slamon et al., 1987). The development of prostatic cancer is also associated with numerous genetic changes (Kallioniemi and Visakorpi, 1996). A large fraction of the early-onset and up to 5 to 10% of all prostate cancer patients may carry an inherited germline mutation that facilitates the initiation of the carcinogenic process. Localized prostate cancer may already show LOH in the region. The transition to metastatic prostate cancer may be associated with a ras gene mutation or with RB loss. Another loss may affect a 16q region, known to carry the E-cadherin gene. This is consistent with the fact that the E-cadherinor catenin-mediated adhesion mechanism is changed in about half of all prostate cancers. Inactivation of p53 is relatively infrequent, whereas amplification of the myc-carrying 8q region is common in progressing tumors. An important and readily rationalizable step has been associated with the transition from hormone-dependent to hormone-independent prostatic cancer. In the recurrent hormone-insensitive tumor, the androgen receptor (AR) gene, carried by the X chromosome, may be amplified by up to 10 gene copies per cell. Hormone-independent prostatic carcinomas may also carry AR mutations, which are rare in primary tumors. Hormone therapy may selectively favor the growth of tumor cells that contain mutant o r amplified ARs genes. These and other examples suggest that the loss of known or presumptive suppressor genes may be more common in multistep carcinogenesis and tumor progression than the activation of known oncogenes. This impression may be biased, however, by the fact that genetic losses are more easily identified by the relatively simple LOH technique. Oncogenes d o not lend themselves to screening but rather require the study of each specific gene, one by one. Only gene amplification can be studied by a similarly generalized technique as gene loss, namely comparative genomic hybridization (CGH). Amplification is a relatively late event in progression, however, and affects only a fraction of tumors. I t may be safely concluded that all four key events-destabilization of the genome, activation of dominant oncogenes by mutations or regulatory changes, functional loss of cell cycle checkpoint (suppressor) genes, and mutational or regulatory changes in genes that protect cells against apoptotic death-may contribute to the development of most tumors. The long-standing prediction that the tumors of adults require five to seven mutations is holding up well. Childhood tumors may require fewer changes. The age-

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incidence curve in retinoblastoma indicates only a single event in the familial cases, in which the germ line allele is already mutated, and two events in the sporadic cases, corresponding to the loss of both RB alleles, as Knudson (1971) originally postulated. Leukemias and lymphomas may be due to a smaller number of changes at all ages than solid tumors, although multistep progression has been clearly demonstrated in Ph 1 ( b c d a b f )translocation-positive chronic myelogeneous leukemia that eventually leads to acute leukemia, designated as blast crisis (Rowley and Testa, 1982). In Burkitt lymphoma, c-myc is constitutively activated in 100% of cases, owing to the juxtaposition of the protooncogene to an immunoglobulin locus by chromosomal translocation (Klein, 1989). In addition, about 60% of the Burkitt lymphoma-derived lines and at least 30% of the in uiuo tumors carry mutated p53 (Gaidano and Dalla-Favera, 1995; Ramqvist et d., 1993; Wiman et al., 1991). This may provide the myc-driven cells with a selective advantage, owing to the protective effect of p53 mutations against apoptosis. Transgenic mice that carry immunoglobulin-enhancer myc constructs provide an experimental facsimile of the Iglmyc translocation (Adams et al., 1985). Such mice develop pre-B and B lymphomas in more than 90% of cases. Most lymphomas are monoclonal, however, indicating that at least one additional change is required for tumor development. With rare exceptions, such as retinoblastoma, most relatively well-known pathways of tumor development thus appear to require mutations in several genes that lead to oncogene activation, suppressor loss, and apoptosis protection. The relative importance of changes in these categories appears to differ between different cell lineages and their developmental stages. Mutations in the first two categories have the derangement of cell cycle regulation as their common denominator. Can the gene categories mentioned account for all phenotypic features of malignant cells? Probably not. Additional properties include the ability of tumor cells to stimulate angiogenesis (Folkman, 1992; 1995), their constitutive telomerase activity (Healy, 1995), and their escape from immune responses (Klein, 1993). With regard to the former, it is particularly clear that the angiogenic switch precedes the progression to invasive cancer in most, if not all, tumor systems. This step may therefore reflect the activation of a yet unknown set of genes (reviewed by Hanahan and Folkman, 1996).

V. STRONG SELECTIVE PRESSURES FAVOR MULTIPLE ESCAPES The mammalian fetus is a protected homograft within the mother's body. Prevention of rejection is absolutely essential for the survival of the species.

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It is therefore not surprising that multiple mechanisms have evolved to pro-

tect the fetus. They include impairment of antigen release, protection from invading maternal lymphocytes, a certain degree of tolerance of the fetal antigens by the mother, specific blocking antibodies, and nonspecific delay of rejection responses. It is no overstatement to say that all conceivable mechanisms sought were actually found. The experimental fantasy of the immunologist appears to be more limited than the protective mechanisms that have been established by selective pressure. A similar point can be made about the escape of the tumor cell from host control. Even if only a single type of host control were analyzed in relation to a single type of tumor cell, multiple escape mechanisms could be identified. This can be exemplified by the escape of the Epstein-Barr virus-carrying Burkitt lymphoma cell from the host immune response. Each of the following mechanisms might have been sufficient to permit the in vivo growth of this cell: downregulation of all immunogenic, virally encoded proteins, except the one that must be regularly expressed to secure the maintenance of the viral episomes (Rowe et al., 1987); sequestration of the latter (EBNA1) from ordinary processing and transport (Levitskaya et al., 1995; Trivedi et a/., 1991); decreased expression of adhesion molecules (Patarroyo et al., 1988);impairment of the TAP-dependent processing mechanism (Khanna et al., 1994; Rowe et al., 1995); and decreased expression of certain major histocompatibility complex class I alleles (Imreh et al., 1995). All of these escape routes have been compounded by the fully fledged Burkitt lymphoma cell that is driven by the translocated c-myc gene and is protected from apoptosis, as already mentioned. It is particularly remarkable that all this modeling can take place in a tumor that develops predominantly in young children! Multiple escape mechanisms are also exemplified by Hanahan’s mouse model of multistage carcinogenesis (Christofori et al., 1995). Transgenic mice that carry the SV40 LT gene under the control of the rat insulin gene regulatory region were generated. This initiates the selective entry of the pancreatic p cells into a multistep pathway that leads them toward islet cell carcinoma. LT is already expressed in the developing pancreas of the 9-day embryo, but hyperplastic islets with a high proliferation index are only seen 3 to 4 weeks after birth. The insulin-like growth factor gene IGF2 is upregulated at this point, indicating that it is functionally involved in the tumorigenic process. Progression does not proceed in the absence of the IGF2 gene. Both the expressed and the imprinted allele of IGF2 are activated during tumor development. The autocrine or paracrine signaling pathway involving IGF2 and its receptors thus appears as an important factor in initiating tumor cell proliferation. The second step is induction of angiogenesis, the activation of the normally quiescent vasculature to proliferate and form new capillaries. The third stage is the development of solid tumors. The three stages are separable. Further progression is accompanied by resistance to the

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immune response against the LT-positive cells and by upregulation of telomerase activity in advanced tumors. Suppressor genes localized on mouse chromosomes 9 and 16 are lost at different stages of progression. The early loss of a gene from chromosome 16 appears to be associated with the appearance of angiogenic activity, whereas the loss from chromosome 9 is linked to the progression from the angiogenic stage to a solid tumor. Protection from apoptosis is another identifiable step that occurs independently of the development of angiogenic activity. The selective pressure, the number of changes involved in progression, and the flexibility of the preneoplastic cell population can thus hardly be overestimated.

VI. CONCLUSION Speaking about “Darwin’s dangerous idea,” Dennett ( 1995) writes: If I were to give an award for the single best idea anyone has ever had, I would give it to Darwin, ahead of Newton and Einstein and everyonc else. In a single stroke, the idea of evolution by natural selection unifies the realm of life, meaning and purpose with the realins of space and time, cause and effect, mechanism and physical law. But it is not just a wonderful scientific idea. It is a dangerous idea. . . . My admiration for Darwin’s magnificent idea is unbounded but, I, too, cherish many of the ideas and ideals that it seems to challenge, and want to protect them. There are many . . . magnificent ideas that are . . . jeopardized, it seems, by Darwin’s idea, and they. . . may need protection. The only good way to do this . . . is to cut through the smoke screens and look at the idea as unflinchingly, as dispassionately, as possible.

The same could be said about Foulds’ dangerous idea. Tumor biologists must welcome and indeed embrace complexity. The sociology of scientists may create some problems here, but the clash is much gentler than that between the evolutionists and the creationists. Some of our clinical colleagues and most of the lay public still expect us to come up with “the solution” and “the cure.” It is sometimes said that the investment into cancer research has been a waste, that science did not live up to expectations, or that cancer supports more people than it kills. For those who have followed in Darwin’s and Foulds’ footsteps, there is no return. Even though tumor development is a tiny piece of evolution, compared with our common mother, it is an evolutionary process, with many subtle, seemingly disconnected selective steps, based on an almost infinite cellular variability. Like each species, each cancer is the result of an algoritm. Fortunately, we d o not have to identify every step in each algoritm to stop the process. The appropriate differentiation-inducing signal, inhibition of vascularization, introduction of a powerful suppressor or apoptosis-

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promoting gene, or construction of a potent immune missile may cut the Gordian knot. At the end of the day, we can, in the words of Dennett, “assess the bargain we get when we trade in pre-Darwinian for Darwinian thinking.. . . showing how what really matters to us . . . shines through, transformed but enhanced by its passage through the Darwinian evolution.

ACKNOWLEDGMENTS I express my gratitude to Drs. Klas Wiman, Douglas Hanahan, and George Vande Woude for their critical reading of the manuscript. The editorial help of Marie Bohm is greatly appreciated.

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Klein G. (Ed.) (1982). I n “Advances in Viral Oncology.” Raven Press, New York. Klein, G. (1989). Genes Chromosomes Cancer, 1 , 3-8. Klein, G. (1993). Gene, 135, 189-196. Klein, G., and Klein E. (1957). Symp. Soc. E x p . Biol., 11, 30.5-328. Klein, G., and Klein, E. (1958).J. Cell Conrp. Physiol., 52, 126-168. Klein, G., and Klein, E. (198Sa). Nature, 315, 190-195. Klein, G., and Klein, E. (1985b). Immunol. Today, 6, 208-215. Knudson, A. G. Jr. (1971). Proc. Natl. Acad. Sci. U.S.A., 68, 820-823. Knudson,A. G. (1993). Proc. Nut/. Acad. Sci. U.S.A., 90, 10914-10921. Kok, K., Naylor, S., and Buys, C. (1996). Adu. Cancer Res., 71, 28-93. Konopka, J. B., and Witte, 0. N. (lY85). Biochim. Biophys. Acta., 823, 1-17. Korsmeyer, S. J. (1992). Annu. Rev. Immunol. 10, 78.5-807. Kovacs, G., Erlandsson, R., Boldog, F., Ingvarsson, S., Mullcr-Brechlin, R., Klein, G., and Sumegi, J. (1988). Proc. Nut/. Acad. Sci. U.S.A.. 85, 1571-1575. Lamhert, W. C., Kuo, H. R., and Lambert, M. W. (1995). Dermatol. Clin., 13, 169-209. Lane, D. P. ( 1 992). Nature, 3.58, 15-16. Lee, E. Y. (1995). Senzirz. Cancer Biol., 6, 119-12.5. Levitskaya, J., Coram, M., Levitsky, V., Imreh, S., Steigcrwald-Mullen, P. M., Klein, G., Kurilla, M. G., and Masucci, M. G. (1995). Nature, 375,685-688. Liebermann, D. A., Hoffman, B., and Steinman, R. A. (1995). Oncogene, 11, 199-210. Little, C. D., Nau, M. M., Carney, D. N., Gazdar, A. F., and Minna, J. D. ( I 983). Nature, 306, 194-1 96. Loeb, 1.. A., and Cheng, K. C. (1990). Mutut. Res., 238, 297-304. Lowe, S. W., and Ruley, El. E. (1993). Genes Dei)., 7, 535-545. Malkin, D. ( 1993). Cancer Genet. Cytogenet., 66, 83-92. McDonnell, T. J., Deanc, N., Platt, F. M., Nunez, G., Jaeger, U., McKearn, J. ,!I and Korsmeyer,S. J. (1989). Cell, 57,7948. Merlo, A., Herman, J. G., Mao, I.., Lee, D. J., Gabrielson, E., Burger, I?C., Haylin, S. B., and Sidransky, D. (1995). Nature Med., 1, 686-692. Milner, J. (1994). Semin.Cancer Biol., 5 , 21 1-219. Mintz, B., and Fleischman, R. A. (1 98 I ) . Adv. Cancer Res., 34, 21 1-278. Noda, M. ( 1993). FASEH J., 7 , 834-840. Nowell, P. C. (1976). Sciwce, 194, 23-28. Okan, I.,Wang, Y., Chen, F., Hu, L. F., Imreh, S., Klein, G., and Wiman, K. G. (1995). Oncogene, 11, 1027-1 03 I . Oltvai, Z . N., Milliman, C. I.., and Korsmeyer, S. J. (1993). Cell, 74. 609-619. Patarroyo, M., Prieto, J., Ernberg, I., and Gahmherg, C. G. (1988). In?./. Cancer, 41, 901-907. Peltomaki, P., and de la Chapelle, A. (1996). Adv. Cancer Res., 71, 94-121. Rabbitts, T. H. (1994). Ndtcrre, 372, 143-149. Ramqvist, T., Magnusson, K. P., Wang, Y., Szekcly, L., Klein, G., and Wiman, K. G. ( I 993). Oncogme, 8, 149.5-1 500. Kous, I).,and Beard, J. ( I 935). /. E x p . Med., 62,523-548. Rowe, M., Khanna, K., Jacob, C. A., Argaet, V., Kelly, A., Powis, S., Belich, M., Croom-Carter, D., Lee, S., Burrows, S. R., et a/. (1995). E m J. Iwimrrnol., 25, 1374-1384. Rowe, M., Ilowe, D. T., Gregory, C:. I)., Young, L. S., Farrell, P. J., Rupani, H., and Kickinson, A. B. ( 1 987). E M A O J., 6, 2743-27.5 I . Rowley, J. D., and Testa, J. R. (1982). Adv. Cuncer Res., 36, 103-148. Selivanova, G., and Wirnan, K. G. (1995).Adu. Cancer Res.. 66, 143-180. Sherr, C. J. (1993). CelI, 73, 1059-1065. Silva, S., Sugiyama, H., Babonits, M., Wiener, F., and Klein, G . (1991). Int. J. Cancer, 49, 224-228.

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Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A,, and McGuire, W. L. (1987). Science, 235, 177-1 82. Strasser, A,, Harris, A. W., Jacks, T., and Cory, S. (1994). Cell, 79, 329-339. Strasser, A., Whittingham, S., Vaux, D. L., Bath, M. L., Adams, J. M., Cory, S., and Harris, A. W. (1991).Proc. Natl. Acad. Sci. U.S.A., 88, 8 6 6 1 4 6 6 5 . Strauss, M., Lukas, J., and Bartek, J. (1995). Nature Med., 1, 1245-1246. Strong, L. C., Williams, W. R., and Tainsky, M. A. (1992). Am. 1. Epidemiol., 135, 190-199. Toyoshima, H., and Hunter, T. (1994). Cell, 78, 67-74. Trivedi, P., Masucci, M. G., Winherg, G., and Klein, G. (1991). Int. 1. Cancer, 48, 794-800. Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, 1.' C., and Croce, C. M. (1984). Science, 226, 1 09 7-1 0 99. Weinbcrg, R. A. (1995). Cell, 81, 323-330. Weintraub, H., Beug, H., Groudine, M., and Graf, T. (1982). Cell, 28, 931-940. Weisenburger, D. D. (1991). Leukemia, 5(Suppl I ) , 26-29. White, E. (1996).Genes Deu., 10, 1-15. Wiels, J., Fellous, M., and Tursz, T. (1981). Monoclonal antibody against a Burkitt lymphomaassociated antigen. Proc. Natl. Acad. Sci. U.S.A., 78, 6485-6488. Wiman, K. G., Magnusson, K. P., Raniqvist,T., and Klein, G. (1991).Oncogene, 6 , 1633-1639. Xiong, Y., Zhang, H., and Beach, D. (1993). Genes Deu.. 7, 1.572-1583. Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).Cell, 85, 537-548. Yanagihara, K., Ciardiello, E, Talbot, N., McCeady, M. I,., Cooper, H., Benade, L., Salomon, D. S., and Bassin, R. H. ( 1990). Oncogene. 5, 1 179-1 186. Yokota, J., and Sugimura, T. (1993). FASEB J., 7, 920-925. 7,, 855-865. Zambetti, G. P., and Levine, A. 1. (1993). FASEB I.

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FOUNDATIONS IN CANCER RESEARCH ~~

Cancer Cells Exhibit a Mutator Phenotype Lawrence A. Loeb The Joseph Gottstern Memorid Cancer Research 1.ahoratory Department of Pathology, Unrurrsrty of Wushmgton. Box 357705 Seattle, Washington 98 19 F-770Y

1. Introduction 11. Historical Perspective 111. Requirement for a Mutator Phenotype A. General Considerations B. Number of Mutations in Human Cancer Cells C. Roles of Clonal Expansion and Somatic Selection D. Coupling of Enhanced Mutagenesis with Somatic Selection IV. Origins of Mutations A. DNA Replication and Mismatch Repair B. DNA Damage and Repair V. Candidate Target Mutator Genes A. DNA Polymerases B. Genes Governing the Cell Cycle C. Genes Involved in DNA Repair and DNA Replication D. Other Target Genes VI. Microsatellite Instability A. Microsatellites B. Microsatellite Instability in Hereditary Nonpolyposis Colon Cancer C. Microsatellite Instability in Sporadic Tumors D. Other Mechanisms for Mutation Accumulation VII. Theoretical and Practical Considerations A. Mutations During Tumor Progression B. Diagnosis C. Prognosis D. Cancer Prevention VIII. Summary and Perspectives References

This review analyzes the concept and evidence in support of a mutator phenotype in human cancer. The large number of mutations reported in tumor cells cannot be accounted for by the low mutation rates observed in normal somatic cells; rather, it must

Advances in CANCER 00hS-L30X/YX $?S.OO

RESEARCH

Copyright 0 199X by Academic Prcs. All rights of reproduction in any form rmervcd.

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be a manifestation of a mutator phenotype present early during the tumorigenic process. The interaction hetween increased mutagenesis and clonal selection provides a mechanism for the selection of cells with increased proliferative advantage. The concept of a mutator phenotype in cancer has gained considerable support from the findings of enormous numbers of somatic mutations in repetitive sequences in human tuniors. Moreover, cell lines exhibiting microsatellite instability demonstrate an increased mutation frequency in expressed genes. A knowledge of mechanisms that generate multiple mutations in cancer cells has important implications for prevention. For many tumors, a delay in the rate of accumulation of mutations by a factor of two could drastically reduce the death rates from these tumors.

1. INTRODUCTION Evidence indicates that human cancers arise from one or a few of the 500 trillion cells in the human body. It frequently takes 20 years or more for a single cell to multiply preferentially and to constitute a mass that can be clinically detected. At the time of diagnosis, most tumors contain about 1 billion cells and exhibit a high degree of intercellular heterogeneity. Yet even these phenotypically diverse tumors display genetic footprints that reveal their clonal lineage (Fialkow, 1974). Karyotype analysis suggests that most tumors contain multiple chromosomal alterations. Although these chromosomal changes encompass large segments of the genome, they may signify an even larger number of smaller changes that may also be present in tumor DNA. A complete description of nucleotide sequence changes in various types of human cancers-their number, nature, and origins-has yet to be obtained. The genetic and phenotypic heterogeneity exhibited by most human tumors has practical implications. The heterogeneity within each tumor could account for o u r repeated failure to delineate common metabolic alterations in cancer cells as well as the failure of chemotherapeutic agents to eradicate cancers. The cellular heterogeneity within a tumor could account for the ability of tumors to develop resistance to both immunotherapy and chemotherapy. Quantitation of genetic heterogeneity within a tumor could provide a marker for the extent of tumor progression. Because genomic stability is under genetic control, mutations in genes that maintain this stability may provide new targets for cancer treatment and prevention. Two overlapping mechanisms have been proposed to account for the multiple mutations in tumor cells. We proposed that these mutations result from an increase in the rate of mutagenesis in cancer cells, that is, that cancer cells exhibit a mutator phenotype (Loeb et al., 1974). The mutator concept states that tumor evolution is driven by genetic instability with the generation of large numbers of random mutations; there is selection for clones that exhib-

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it malignant properties. Also, Nowell (1976) analyzed chromosomal aber-

rations in tumors and proposed that these mutations result from multiple rounds of clonal selection. Acquired genetic variability permits stepwise selection of variant sublines and underlies tumor progression. Because these concepts were proposed some 20 years ago, it has become increasingly apparent that both increased mutagenesis and clonal selection are important factors in tumor progression. These two mechanisms are not necessarily mutually exclusive and in fact may be tightly coupled. I have argued that the large number of mutations already reported in tumor cells cannot be accounted for by the low mutation rates observed in normal somatic cells, but instead must be a manifestation of a mutator phenotype (Loeb, et al., 1974; Loeb, 1991 [expanded]). The number of mutations observed in individual tumor cells exceeds that which would be expected from the data on spontaneous mutation frequencies in normal cells, even when clonal expansion is taken into consideration. The concept of a mutator phenotype in cancer has gained considerable support from findings of enormous numbers of somatic mutations in repetitive sequences in human tumors (Ionov et al., 1993).Moreover, cell lines exhibiting microsatellite instability also display more than 100-fold increases in mutations in expressed genes (Bhattacharyya et al., 1994; Phear et al., 1996), and these mutations include single base substitutions (Malkhosyan et al., 1996).The central question may no longer be whether cancer cells exhibit a mutator phenotype, but whether the multiple mutations that accumulate in cancers are rate limiting for the carcinogenic process. Is genetic diversity a driving force in tumor progression, or is it simply one of many manifestations that underlie this process? The purposes of this review are to summarize the evidence that the progression of malignancy is associated with increased mutagenesis and to consider whether a mutator phenotype is required for the evolution of tumors. For a historical perspective on chromosomes and cancer, refer to the review by Nowell ( 1 993). The perspective in the current review is at the level of molecular biology. I consider the following concepts: ( 1 ) that cancers exhibit a mutator phenotype; (2) that DNA is turned over in human cells; (3) that there are multiple potential targets for a mutator phenotype; and (4)that the multiple mutations present in cancer cells have important theoretical and practical implications. Many other enticing phenomena, including the regulation of transcription, epigenetic changes, and cell-cell interactions, that may be equally important for the evolution of tumors (Prehn, 1994), are not yet opened to mechanistic experimentation and are not discussed here. Thus, this review is biased and personal in that it focuses on the concept of a mutator phenotype. Evidence supports the hypothesis that sometime during the evolution of a tumor, cancer cells must have exhibited a mutator phenotype. The new mol-

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ecular techniques have made it possible to dissect the human genome and to delineate genes that are mutated during tumor progression. We hope that methods will be developed to identify random mutations in cells; it may then be possible to quantitate the number of mutations within individual cancer cells. Most important, we need to determine whether an increase in the mutation rate of cancer cells is an early event in the evolution of a tumor and whether the accumulation of mutations limits events in tumor progression.

II. HISTORICAL PERSPECTIVE Our initial hypothesis that cancer cells exhibit a mutator phenotype was based on the infidelity of DNA synthesis in vitro (Loeb et al., 1974). It was grounded on extensive laboratory experimentation that indicated that DNA polymerases and reverse transcriptases frequently incorporate noncomplementary nucleotides (Battula and Loeb, 1974; Fry and Loeb, 1986). Mutations in DNA polymerases might render them even more error prone. Mutant DNA polymerases could be a source of random mutations throughout the genome. We proposed that “infidelity of DNA replication may be responsible for tumor oncogenesis and progression” (Loeb et al., 1974). Our initial hypothesis was supported by multiple phenotypic changes occurring during tumor progression, in particular (a) the continuous evolution of new cell variants; ( b )the increasing ability of tumor cells to escape the host mechanism for regulating cellular proliferation; (c) the progressive accumulation of chromosomal aberrations; and ( d ) resistance to chemotherapeutic agents. We pointed out that infidelity of DNA replication could result from increased misincorporation either by an altered DNA polymerase or by deficits in the repair of misincorporated nucleotides (Loeb et a/., 1974).The concept, diagrammed in Figure l, is that tumor progression proceeds by enhanced mutagenesis resulting from mutations in key enzymes (DNA polymerases) that render them error prone. The concept of a positive feedback loop, in which subsequent mutations occur in many genes, including those that encode DNA polymerases, was in accord with the concept of an error catastrophe involved in aging (Murray and Holliday, 1981; Orgel, 1973). In our model, tumors would grow as a result of a mutation cascade, with the production of new mutants that exhibited a proliferative advantage. From an analysis of chromosomal changes in tumors, Nowell (1976) proposed that tumor progression occurs by clonal evolution, in which a single cell expands by stepwise selection to populate a tumor. Tumor progression results from acquired genetic variability within the original clone, allowing sequential selection of more aggressive sublines. Successive waves of clonal expansion drive tumor progression and result in multiple chromosomal

29

Cancer Cells Exhibit a Mutator Phenotype

VIRUS CHEMICAL CARCINOGEN

1 Neoplastic

Tumor Progression

I Frequency

Proliferation

4 Phenotypes with Selective Value for Proliferation in Host

]

Genotypes

I I

I

Parental

Fig. I Accumulation of genetic errors during tumor progression. The model indicates that an early event in neoplastic transformation is a mutation in DNA polymerase resulting in an increase in mutation during tumor progression. Included is a positive feedback mechanism in which additional mutations could occur in DNA polyrnerases, rendering them more error prone (Loeb, et al., 1974).

changes within individual tumors. Nowell's model for clonal evolution is diagrammed in Figure 2. Human tumors with minimal chromosome changes, such as diploid acute leukemias, are considered to be early in clonal evolution, whereas highly aneuploid solid tumors have undergone multiple clonal selections. Inherent in this model is a mechanism for drug resistance and metastasis based on the selection of mutant variants. Even though the concept of cancer exhibiting a mutator phenotype and evolving by clonal selection was introduced in 1974 to 1976, it only recently has been studied experimentally. The idea that cancer results from random mutagenic events has always met resistance, in part because of the direct implication that carcinogenesis would then be an irreversible mutagenic process-once a cancer cell, always a cancer cell. The reversion of any mutation is an infrequent event. Embryonal carcinoma cells, however, populate tissues on transplantation into a developing blastocyst and result in a mosaic mouse (Illmensee and Mintz, 1976). Thus, there are situations in which a tumor can assume a normal phenotype, but it may still harbor mutations. The attractiveness of the concept of cancers having an epigenetic origin is the

Lawrence A. Loeb

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mm Diploid Acute Leukernia

Early Solid

E l Human Solid Malignancies

Fig. 2 Model of clonal evolution in neoplasia. Carcinogen-induced change in progenitor normal cell ( N ) produces a diploid tumor cell ( T I ,46 chromosomes) with growth advantage, permitting clonal expansion to begin. Genetic instability of T , cells leads to production of variants (illustrated hy changes in chromosome number, T, to T6). Most variants die, owing to metabolic or immunologic disadvantage (hutched circles); occasionally, one has an additional selective advantage (e.g., T,, 4 7 chrornosomes), and its progeny heconie the predominant subpopulation until a n even morc favorable variant appears (c.g., T4).The stepwise sequence in each tumor differs (being partially determined by environmental pressures on selection) and results in a different, aneuploid karyotype in each fully developed malignancy (T,) (Nowell, 1976).

hope of turning off the carcinogenic stimulus and converting cancer cells to normal cells. In the past 15 years, a major emphasis in cancer research has been on identifying pathognomonic chromosomal changes that delineate particular cancers; more recently, emphasis has been on identifying specific mutated genes within those altered chromosomal segments. This reduction approach has yielded a bewildering number of mutated genes, many of which are associated with specific cancers. Multiple dominant oncogenes and tumor suppressor genes have been identified in most cancers (Weinberg, 1991). Interestingly, in no cancer can a single mutated oncogene be found in 100% of the tumors. I have argued that this multiplicity of mutated oncogenes is a manifestation of earlier mutations that increase mutagenesis (Fig. 3 ) . The reductionist approach may still be fruitful; but, rather than trying to identify specific oncogenes, it might be more productive to focus o n a specific pathway that underlies malignancies. An attractive possibili-

Sources of a Mutator Phenotype TARGET MUTATOR GENES

EXOGENOUS Chemical X-ray U.V.

ENDOGENOUS Oxygen Replication Error

Sporadic Cancers DNA Repair Genes X.P., A.T. Helicases Bloom, Werner Syndrome DNA Polymerases Nucleotide Synthesis Cell Cycle Gene

MALIGNANT PHENOTYPE ONCOGENES Tumor Suppressor Gene Metastasis Genes

. { } Metastasize

Chromosome Segregation

Fig. 3 Mutations during tumor progression. The mutator phenotype hypothesis proposes that genes involved in maintaining chromosomal stability are mutated early during tumor progression. Mutations in these genes would result in other mutations throughout the genome. Mutation in oncogenes would be a later manifestation and result in the ability of tumors to divide, invade, and metastasize.

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ty is that tumors evolve by random mutations and that changes observed in oncogenes represent later alterations that determine many of the properties of the tumor but may not be the principle driving force in tumor progression. Current research emphasis is on the concept that mutations in mismatch repair genes account for the multiple mutations observed in many human tumors. I would argue, however, that this is but one of the many mutator pathways that will be found to be altered early in tumor progression. I t is likely that neither a mutator phenotype nor clonal selection is adequate to account for the multiple mutations that are present in cancer cells. Instead, there is probably extensive interplay between the two processes. Repetitive selective events during the course of tumor progression may simultaneously select for cells exhibiting a mutator phenotype.

Ill. REQUIREMENT FOR A MUTATOR PHENOTYPE A. General Considerations Many studies indicate that the spontaneous mutation rate of human cells is about lo-’ mutations per single copy gene per cell generation (Meuth, 1990) or, for an average gene, 1.4 X l O - ’ O mutations per base pair per cell generation (Loeb, 1991). For mutations in autosomal genes to result in a change in phenotype, two independent events may be required (Phear et al., 1996). Based on the total number of cell divisions during an average human life span, we estimated that the spontaneous (background) mutation rate would produce at most two or three mutations in each normal cell. Epidemiologic studies, however, suggest that many events are required for the production of a cancer (Armitage and Doll, 1954; Renan, 1993; Stein, 1991). Although plots of the incidence of retinoblastoma and Wilms’ tumors as a function of age indicate a two-hit mechanism and confirm Knudson’s hypothesis, similar plots of age versus incidence for adult tumors suggest a large number of events. Estimates of the number of events associated with adult tumors vary: 3 for early-onset bone tumors, 6 for cancer of the cervix, and as many as 12 for cancer of the prostate (Renan, 1993). If we assume that these events correspond to mutations, then the spontaneous mutation rate can account for only two or three of these events. If it is true that cancers arise only in a fraction of the cell population (i.e., stem cells), then even fewer cancer-causing mutations could result from normal mutation rates. Furthermore, many tumors contain as many as 50 chromosomal rearrangements. This discrepancy between the rarity of mutations in normal cells and the large number of mutations in cancer cells led us to propose that acquired

Cancer Cells Exhibit a Mutator P h e n o t y p e

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genetic instability o r a mutator phenotype must be an early event in the evolution of a tumor (Loeb, 1991).

B. Number of Mutations in Human Cancer Cells During the past 20 years, there has been an increasing number of reports on chromosomal alterations in human tumors (Balaban et al., 1986; Nowell, 1993) that could result from a mutator phenotype. First, karyotype analysis revealed multiple chromosome translocations (Erikson et al., 1992; Rowley, 1975) and deletions (Yunis and Sanchez, 1975) as well as the presence of amplified portions of chromosomes (Biedler and Spengler, 1976). Second, multiple chromosomal alterations have been reported in specific tumors. Twenty-five to 50% of colon adenocarcinomas have been shown to contain more than nine chromosomal alterations (Fearon and Vogelstein, 1990). Third, loss of heterozygosity is a common feature in tumors. It occurs as a result of the deletion of a large piece of a chromosome, and its presence in tumor DNA has been considered a marker for the deletion of tumor suppressor genes. In ovarian cancers, 23 different chromosomal segments have been shown to exhibit loss of heterozygosity a t a frequency of greater than 3 0 % (Chigira et al., 1993). Fourth, multiple mutations are present within single human cancer cells. Cancers exhibiting multiple chromosomal alterations include small cell cancer of the lung (Naylor et al., 1987), adenocarcinoma of the colon (Fearon and Vogelstein, 1990; Stanbridge, 1990),malignant melanoma (Balaban et al., 1986), and glioma (James et al., 1988). Large deletions and losses of entire chromosomes could be the result of mutations in genes that govern the overall mechanism of chromosomal segregation, including DNA helicases, topoisomerases, and genes not yet identified that are involved in recombination, spindle formation, and chromosomal partitioning during cell division. In addition to multiple chromosomal changes reported in tumors, gene amplification may also be a diagnostic marker for malignant cells. Gene amplification is an increase in the number of copies of a particular gene at a specific locus and thus in a broad sense can be classified as a mutation. Tlsty and colleagues reported that resistance to the drug PALA occurs by gene amplification. It is undetectable ( < l o p 9 ) in primary diploid cells and ranges from l o p 3 to lo-” in transformed cells (Tlsty et al., 1992; Livingstone et al., 1993). Moreover, in cell fusion experiments, it behaves as a recessive trait, suggesting that it is suppressed in normal diploid cells. The fact that gene amplification occurs at high frequency in neoplastic cells from diverse sources and is undetectable in the corresponding normal diploid cells provides strong evidence for genetic instability as a hallmark of neoplastic cells in culture (Otto et al., 1989).

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Frequently, a sequential order of chromosomal alterations occurs during tumor progression. Vogelstein and colleagues ( 1988) delineated an ordering of mutations in colon cancer that parallels the evolution of benign polyps to invasive carcinomas. Among the genes mutated are APC (the gene defective in the hereditary adenomatous polyposis coli), K-ras (a gene involved in signal transduction), DCC (a gene that encodes a protein involved in cell adhesion), and p.53 (a transcription factor involved in apoptosis and in governing checkpoints during the cell cycle). Similar studies that assign an order to chromosomal mutations during tumor progression have been reported for gliomas (James et a/., 1988), carcinomas of the breast and lung (Birrer and Minna, 1988) and malignant melanomas (Balaban et al., 1986). These alterations have been detected cytologically and involve large segments of chromosomes. The ordering of mutations with time could reflect the selection of clones with different phenotypes during tumor progression. These mutations probably are a reflection of an even much larger number of smaller mutations, such as small deletions, rearrangements, additions, and single base substitutions, each of which could involve one or a few nucleotides (Loeb, 1991).Thus, the multiple chromosomal aberrations in cancer may be only the tip of an iceberg. As we determine the sequence of nucleotides in tumor DNA, we likely will discover tens of thousands of mutations in each cancer cell (Loeb and Cheng, 1990).

C. Roles of Clonal Expansion and Somatic Selection Clonal expansion of some cells in a tumor is likely to account for an increased number of mutations by increasing the number of cell replications during tumor progression. A mutation confering a proliferative advantage would result in an expansion of cells with that mutation within the tumor. Each successive round of clonal expansion would yield a large number of cells having N mutations, and thus increasing the probability of the N + 1 mutation. We have estimated that in the absence of clonal expansion, the spontaneous mutation rate in normal human cells can account for only two or three mutations. If we assume that each mutation confers a proliferative advantage that leads to a clonal expansion to los cells before the next mutation, then the chance of a cell getting two mutant genes within that clone would be increased from (10-7)(10-7) to (10p7)(10-7X lo5).Thus, without an increase in the mutation rates, successive rounds of clonal selection could account for as many as five o r six mutagenic events. In the absence of a mutator phenotype, clonal selection could account for the increased incidence of some cancers as function of age (Armitage and Doll, 1954). Despite this theoretical argument, it is unlikely that clonal expansion in the absence of enhanced mutagenesis accounts for the multiple mutations in

Cancer Cells Exhibit a Mutator Phenotype

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cancer cells. First, each mutation would have to exhibit a proliferative advantage. Even a mutation in one of the two alleles of a tumor suppressor gene would have to confer a proliferative advantage to cells harboring that mutation. Second, most mutations found in cancer cells are recessive for tumorigenesis (Harris, 1988), and thus it is unlikely that a single mutation would result in a proliferative advantage. In tumor suppressor genes, usually both alleles have to be inactivated for expression of the malignant phenotype (Knudson, 1971). Third, for the mutant clones to populate the tumor rapidly, the clones must have an exceptionally high selective advantage, or they must prevent DNA synthesis in other cancer cells within the tumor. Most important, although clonal expansion could conceivably account for the five or six mutations in specific cancers, it cannot account for the much larger number of mutations that are likely to be present in many cancers. On the other hand, mutations in genes involved in maintaining chromosomal integrity could occur early during tumorigenesis, accounting for the multiple mutations in individual cancer cells. It is still conceivable that the multiple mutations in cancer cells have nothing to d o with malignant progression, but instead are a parallel occurring event (i.e., the accumulation of mutations is not rate limiting for tumor growth or tumor progression). We have postulated that a mutator phenotype in cancer cells could account for a large number of mutations. Among the random mutations generated would be some that yield a proliferative advantage and that result in clonal expansion and others that are responsible for the cancer phenotype. A mutator phenotype alone does not account for the clonal nature of most human cancers, and thus selection must also be an important determinant in tumor progression.

D. Coupling of Enhanced Mutagenesis with Somatic Selection It is likely that the large number of mutations generated in cancer cells is the result of both enhanced mutagenesis and somatic selection. Mutations may not always be disadvantageous. Competition experiments with bacteria indicate that there are conditions in which bacteria with a higher mutation frequency have a growth advantage over the wild type (Cox, 1976).The question of whether selection for mutants would concomitantly select for mutators has been addressed by Miller, Mao, and colleagues in a series of telling experiments in Escherichiu coli (Ma0 et af., 1996; Mao et ul., 1997; Miller, 1996).The selection of spontaneous Lac-positive revertants in a Lacnegative population resulted in a parallel enrichment of mutator mutants. In this system, reversion occurred by frameshift mutagenesis, and each of their mutators was deficient in mismatch repair, a system that corrects frameshift mutations. The implication is that a fraction of spontaneous mutations is the

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result of mutator mutations; the selection for mutants would increase the fraction of mutators in the population. Moreover, these investigators demonstrated that exposure to a mutagen followed by multiple selections can result in a population of cells consisting nearly entirely of mutators. This situation could be analogous to repetitive rounds of clonal selection in tumor progression. Selection for invasion, nutrients, and drug resistance during the life of a tumor would also result in selection for mutators. As a result, somatic selection could be concatenated with the selection of mutators.

IV. ORIGINS OF MUTATIONS In cells, DNA is damaged by both exogenous and endogenous processes. DNA damage, if unrepaired, can cause mutations when the damaged DNA is copied at the time of DNA replication. The fact that spontaneous mutations occur infrequently in human cells does not necessarily imply either that DNA synthesis is exceptionally accurate or that DNA is not damaged during the life of a cell. Although rare mutations are needed in germline cells for a species to respond to environmental changes, there is no obvious scenario indicating that somatic mutations would be advantageous. One can envision an overall DNA homeostasis, encompassing a multitude of mechanisms, that evolved to govern the mutation rate in normal somatic cells. Either an increase in errors during DNA replication or a decrease in the efficiency of DNA repair could shift the balance of the system and result in an increase in mutations.

A. DNA Replication a n d Mismatch Repair During each cell cycle, every human cell replicates 6 x lo9 nucleotides of nuclear DNA. Based on a background mutation rate in human cells of about 1.4 x mutations per base pair per cell generation, we have estimated that each normal cell replicates its genome with about one error per cell generation (Loeb, 1991). The frequencies of misincorporation by DNA polymerases in vitro is much higher, however. Studies on the fidelity of DNA synthesis in vitro indicate that the frequencies of misincorporation by homogeneous eukaryotic DNA polymerases vary from 1 in 5000 for DNA polymerase-P (Kunkel, 1985; Kunkel and Loeb, 1981) to 1 in l o 7 for DNA polymerase-€ (Kunkel, 1992). Even though other proteins may function to increase the accuracy of DNA synthesis, it is unlikely that the accuracy of the DNA-replicating complex is adequate to account for the low frequency of observed spontaneous mutations in cells. Therefore, human cells must

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possess powerful mechanisms to excise noncomplementary nucleotides that are incorporated as a result of DNA polymerization, a process referred to as mismatch correction. The genetics and biochemistry of mismatch correction have been elegantly delineated in E. coli (Modrich, 1987), and homologous proteins have been identified in yeast and human cells (Modrich and Lahue, 1996), although we still lack a n understanding of the biochemistry and mechanisms to distinguish between the template and newly synthesized, error-containing strand in eukaryotic cells. The enhanced mutation rates exhibited by cancer cells with deficiencies in mismatch correction have provided the strongest evidence for a mutator phenotype.

B. DNA Damage and Repair DNA in both dividing and nondividing cells is subjected to a variety of modifications. DNA is damaged by both endogenous (Loeb and Cheng, 1990)and exogenous sources (Ames etal., 1990). Even small chemical modifications of nucleotides in DNA, such as the addition of methyl and ethyl groups to either purine or pyrimidine bases, alters the coding properties (Singer and Essigmann, 1991; Singer and Grunberger, 1983).Thus, damage to DNA, if not repaired, is likely to cause misincorporations at the time of DNA replication. Many environmental chemicals damage DNA; epidemiologic data indicate that many chemicals that damage DNA are carcinogens (Ames, 1983). In addition, normal cellular metabolic processes generate reactive chemical intermediates with the potential to damage DNA, these might also be a source of spontaneous mutations and cancers. Among such endogenously generated reactions is the hydrolysis of the glycosylic bond in DNA by water molecules. This results in depurinations, with the formation of an abasic site in DNA. Based on extrapolations from rate constants obtained in vitro, it has been estimated that depurination of DNA is an exceptionally frequent occurrence; about 10,000 depurinations occur per cell per day (Lindahl and Nyberg, 1972). The resultant abasic sites are mutagenic (Loeb, 1985; Schaaper et al., 1983). Deamination of cytosine to thymidine residues in DNA is less frequent, but the end product is a change in the nucleotide sequence of DNA (Lindahl and Nyberg, 1974). Equally extensive is damage by oxygen free radicals and related active molecules. Based on the urinary excretion of two major products of DNA damage by oxygen free radicals, thymine glycol and 8-hydroxyguanosine, it has been estimated that reactive oxygen species introduce 20,000 lesions in DNA in each cell per day (Cathcart et al., 1984; Shigenaga et al., 1989). Many of the alterations in DNA produced by oxygen free radicals have been shown to change the coding properties of nucleotides in DNA during copying by DNA polymerases (Basu etal., 1993; Cheng et al., 1992; Shibutani et al., 1991). Other sources

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of endogenous DNA damage being investigated include: methylation (Erlich and Wang, 1981; Shen et al., 1992), attack by lipids (Chaudhary et al., 1994), and glycosylation (Bucala et al., 1984). Each of these processes is likely to result in many altered bases in DNA. Considering the multiplicity of lesions produced in cellular DNA and the compact structure of chromatin, it is unlikely that all lesions in DNA would be repaired before DNA replication. Thus, DNA damage by both endogenous and exogenous processes is likely to contribute to mutagenesis.

V. CANDIDATE TARGET MUTATOR GENES Mutations can be generated by many processes, including errors in DNA replication and DNA damage by endogenous and exogenous sources. Conversely, diminution in the efficiency of mismatch repair or DNA damage repair can also result in increased mutagenesis. Thus, a t a minimum, mutations in genes that govern DNA synthesis and DNA repair could yield cells that exhibit a mutator phenotype. In a larger sense, many other genes are involved in recombination, transcription, cell cycle control, and chromosomal segregation that ensure the genetic stability in normal cells; mutations in these genes could also confer on cells a mutator phenotype (Cheng and Loeb, 1997). As examples, I consider recent studies on a few of the potential target mutator genes.

A. DNA Polymerases Misincorporation by DNA polymerases may be a major contributor to spontaneous mutations. Presumably, misincorporation by mutant DNA polymerases could exceed the capacity of the mismatch repair system. Schaaper (1993)demonstrated that the spontaneous mutation rate of E. coli harboring antimutator DNA polymerases is two- to three-fold lower than that of wild-type cells. This implies that half of spontaneous mutations in E . coli are the result of errors by DNA polymerases. Mutations in DNA polymerases have been shown to alter the fidelity of DNA synthesis and to increase the mutation rate of cells (Beard et al., 1996; Copeland et al., 1993; Sweasy and Yoon, 1995). Most dramatically, a single substitution of an alanine for an arginine at position 283 of DNA polymerase+ decreases the fidelity and catalytic efficiency of DNA polymerase+ by 160-fold and 5000fold, respectively (Beard et al., 1996). Even though it is unlikely that an enzyme with such a marked reduction in catalytic efficiency could function in vivo, these experiments demonstrate how mutations in DNA polymerases can dramatically affect the fidelity of DNA synthesis.

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Only fragmentary studies have been reported o n DNA polymerase genes in cancer cells. Most of these studies have been on DNA polymerase-P. DNA polymerase-P is located on the short arm of chromosome 8; in cancers, this segment is subjected to loss of heterozygosity and microsatellite instability (Patel et al., 1994). Alterations in DNA polymerase-@ mRNA was detected in 5 of 6 cases of colon cancer (Wang et al., 1992),in 2 of 12 cases of prostate cancer (Dobashi et a/., 1994), and in 4 of 24 cases of bladder cancer (Matsuzaki et al., 1996). The DNA polymerase+ alterations in colorectal cancers consist of a 87-base-pair deletion in the mRNA transcript and are likely to encode an inactive enzyme as a result of an alternative slice site. If this mutation is a dominant negative, however, it might yield a mutator phenotype by preventing DNA repair by the unaltered allele. Among polymerasep mutations in bladder cancer are missense mutations that could alter the catalytic properties of the enzyme. Because the polymerase+ mutations in bladder cancer were observed in early tumors, it has been suggested that polymerase-P mutations contributed to the development of these cancers (Matsuzaki et af., 1996). Thus, polymerase-P remains a candidate target for a mutator phenotype. Mutations in other DNA polymerases that synthesize larger portions of the genome during DNA replication are more likely to generate a mutator phenotype in cancer cells. The only other polymerase mutant that has been identified is a mutation in the conserved exonuclease Ill motif of DNA polymerase-6 in DLDlIHCTlS, a human colon cell line. I t is not known if this mutation reduces proofreading o r enhances slippage by the polymerase (Minnick and Kunkel, 1996). Studies on infidelity of DNA synthesis in cancers have been limited mainly to experiments with crude extracts. These studies measure the frequency of misincorporation by DNA polymerases using either polynucleotides or plasmid DNA as a template. In early studies, we demonstrated that cell extracts from human leukemic cells misincorporated noncomplementary nucleotides more frequently when copying polynucleotides than did similar extracts from phytohemagglutinin-stimulated normal lymphocytes (Loeb et al., 1974). Recently, experiments carried out using chemically induced mouse primary thymic lymphomas also demonstrated a reduction in fidelit y using natural DNA templates (Kubota et al., 1995). Control experiments in both studies provided evidence that the increase in errors is not correlated with the presence of terminal deoxynucleotidyl transferase that adds nucleotides onto DNA without instruction from the template strand. In contrast, studies of the fidelity of DNA synthesis using the SV40 replication complex failed to reveal a difference between normal and malignant cells in culture (Boyer et al., 1995) and thus d o not support the concept of an errorprone polymerase in cancer cells. In principle, fidelity studies with purified DNA polymerases from normal and tumor tissues should provide definitive information on whether any of these enzymes is mutated. Mutant enzymes,

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however, are likely to be thermosensitive (Munir et al., 1993), exhibit different chromatographic properties, and may be lost during extensive purification. Thus, the most definitive studies in this area will be those that identify alterations in the DNA sequence of polymerase genes in cancer cells and then demonstrate that these same alterations enhance the frequencies of misincorporation by the encoded DNA polymerase. In the absence of these studies, it is difficult to determine whether mutations in DNA polymerase result in a mutator phenotype in specific cancers.

B. G e n e s Governing the Cell Cycle Genes that control the cell cycle are likely targets for the production of a mutator phenotype (Hartwell and Kastan, 1994). Mutations in G,/S checkpoint genes allow DNA replication in the presence of unrepaired lesions (Crook et al., 1994) and thus result in increased mutagenesis. For the purpose of this review, I focus on p53. Refer to recent reviews on the cell cycle for possible mutations in other proteins (Stillman, 1996; Elledge, 1996). In human cancers, p53 has been reported to be mutated more frequently than any other single gene (Greenblatt et al., 1994). It encodes a multifunctional protein involved in DNA repair, cell cycle control, and genomic stability (Harris, 1996). Mutations in p53 have been postulated to permit cells to enter S phase before the completion of DNA repair. As a result, unrepaired lesions could cause mutations during DNA replication. Also, mutations in p53 in murine cells can destabilize the genome and result in amplification of drug-resistant genes (Livingstone et al., 1992) and presumably other genes. Even though functional studies on mutants in p53 have yielded a wealth of information, we still lack knowledge of a definite role for p53 in malignant transformation. The p53 mutation spectrum, however, has provided unique clues about the relation of DNA damage to mutations in tumors (Weinstein, 1995). In three common human cancers, p53 mutations are the same type as those of the agents causally associated with that tumor. In liver cancer, in geographic areas with a high level of exposure to aflatoxin, p53 mutations are localized to codon 249 and are predominantly G+T transversions. The same G+T transversions are produced in livers of animals exposed to aflatoxin. In human skin tumors (basal and squamous cell carcinomas and melanomas), presumably caused by ultraviolet ray exposure, p53 mutations include tandem CC+TT substitutions. These mutations have been reported only after exposure of DNA to ultraviolet irradiation (Miller, 1985) or to oxygen free radicals (Reid and Loeb, 1992). In lung cancers, three codons in p53 are mutated a t an unusually high frequency (Hollstein et al., 1996). One of these codons, 157, is not a frequent site for mutations

Cancer Cells Exhibit a Mutator Phenotype

41

in other tumors. Mutations at this hot spot in lung cancers are predominantly G+T transversions. The distribution of adducts and mutations in p53 in HeLa cells or bronchial epithelial cells after exposure to the tobacco carcinogen, benzo[a]pyrene diol epoxide, corresponds to the distribution in lung cancers. These studies imply that adduct formation in p53 is targeted by some unknown mechanism and is an early and perhaps initiating event in the carcinogenesis by these agents. Before designating mutations in p53 as the primary event in the pathogenesis of these cancers, however, we should recall the situation with the ras oncogene in methylnitrosourea-induced breast cancer. Without exception, G+A transitions were observed in the induced tumors; this mutation mimics the mutagenic specificity of methylnitrosourea in vitro (Burns et al., 1988) and thus provides compelling evidence that the tumors result from the corresponding mutation in the ras oncogene (Barbacid, 1987). The presence of the mutation in normal cells and the dependency of the tumors on hormonal stimulation, however, have brought this concept into question (Cha et al., 1996). The mutation spectrum of p53 has also provided support for a mutator phenotype in cancer. Strauss (1996, and unpublished results) analyzed the reported sequences of p53 mutations in human cancers. The percentage of silent mutations (i.e., mutations that encode an amino acid identical to the wild type) in mutated p53 is at least 20-fold greater than anticipated. There is no evidence to indicate that these silent mutations are selected, nor d o these mutations correspond to the p53 polymorphisms in the human population. Therefore, these results provide strong evidence that p53 is hypermutable, at least a t some stage during the progression of these tumors. A demonstration of an increase in silent mutations in other genes or in pseudogenes would provide exceptionally strong evidence in support of a cancer mutator phenotype.

C. Genes Involved in DNA Repair and DNA Replication Several rare inherited human diseases are characterized by deficits in DNA repair and also exhibit unusually high incidences of cancer (Cleaver and Kraemer, 1989). There appears to be direct associations among the source of exogenous DNA damage, the mutation that renders the DNA repair pathways ineffective, and the type of mutations produced in the resultant tumors. These associations may provide evidence that deficits in DNA repair and mutations are the initiating events in human cancer. The two most extensively investigated mechanisms for the repair of chemically modified DNA are nucleotide excision repair and base excision repair pathways (Hanawalt et a/., 1979). Nucleotide excision repair removes a diverse spectrum of chemically modified nucleotides in DNA. Genes involved

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in this repair pathway are mutated in xeroderma pigmentosum, and cells harboring these mutations are sensitive to ultraviolet irradiation and carcinogens. Considering the wide spectrum of modified nucleotides repaired by this mechanism, it is surprising that susceptibility in xeroderma pigmentosum is restricted to skin cancer. Based on this anomaly, it has been suggested that many common human cancers are not caused by the DNA lesions that patients with xeroderma are unable to repair (Cairns, 1981). Base excision repair appears to be more specific for certain modifications. Mutations in this pathway have not been identified in human diseases. Studies are in progress in many laboratories to knock o u t these genes in mice and to determine if there is an increased incidence of malignancies. Despite the importance of these inherited diseases in defining the associations between DNA damage and cancer, mutations in these DNA repair genes have not been associated with common human cancers. Possible exceptions are the increased incidence of breast, lung, bladder, and stomach cancer in blood relatives of patients with ataxia telangiectasia (Swift et al., 1991). The DNA repair protein Oh-methylguanine-DNA methyltransferase (MGMT) removes highly mutagenic 06-alkyl groups from Oh-alkylguanine in DNA. It has been reported that histologically normal brain adjacent to most primary human brain tumors lacks detectable levels of this important DNA repair activity (Silber et al., 1996). These authors suggested that the mutator phenotype arising from lack of M G M T may be involved in generation of the genomic alterations characteristic of gliomas.

D. Other Target G e n e s Less well defined targets for the generation of a mutator phenotype are DNA helicases. These enzymes separate the two strands of a DNA helix ahead of the DNA replicating proteins. They are involved in a variety of DNA synthetic processes, including DNA replication, repair, transcription, and recombination. Decreased strand separation could cause pausing by DNA polymerases and increased misincorporation (Fry and Loeb, 1994). Two inherited diseases, Werner and Bloom syndromes, have been shown to contain mutations in genes that have all the signature nucleotide motifs of a helicase (Ellis et al., 1995; Yu et al., 1996). In both syndromes, cells from patients exhibit spontaneous genetic instability in culture (German et al., 1977; Fukuchi et al., 1989). Patients with Bloom syndrome are immunologically deficient and usually die of leukemia or lymphomas in early adulthood (German et al., 1977). Patients with Werner syndrome prematurely exhibit nearly all of the stigmata of aging and have a wide spectrum of unusual tumors at a young age (Epstein et al., 1966; Goto et al., 1996). Many proteins participate in DNA replication. DNA polymerases are just

Cancer Cells Exhibit a Mutator Phenotype

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one component of a complex DNA-replicating apparatus to ensure the fidelity of DNA synthesis. Among these proteins is proliferative cell nuclear antigen (PCNA), a processivity factor for DNA polymerase-6 that has been shown to interact with human mutL (Umar et al., 1996). These findings suggest a direct linkage between complexes of proteins that replicate DNA and that repair errors in DNA replication. Based on these associations, it is likely that mutations in PCNA would alter the fidelity of DNA synthesis. In a wider context, it is reasonable to assume that many of the genes that function to maintain genomic stability in normal cells would be targets for a mutator phenotype. Among these candidate target genes are those involved in nucleotide metabolism, recombination, and chromosomal segregation as well as genes yet to be identified that determine chromosomal localization, gene copy number, and the mitotic cycle (Cheng and Loeb, 1993; Hartwell and Kastan, 1994).

VI. MICROSATELLITE I NSTABlLlTY Until recently, experimental support for the concept of a mutator phenotype has been equivocal. There was insufficient experimental evidence to establish that any of the postulated target genes are mutated in tumors or that these mutations generate a mutator phenotype. Analysis of mutation frequencies in cell lines derived from human tumors yielded variable results, as have most studies on the error rates of DNA polymerases or crude DNA replicating complexes (Loeb, 199 1).This situation has dramatically changed. Recent studies on microsatellite instability have provided strong evidence for a mutator phenotype in human cancers. Microsatellite instability has been detected in colon cancers, both sporadic and those associated with hereditary nonpolyposis coli, and it was then shown to be a general property of a variety of human malignancies.

A. Microsatellites Within the human genome are a multitude of repetitive nucleotide sequences. Among these are as many as 50,000 noncoding microsatellites, consisting of repeat units 1 to 4 bases long, with a n average of 15 to 60 tandomly linked repeat units. For the most part, they are found in introns and presumed to be noncoding. The most frequent repetitive sequence is a homopolymeric tract of adenosine residues [(A),];the second most frequent are alternating cytidine and adenosine residues [(CA),] (Weber and May, 1989). The variations in these sequences among individuals has been extensively ex-

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ploited for gene mapping. The pattern of sequences within all tissues in an individual is invariant. Within tumors, however, there are marked variations in many of these sequences generated by slippage during copying by DNA polymerases. In vivo, most of the slippage errors by DNA polymerases are corrected by the mismatch repair system. In tumors, this mechanism is defective, and as a result, variations in the lengths of microsatellites in tumor DNA occur frequently in association with mutations in mismatch repair genes.

B. Microsatellite Instability in Hereditary

Nonpolyposis Colon Cancer In seminal papers, Peinado and colleagues ( 1 992), and Ionov and associates (1993) detected multiple deletions in repetitive nucleotide sequences in DNA by comparing DNA fingerprints in colorectal tumors using polymerase chain reaction (PCR) primers with nucleotide sequences that were arbitrarily chosen. Because this method screens a large fraction of the human genome, the authors were able to conclude that these tumors carry more than 100,000 such mutations. They surmised that these deletions may represent an inherited form of colon cancer and might be mediated by a reduction in the fidelity of DNA replication or a decrease in the efficiency of DNA repair. Moreover, the presence of these mutations in adenomas suggested that these mutations were an early event in the carcinogenic process. At the same time, it was reported that many types of repetitive sequences within the genome, including specific microsatellites, were altered in hereditary nonpolyposis colon cancer (HNPCC) (Thibodeau et al., 1993). It was recognized rapidly that expansion of repetitive sequences in DNA could be the result of defects in the mismatch repair pathway. A historical perspective of these findings has been presented (Marra and Boland, 1995). Four different mismatch repair genes have been demonstrated to be mutated in about half of the different HNPCC families. Sixty percent of these HNPCC families have a mutation on chromosome 2p (Aaltonen et al., 1993; Peltomaki etal., 1993).The chromosome 2p HNPCC protein was soon identified as a homolog of the bacterial mutS protein, involved in mismatch repair in E. coli, and was named MSH2 (human mutS homolog) (Fishel et al., 1993). Thirty percent of these HNPCC families segregate with a locus o n chromosome 3p (Lindblom et al., 1993) that has been established to encode a different mismatch repair homolog, MLH1, which is homologous to the bacterial mutL protein and the yeast MLH protein (Bronner et al., 1994; Papadopoulos et al., 1994). Also, the remaining mutations have been detected in the human mutL homologs, PMSl and PMSZ (Nicolaides et al., 1 994); PMSZ has been shown to be functionally involved in mismatch repair (Risinger et al., 1995a).In each of these

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families, one mutant allele is inherited, and the other arises as a result of differences in somatic mutation rates in different tissues. Systematic biochemical studies of E. coli have established the function of each of these genes in E . coli mismatch repair. The system has been reconstituted in vitvo with purified proteins (Modrich, 1995). mutS is involved in the recognition of the mismatched nucleotides in DNA (Modrich and Lahue, 1996), and mutL is involved in the interaction of mutS with other proteins. In human cells, there appear to be multiple components for recognition of specific mismatches as well as insertions and deletions. In E. coli, mutH recognizes the methylated sequence on the template strand and thus is responsible for determining which of the strands is repaired. The fact that there is no human homolog for mutH suggests that the signal for strand discrimination in eukaryotic cells is different from that in bacteria. Although there is some evidence that single-strand breaks act as a signal to designate the newly replicated lagging strand in eukaryotes, further studies are required. The concept that microsatellite instability is an indicator of a more general mutator phenotype (Loeb, 1994) in cancer is receiving increasing experimental support. First, alterations in repetitive sequences do not appear to be confined to introns. Human colon cancers with high rates of microsatellite instability were found to harbor mutations in repetitive sequences in transforming growth factor-p receptor (Lu et al., 1995). These mutations inactivate the receptor, allowing the escape of cancer cells from transforming growth factor-p mediated growth control. This finding links studies on a mutator phenotype with mutations in tumor suppressor genes. Second, cell lines derived from colon cancers that exhibit a high degree of microsatellite instability also exhibit a 200-fold to 600-fold increase in mutations that render them resistant to 6-thioguanine and ouabain (Bhattacharyya et al., 1994; Phear et al., 1966; Eshleman et al., 1995). Thus, microsatellite instability is linked to and provides a sensitive indicator of a mutator phenotype.

C. Microsatellite Instability in Sporadic Tumors Microsatellite instability has been reported in a large number of tumors not associated with HNPCC, including cancers of the colon (Ionov et al., 1993), endometrium (Risinger et al., 1993), breast (Patel et al., 1994), lung (Merlo et al., 1994), stomach (Dos Santos et al., 1996), ovary (Quinn et al., 1995), and prostate (Egawa et al., 1994). Even though microsatellite instability in many of these sporadic cancers is not as robust as that reported in HNPCC, the identification of this instability in multiple laboratories is impressive. A detailed compilation of the percentages of different sporadic tumors exhibiting microsatellite alterations is given in a review (Eshleman and Markowitz, 1995). In addition to sporadic tumors, there is an exceptional-

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ly high incidence of microsatellite instability in familial gastric cancers in Japan (Akiyama et al., 1996). These families have been shown to lack mutations in the known human mismatch repair genes. Furthermore, defects in known mismatch repair genes have been detected in only about half of sporadic colorectal tumors that exhibit microsatellite instability (Huang et al., 1996; Papadopoulos et al., 1995). This implies that other changes, presumably other mutations, are responsible for the observed microsatellite instability in many of these tumors. Thus, it is likely that other genes still to be identified are involved in mismatch repair in humans, o r that other genes involved in DNA replication are responsible for these mutations (Loeb, 1994). The source of microsatellite instability in many sporadic tumors is not clear. The presence of microsatellite instability in sporadic tumors has been verified in studies of tumor cell lines. As far as is known, most of these cell lines are derived from sporadic tumors and not from HNPCC-associated tumors (Boyer et ul., 1995). Tumor cell lines have been shown to contain mutations in MSH2 (Umar et al.. 1994), GTBP (Papadopoulos et al., 1995), M L H l (Li and Modrich, 1995), and PMS2 (Risinger et al., 1995a). Cornplementation studies with cell free extracts from endometrial and colorectal cell lines indicate at least four different subsets that generate microsatellite instability (Umar et al., 1994).The finding that microsatellite instability can be complemented with wild-type mismatch repair proteins provides support that the mutations in mismatch repair genes in some of these cell lines are responsible for the associated microsatellite instability (Drummond et al., 1995; LI et al., 1994). It is possible, however, that these mutations in mismatch repair genes were produced during repetitive passages in cell culture. Nevertheless, the facts that the defect exhibited in sporadic tumors is also observed in cell lines derived from these tumors and that the defect can be corrected by the introduction of specific chromosomes containing the wildtype genes (Koi et al., 1994; Risinger et al., 1995b) establish that microsatellite instability in at least some sporadic tumors is the result of deficits in mismatch repair.

D. Other Mechanisms for Mutation Accumulation The concept of a mutator phenotype is based on the assumptions that mutations arise in dividing cells and that the mutation rate (mutations per cell generation) in these cells is insufficient to produce the large numbers of mutations found in human cancers. An alternative hypothesis has been proposed, namely, that mutations accumulate in nondividing cells (Strauss, 1992). Simply expressed, the correct denominator in this formulation is not cell generation, but time. The implication is that cancers arise in nondividing cells that have accumulated large numbers of mutations. In support of

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this concept, E. coli and yeast maintained in stationary phase exhibit high mutation frequencies in specific selectable markers (Cairns et al., 1988; Hall, 1992; Hall, 1995). Furthermore, many of the mutations that arise in stationary phase occur in runs of similar nucleotides, suggesting slippage by DNA polymerases. MacPhee (1995) has set forth the interesting hypothesis that mutations in nondividing cells can arise by mismatch repair in the absence of strand discrimination. In this situation, half of mismatches would be repaired correctly (by replacement of the altered nucleotide) and half would be converted to mutations. This is at variance with the findings of mutations in mismatch repair genes in some cancers but is compatible with cancers that exhibit microsatellite instability in the absence of mutations in mismatch repair genes.

VII. THEORETICAL AND PRACTICAL CONSI DERATI ON S A. Mutations during Tumor Progression For a mutator phenotype to be a driving force in tumor progression, it would have to be an early event. The initial mutation in a key enzyme involved in DNA replication or DNA repair could decrease the fidelity or efficiency of these processes. With each subsequent round of DNA replication, there would be an increase in mutations throughout the genome. Among these mutations would be mutations in other genes involved in maintaining genetic stability. The timing of mutations in mismatch repair genes is controversial. Some data suggest that mutations in mismatch repair genes are not early events in the progression of colorectal cancer. For example, mutations in mismatch repair genes and microsatellite instability were considered to occur during the transition from late adenoma to early carcinoma stages (Tomlinson et al., 1996). Moreover, computer simulation of a mathematical model for colon cancer suggested that selection without increased mutation rates is sufficient to explain the evolution of tumors (Tomlinson et al., 1996). In contrast, the higher prevalence of microsatellite instability in cancers from patients with multiple independent sites than in cancers from patients with solitary tumors suggests that microsatellite instability is a much earlier event (Shinmuv c l al., 1995). Shibata and colleagues (1996) microdissected different Seb;fl

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gCrkh .

I

is d

/

I I I

E

I I

I

. I

6'

I

Senescence

Pre-Crisi Fibroblasts

Immortnlized Fibroblasts

Fig. 2 Summary of experimental data for relations of DNA-MTase activity to fibroblast aging and infection with SV40. The graph depicts the fold changes in DNA-MTase activity relative to 1.O as the level in normal young cells (about passages 0 to 2 5 ) , as a function of cell passage for fetal human fibroblasts (young, about passage 25; senescent, about passage 50) and the same cells infected with SV40 virus at about passage 30 (infected cells in the precrisis stage from about passage 50 to 70; crisis phase cells from about passage 70 to 90; immortalized cells from about passages 130 to 200). The data are from Vertino et al., 1994, as discussed in the text, and relate closely to findings for overall DNA methylation by others in similar studies (Matsumura et al., 1989a, 1989b).

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which DNA-MTase gene mRNA levels were found to be substantially higher in cultured cancer cells of several types than in cultured nontumorigenic cells (El-Deiry et al., 1991). Matsumura and coworkers (1989a, 1989b) have noted a fundamental difference in the capacity of normal versus transformed human fibroblasts to methylate DNA, which fits well with the previous data. In nontransformed fibroblasts in culture, overall levels of genomic methylation fall as the cells approach a final state of senescence. When young cells are infected with the SV40 virus, however, they bypass the normal cell senescence point and maintain an increased life span during which overall levels of DNA methylation d o not decrease. Moreover, in these pretransformed cells and in those that actually survive a later crisis period and emerge as immortalized cells, it is more difficult to inhibit active DNA methylation with the drug 5-AZA-cytidine than in normal fibroblasts (Matsumura et al., 1989b). More recently, we have mapped the expression of DNA-MTase mRNA and activity in this same experimental system and revealed a pattern, summarized in Figure 2, which well fits with the previous data (Vertino et al., 1994).In normal senescing fibroblasts, levels of DNA-MTase gene expression fall to 5 0 % of those seen in young fibroblasts. When the cells are infected with SV40, however, these levels fail to fall because the cells bypass the senescence checkpoint and enter an extended replication period before entering cell crisis. At crisis, levels of DNA-MTase expression are actually as high or higher than those in young normal fibroblasts, and these high levels are maintained in those few cells that escape crisis and become immortalized (Vertino et al., 1994). An association between increased DNA-MTase activity and early events leading toward the transformed state has been further reinforced by two recent studies. First, nontumorigenic immortalized hybrid cells, which have been produced from fusion of normal human fibroblasts with malignant fibrosarcoma cells that have a seven-fold increased DNA-MTase activity, retain the high levels of the parent tumor cells (Kuerbitz and Baylin, 1996). This finding, in addition t o those from SV40 work described earlier, indicate that increases in DNA-MTase activity can occur before appearance of the fully transformed phenotype. Second, in a strain of mice genetically susceptible to lung carcinogenesis, but not in a resistant strain, exposure to the major tobacco carcinogen, NNK, leads to a three-fold increase in DNA-MTase activity, specifically in the lung type I1 pneumocytes that later give rise to adenocarcinomas (Belinsky et al., 1996).The levels initially peak within 3 days, return to baseline by 7 days, and then increase again several weeks later in the early benign lung tumors and progressively thereafter as these form carcinomas (Belinsky et al., 1996). These findings reinforce the concept that DNA-MTase gene expression begins early in, and is thus in a position to contribute to, tumorigenesis. Findings for DNA-MTase gene expression in natural settings for human

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tumor progression have paralleled some of the previously described findings for experimental systems. In initial studies of expression of the human DNAMTase gene in the colon, using reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we found evidence for progressively higher gene expression from normal mucosa to premalignant adenomas to frank carcinomas (El-Deiry et ul., 1991). Additionally, in this study, we found higher DNA-MTase transcript levels in normal-appearing mucosa from patients with colon neoplasms than from patients without these tumors (El-Deiry et al., 1991). The PCR procedures in this first study may have led to overestimates of the differences between each progression stage (15-fold for cancer versus normal mucosa from the same patient). Other workers more recently have shown 1.5- to +fold increases in mRNA for DNA-MTase in colon cancer versus adjacent colon (Lee et al., 1996; Schmutte et al., 1996). Our own subsequent study of DNA-MTase activity in these same settings again revealed increasing levels from normal mucosa to polyps to carcinoma and an average increase of about three-fold in colon cancers versus normal mucosa from the same patient (Issa et d.,1993). The factors underlying increased DNA-MTase activity in tumor progression have not been clarified. As mentioned earlier, there are reports that control regions within the murine DNA-MTase gene mediate increased transcription by response to activation of the ras signal transduction pathway (MacLeod et al., 1995). The region of the DNA-MTase gene identified as the rus responsive area, however, initially thought to be in the 5' flanking promoter, actually lies far downstream of the transcription start site and has weak basal transcriptional activity (Tucker etal., 1996b; Yoder et al., 1996). Thus, further studies are required to understand fully the role for the ?as pathway, or for other pathways, in modulating DNA-MTase activity during the various steps in cell transformation. As mentioned earlier, DNA-MTase activity is regulated as a function of DNA synthesis and cell cycle activity, and some have considered that this relation might be instrumental for producing any differences seen between normal and neoplastic cells. In fact, in a recent study, Lee and colleagues (1996) challenged, on this basis, the significance of the reported increases in DNAMTase gene transcripts in human colon tumor progression. When they use RNAase protection assays for DNA-MTase expression, they found only a 1.8- to 2.5-fold increase between normal colon and colon tumors (Lee etal., 1996). Furthermore, these differences were abolished if they normalized the data for expression of histone H4, which is a measure of S-phase activity of cells. These authors concluded that any differences between the tumor and normal cells are, then, solely due to the increased numbers of proliferating cells in the cancer (Lee et al., 1996). Although the results of the above study must be taken carefully into account, we think there is much evidence that such a simple relation between

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increased numbers of proliferating cells in tumor and normal tissue cannot account for much experimental data linking increased DNA-MTase and neoplasia. First, in our hands and others (Kautiainen and Jones, 1986), both in cell culture systems and in natural tumors, the fold increases for DNA-MTase activity in neoplastic cells are often too high for simple attribution to increased cell proliferation. In fact, in many tumors, such as colon cancer, the proliferative index is only marginally, or not at all, increased compared with corresponding normal cells or tissues (El-Deiry et al., 1991; Issa et al., 1993). Furthermore, when we employed in situ hybridization techniques for visualization of DNA-MTase mRNA in the murine lung cancer model discussed previously, virtually every type I1 cell, from the stages of hyperplasia to frank carcinoma, had markedly increased staining compared with normal cells (Belinsky et al., 1996). No previous data would predict, at a given point in time, such universal participation of entire cell populations in the modestly increased proliferative indices found for neoplastic tissue. Indeed, we found n o such measurable proliferative increase in the type I1 cells that had responded early to NNK exposure with a n increase in DNA-MTase activity (Belinsky et al., 1996). Given all of the above data, can some of the controversies about the increase of DNA-MTase in neoplasia be reconciled? It is well possible that entrance of cells from a resting Go stage into cell cycle could be fundamental t o initiating increased DNA-MTase activity in the process of neoplastic evolution. As discussed previously, however, in immortalized and transformed cells, this increase appears to persist, and even accumulate, when such cells are not engaged in cycling activity and S-phase-mediated DNA synthesis. This fact may underlie the accumulating evidence, discussed later, that the increases in DNA-MTase gene expression in tumorigenesis have a profound effect on DNA and play a fundamental role in the neoplastic process rather than serving as a simple index of cell proliferation.

2. WHAT IS THE EVIDENCE THAT INCREASES IN DNA-METHYLTRANSFERASE PLAY A FUNDAMENTAL ROLE IN TUMOR PROGRESSION? Although, as presented previously, there is a growing body of evidence associating increased DNA-MTase activity with the neoplastic state, it remains to be proved formally that this change is directly responsible for mediating specific steps in tumor progression. A major conundrum that must be resolved is why, frequently, the cancer cell genome simultaneously harbors overall hypomethylation and increased DNA-MTase activity. Several possibilities have been entertained in this regard. First, there could be a decreased efficiency of maintenance methylation because of chromatin changes that block the normal access of DNA-MTase to DNA. In this regard, the increased

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DNA-MTase might actually be stimulated, early in tumorigenesis, by the decreased overall genomic methylation, but the increase is insufficient to restore normal sites of methylation. Support for such a concept comes from the fact that folate deficiency induces an evolution of hepatic carcinogenesis during which both DNA hypomethylation and increased DNA-MTase activity occur very early (Christman et al., 1993; Pogribny et al., 1995). Alternatively, there is some evidence that active mechanisms are increased in tumor cells t o demethylate DNA directly, as opposed to loss of methyl groups resulting from simple decreases in maintenance methylation (Syzf et al., 1995). In this regard, an RNA species with apparent demethylation capacity has been reported (Weiss et al., 1996) and could play a role through increased activity in neoplastic cells. These possibilities and others remain to be verified, but for now, the explanation for the coexisting DNA-MTase increases, and genomic hypomethylation in tumor cells remains a mystery. Despite this dilemma, several studies suggest that the levels of increased DNA-MTase activity found in neoplastic cells can profoundly affect DNA and could well be involved with regional hypermethylation. Multiple lines of experimental evidence indicate that acute, but modest, increases in DNAMTase activity can transiently increase overall DNA methylation. First, twoto three-fold increases of DNA-MTase activity in murine NIH 3T3 cells expressing an exogenous DNA-MTase gene are accompanied by significant increases in overall DNA methylation (Wu et al., 1993). Second, in the previously discussed murine model for carcinogen-induced lung carcinoma, increased overall DNA methylation accompanies the transient increases in type I1 pneumocyte DNA-MTase activity induced by acute exposure to NNK (Belinsky et al., 1996). Third, studies from the Jaenisch laboratory in mouse cells homozygously deleted for the endogenous DNA-MTase gene (Tucker et al., 1996b) again reflect significant effects of modestly increasing DNA-MTase activity. In these cells, constitutive expression of the originally characterized murine cDNA for DNA-MTase from a strong exogenous promoter is poorly tolerated. In those few surviving clones in which the cDNA was expressed, however, levels of DNA-MTase activity much lower than those seen in wild-type cells were sufficient to effect restoration of much of the methylation normally seen in endogenous repeat viral sequences (Tucker et al., 199613). In addition to these studies, there are also experimental data indicating how the more long-term effects of increased DNA-MTase activity may profoundly effect DNA and influence tumor progression. The work suggests that during tumor progression, transformed cell types could be selected that have only a modest increase for DNA-MTase expression, whereas cells with higher increases d o not survive. For example, expression of a de novo bacterial cytosine DNA-MTase in murine cells is toxic except when low levels are produced from a leaky, inducible promoter or from a constitutive pro-

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moter in occasional selected cell clones (Wu et al., 1996). Similarly, constitutive overexpression of the human DNA-MTase gene is not well tolerated in human cells (Vertino et al., 1996). Also, even though it is not particularly difficult to select for murine NIH 3T3 cells expressing a constitutively regulated exogenous murine DNA-MTase gene cDNA, all clones obtained had not more than two to three times the activity of the parent cells (Wu et al., 1993). This increase is comparable to those discussed previously for colon carcinoma and other types of cancer cells. Thus, increases in DNA-MTase activity of more than three-fold may lead to cell death, whereas cells with more modest DNA-MTase gene overexpression are selected. Importantly, in the previously discussed murine systems, these latter cells have become tumorigenic and have evidence for either overall or regional increases in DNA methylation (Wu et al., 1993, 1996; Vertino et al., 1996). These and other discussed overall relations between various levels of DNA methylation and experimental manipulation of DNA-MTase activity are all summarized in Figure 3. The associations depicted may provide insight into how DNA methylation changes are of fundamental importance for tumor progression. Relative to the data in Figure 3, there is an important caveat for the biologic importance of the studies suggesting that cells may only tolerate modest increases in DNA-MTase activity. In the previously mentioned study of Tucker and colleagues (1996b), although expression of the short original cDNA for murine DNA-MTase was not tolerated by cells, expression of the longer DNA-MTase gene, now known to encode for the entire DNA-MTase protein of humans and mice (Tucker et al., 1996b; Yoder et al., 1996), was well tolerated and restored wild-type DNA-MTase activity and DNA methylation levels. The authors attribute this tolerance to inclusion of the new 5’ sequences in the protein (Tucker et al., 1996b). It is critical to note, however, that the longer DNA-MTase gene was inserted in a genomic fragment rather than as a cDNA driven from an exogenous constitutive promoter. In this setting, expression was achieved solely from an endogenous promoter located either in the 5’ flanking region of the genomic piece or in the homologous DNA-MTase site where this fragment was documented to have inserted (Tucker et al., 1996b). Thus, tolerance for the gene could well be due to the lower levels of expression for this genomic construct than found in those achieved with the constitutively driven shorter cDNA. If, as we are suggesting, increased DNA-MTase activity contributes directly to cell transformation, what are the underlying mechanisms? Although this remains to be determined, experimental evidence is accruing to suggest that this change could contribute to the CpG island hypermethylation associated with transcriptional inactivation of key tumor suppressor genes. First, when human fibroblasts are infected with SV40, and DNA-MTase activity levels fail to decrease as cells bypass the senescence checkpoint, aberrant CpG island methylation begins to appear by the time cells enter the crisis pe-

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riod (Vertino et al., 1994). This change is further apparent in those cells that survive crisis, and the immortalized progeny have multiple densely methylated CpG islands (Vertino et al., 1994). Second, in SV40-transformed human fibroblasts engineered to overexpress an exogenous human DNA-MTase gene, selected C p C islands become progressively hypermethylated over successive numbers of cell passage (Vertino et al., 1996). In general, the islands affected were those that showed some initial hypermethylation in the parent transformed cells. Thus, the single human DNA-MTase enzyme characterized could, during the prolonged course of neoplastic evolution, be permissive for both aberrant de nova methylation events and their perpetuation and extension. Alternatively, the initial de nova methylation events could be mediated, during tumorigenesis, by another DNA-MTase, such as that proposed by the Jaenisch group (Lei et d., 1996; Tucker et al., 1996a). This methylation could then be readily extended by the increases in the well-characterized DNA-MTase because this enzyme is known to perform de N O V O methylation more efficiently in regions of DNA in which fully methylated CpC sites already exist (Tollefsbol and Hutchison, 1995). All of the data linking increased DNA-MTase activity with tumor progression may underly findings from the Jaenisch laboratory, which have focused much recent attention on the role of DNA methylation changes in cancer. These workers performed a genetic experiment in which phenotypically normal mice heterozygous for deletion of the DNA-MTase gene and having 5 0 % of the wild-type levels of DNA-MTase activity, were bred with mice harboring a mutant allele of the APC gene (Laird et al., 1995). This latter mutation, when combined with somatic loss of the other APC allele, results in progressive formation of adenomas throughout the gastrointestinal tract

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Fig. 3 A model summary of experimental data, discussed in the text, relating relative levels of DNA-MTase activity to cell phenotype, in systems manipulated to overexpress o r undcrexpress the DNA-MTase gene. The graph assumes a valuc of 1.0 for a normal parent cell and relative values for experimental maneuvers that raise o r lower steady-state enzyme activity from this value. As discussed in detail in the text, levels below about 10 to 15% are shown to be associated with cell death, such as is seen in mouse embryos homozygous for knockout of the DNA-MTase gene (Li et a/., 1992), or after attempts to differentiate cultured embryonic stein cells completely lacking the gene (Tucker et d,1996a). Levels from about 15 to 2 0 % arc depicted associated with chromosome instability changes, as reported in studies of cells cultured in 5-AZA-cytidine (see text). Cells with levels from 25 to 100% are assumed to grow with a normal phenotype (Li et a/.. 1992; Tucker e l al., 1996a). Manipulations that chronically increase DNA-MTase activity up to 3-fold are associated with cell transformation (Wu et nl., 1993, 1996), whereas increases above this range are highly correlated with cell death (Vertino et ul., 1996; Wu et al., 1996). The asterisk refers to the fact that some rare ccll subpopulations survive levels higher than threc-fold and have CpG island hypermethylation and/or a transformed phenotype (Vertino et al., 1996).

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of newborn animals. Offspring mice harboring both the APC and DNAMTase gene mutations had a 50% reduction of intestinal adenomas during a 6-month period following birth, and these lesions could be essentially eliminated if treatment with the demethylating agent, 5-deoxy-AZA-cytidine, was combined with the DNA-MTase allelic deletion. The mechanisms underlying this striking reduction of tumor progression are not yet apparent, although two leading possibilities have received much discussion (Hopkin, 1995; Laird et al., 1995). The Jaenisch group initially favored the proposal that the lowering of DNA-MTase activity reduces rates of DNA mutations during adenoma formation in the newborn animals (Hopkin, 1995; Laird et al., 1995). The rationale underlying this theory is that the reduced levels of DNA-MTase activity in the heterozygous knockout mice might result in appearance of less methylated C p C sites, which could undergo deamination to thymidines. Also, in vitro, a bacterial DNAMTase has been shown to directly cause deamination of cytosine to form uracil when levels of the methyl donor, S-adenosylmethionine, are extremely limiting (Shen et al., 1992). When repaired, these uracils would be converted to thymidines. Such low levels of S-adenosylmethionine, however, have not been found in vivo (Schmutte et al., 1996), and subsequent studies of the APC and DNA-MTase heterozygous animals have shown no evidence for decreased mutational rates (Jackson-Grusby et al., 1997). The second leading possibility for the results from the Jaenisch laboratory is that lowering of DNA-MTase activity might reduce rates of accrual for aberrantly methylated CpG islands and thus reduce the rate of tumor suppressor gene inactivation over the course of adenoma formation (Hopkin, 1995; Laird et al., 1995). We think this is the most attractive possibility, but one that must await verification from more formal testing in the Jaenisch and other models. In summary, a growing body of evidence is pointing to an important role for increased DNA-MTase activity in tumorigenesis. Much work remains to document this possibility and to elucidate the exact mechanisms involved. The evidence has been crystallized by the findings of the Jaenisch group, but the generality of their findings must be studied in other systems. The intestinal tract of the mouse undergoes profound differentiation changes 2 weeks after birth (Crane, 1968; Koldovsky, 1969), and some of the effects of lowering DNA-MTase on adenoma formation in this setting might be linked to alterations in this process. Studies of adult carcinogenesis models will then be particularly important before the results from the Jaenisch group can be placed into perspective for neoplasia in general. The next few years should see much research activity regarding the role of DNA-MTase in neoplasia, and the possibility of targeting this enzyme as an anticancer strategy is discussed at the end of this chapter.

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B. What Additional Mechanisms Underlie the Aberrant

Methylation of CpG Islands in Neoplasia? Among the methylation changes that occur in the evolution of neoplasia, hypermethylation of CpG islands is now best functionally linked to tumor progression, so further discussion of the mechanisms underlying this process seem warranted. Although, as noted previously, increases in DNA-MTase activity may play a critical permissive role in the appearance of this change, it is likely that other factors are equally as important, or in a gene-specific manner, more important. These potential events are considered in this section. I . DOES CpG ISLAND HYPERMETHYLATION TRULY REFLECT A DE NOVO PROCESS IN TUMOR PROGRESSION?

For classic gene mutations in noninherited neoplasms, one usually does not question whether the specific mutation preexisted in the normal cells from which the neoplasm arose. The special nature of DNA hypermethylation as an inactivating event, however, particularly the potential reversibility of the process, raises the issue of whether some of the patterns now associated with neoplasia could reflect clonal expansion of cells normally harboring such methylation. For example, could stem cells in selected tissues, which may be the initial targets of cell transformation, normally contain densely methylated CpG islands in the promoter regions of genes that are subsequently unmethylated in all the more mature progeny of these precursor cells? Such a mechanism might help maintain the associated gene in a silent state before required activation for steps in cell differentiation. We have addressed this possibility for several genes by using a sensitive new PCR technique that can monitor, in DNA from small numbers of cells, the methylation status of minor allele populations in complex cellular backgrounds. This procedure, which we have termed methylation-specific PCR (MSP) (Herman et al., 1996a), depends on initial exposure of DNA to sodium bisulfite under conditions in which all unmethylated cytosines are deaminated to form uracil, but methylated cytosines are resistant to this change (Frommer et al., 1992; Myohanen et al., 1994).During subsequent PCR amplification of the treated DNA, the uracils are converted to thymidine. The PCR primers are designed to detect these sequence differences and specifically to amplify sequences that did or did not initially have selected CpG sites methylated (Herman et al., 1996a). This technique can detect the methylation of 1 among 1000 unmethylated alleles (Herman et al., 1996a). Using MSP analysis for study of the p l 5 and p16 genes, which are frequently hypermethylated in multiple types of hematopoietic malignancies, we did not detect this change in highly purified stem cell populations from the bone mar-

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row of normal individuals (Herman et al., 1997). Similarly, the MSP technique detected no hypermethylated alleles for the E-cad gene CpG island in purified normal breast epithelial cells (Graff et al., 1997), even though hypermethylation of this gene is a frequent event in breast carcinomas. We concluded from these initial studies that there are tumor suppressor genes for which C p C islands truly become de nouo methylated during the process of tumor progression.

2. AGING IS ASSOCIATED WITH PROMOTER REGION CpG ISLAND METHYLATION IN SELECTED G E N E S In contrast to the previous findings for several tumor suppressor genes, we have detected a class of genes that appear to have hypermethylated patterns in neoplasia, which may reflect the normal cell populations, giving rise to the involved tumors. These genes may provide one fundamental link between cancer and perhaps the most important risk factor for the development of most human neoplasms, aging. The role of aging in cancer has generally been attributed to cumulative exposure to carcinogens over time as well as to the long period required for the development of a clinically detectable tumor in uiuo. Physiologic aging, however, is accompanied by profound changes in gene expression that cannot be explained on the basis of accumulated mutations (for review, Holliday, 1991; Smith and Pereira-Smith, 1996). Because, as discussed previously, DNA methylation changes have great potential to affect gene expression by epigenetic mechanisms, altered DNA methylation patterns have long been suspected to play a role in aging and the associated increased incidence of neoplasia (Holliday, 1987). Many of the patterns observed fit well with components of the methylation imbalance observed in neoplasia, and hypermethylation changes in gene promoter regions are proving most intriguing. First, both overall genomic hypomethylation and hypomethylation of specific genes have been associated with aging cells. Aging human fibroblasts in culture have progressive loss of methylcytosines as they approach senescence (Holliday, 1986; Wilson and Jones, 1983), and this decrement has even been proposed as a “counting mechanism” used by the cells to monitor the number of divisions allotted over their lifetime (Holliday, 1990b). It is has been suspected that this hypomethylation could play a role in altered gene expression associated with aging because this change has been reported for genes such as c-myc (Ono et al., 1990) and actin (Slagboom et al., 1990). The position of the change relative to gene regulatory regions, however, has generally not been well correlated, and the role of methylcytosine loss in aging-associated changes in gene expression remains unknown. Second, regional areas of hypermethylation have also been associated with aging cells (Schmookler Reis and Goldstein, 1982; Swisshelm et al., 1990; Uehara et al., 1989), although until recently, the implications of this change

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during the aging process and its link to cancer have not been clear. Hints that these changes could help predispose cells to neoplasia had emerged from experimental systems. As mentioned earlier, Matsumura and colleagues (1989a, 1989b) have observed that induction of human fibroblasts by SV40 to escape the senescence checkpoint is associated with abrogation of the methylcytosine loss seen during senescence of normal cells. Subsequently, we have shown that this SV40 effect is paralleled by the emergence of hypermethylation changes in selected promoter region CpG islands (Vertino eta/., 1994). Most important, however, has been the association, in the promoter regions of specific genes, of hypermethylation patterns common to aging cells and neoplasms. Our initial observations of these associations stemmed from the serendipitous observation that, in colon neoplasms, while all benign and malignant tumors examined displayed hypermethylation of the ER gene promoter and a decreased expression of this gene relative to normal colon, distinct partial methylation could also be observed in adjacent, normal-appearing colonic tissue (Issa et a/., 1994). O n further investigation, we determined that this methylation in normal colon was an age-related event, almost undetectable in young individuals and progressively more prominent in older individuals (Issa et ~ l . ,1994). Because 100% of colonic tumors examined had extensive ER methylation, including small adenomatous polyps, and the aging changes are greatest in colon regions where tumor incidence is highest (Issa et al., 1994), we hypothesized that the cell of origin of these tumors was one in which the ER gene was hypermethylated. Based on these data, and o n the fact that introduction of the ER gene into colon cancer cells resulted in marked growth suppression (Issa et al., 1994), it may be envisioned that the aging-associated methylation of the ER gene provides a selective growth advantage to subpopulations of colon mucosa cells that harbor this change. We discuss later how such cells could constitute the field defect that may predispose groups of cells to the most common forms of colonic neoplasia. The prevalence of these types of age-related gene methylation changes remains to be determined. Our initial observations from studying randomly selected genes suggest that such hypermethylation is a relatively rare event. Genes such as p16, which are hypermethylated in 20 to 4 0 % of all colonic tumors, are not detected by MSP to have any methylation in normal colon from older people (Herman, unpublished data). Nevertheless, the age-related events seen for the ER gene in normal colon d o occur for other genes, and the observations for one of these, the insulin-like growth factor-2 (IGF2) gene, possibly even hold clues to the events that underlie the process. The IGF2 gene is a fetal growth factor that is imprinted in rodents and in humans, and is silent on the maternal allele and expressed exclusively from the paternal allele (Barlow, 1995). This gene is highly expressed in several forms of adult tumors, including colon cancer, and this growth factor is

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thought to play a potential role in the evolution of these neoplasms (Christofori et al., 1994). In humans, the IGF2 gene, located on chromosome 1 lp, has four promoters (P1 to P4), with four different leader exons (for review, Sussenbach et al., 1993). P2 through P4, which are relatively close to each other, are contained in a CpG island and are coordinately imprinted. By contrast, P1, which is located more than 20 k b upstream of P2, is not contained in a CpG island and is expressed biallelically in human tissues, suggesting that it escapes imprinting (Vu and Hoffman, 1994). In adult humans, the liver is the major source of IGF2 production, and P1 is preferentially used in this tissue. The imprinted status of IGF2, in part, may be determined by interaction with enhancers for the H19 gene, which resides nearby on chromosome 1l p . The H19 gene is reciprocally imprinted to IGM and is thus expressed exclusively from the maternal chromosome (for review, Barlow, 1995). The gene also encodes for an RNA species only, which could play a key role in imprinting of IGF2 (Barlow, 1995). We hypothesized that in addition to the previously proposed regulation, promoter methylation might also play a role in the allele-specific expression of IGF2. We investigated methylation around P2 to P4 in various tissues, in aging colon, and in various neoplastic tissues and found that, in young people, the P2 to P4 CpG island is methylated only on the silenced maternal allele (Issa et al., 199613).Strikingly, P2 to P4 methylation increases progressively with age, and in the colon, this increased methylation can be accounted for, in part, by methylation of the CpG island on the previously unmethylated paternal allele. Most common adult neoplasms studied had even more extensive P2 to P4 methylation, and in tumor cell lines, hypermethylation at P2 to P4 resulted in a marked reduction of IGF2 transcription from P3. By contrast, P l y the upstream promoter that is not contained in a CpG island, was used in most cell lines examined (Issa et al., 1996b). In this setting, we then have the ironic situation in which promoter hypermethylation is associated with possible overexpression of a gene but in which a promoter switch is associated with this event. We believe that these age-related methylation changes in IGF2 are particularly important not only for their potential to enhance tumor growth but also because they provide a potential clue to the mechanism of age-related hypermethylation. The data suggest that areas of parentally determined allele-specific methylation may be at special risk for this phenomenon because, during aging, methylation could spread from the initially methylated allele to the opposite allele. Such spreading of methylation across alleles previously has been suggested to account for a process in plants termed cosuppression (Flavell, 1994), in which a transgene, which has become methylated and inactivated, results in age-dependent methylation and inactivation of the homologous endogenous gene. To explain this phenomenon, it has been proposed that the methylated transgene and the endogenous gene pair, transiently, by homologous recombination and that DNA-MTase recognizes the paired strands as hemimethylated DNA and spreads methylation from the

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transgene to the endogenous gene. Such homologous pairing has been observed at imprinted loci in humans (Lasalle and LaLande, 1996). In addition, methylation spread across alleles has now been shown to occur in Ascobolus sp., in which it appears to be mechanistically related to homologous recombination (Colot et al., 1996).Interestingly, homology-dependent spreading of methylation with age may not always require prior methylation of one allele. Many human genes have pseudogenes which are inactive and usually hypermethylated. If these pseudogenes share significant homology to the 5’ region of an expressed gene, then one can envision homologous pairing and spreading of methylation during the aging process.

3. D O OTHER RISK FACTORS FOR TUMOR DEVELOPMENT PLAY A ROLE IN C p C ISLAND HYPERMETHYLATION? In addition to aging, many other events contribute directly to tumor development. Environmental exposure and specific carcinogens are perhaps the most prominent of these. One group has shown that the metal carcinogen, nickel, which localizes to the cell nucleus, can evoke reversible tumorigenic stimuli that are apparently associated with hypermethylation changes surrounding an inserted DNA construct containing a selectable marker (Lee et al., 1995). These authors postulated that the metal evokes an alteration in chromatin structure associated with the methylation change and that this is a model for how carcinogens might lead to inactivation of certain tumor suppressor genes. In keeping with this model, our studies in collaboration with the Belinsky laboratory have shown that different carcinogens evoke strikingly different patterns of ER gene hypermethylation in murine and rodent models of lung carcinogenesis (Issa et al., 1996a). In this setting, the major tobacco metabolite, NNK, causes tumors with a low incidence of ER gene hypermethylation, whereas this change is virtually universal in the same tumor type induced by irradiation or when arising spontaneously in the animals. In contrast, in rats, there is a high incidence of p16 gene hypermethylation in the NNK-induced tumors (Swafford et al., 1997). Thus, different carcinogens may evoke specific changes in different regions of the genome, which may play a major role in the hypermethylation changes under discussion. Likely, defining these interactions will become a n active area of research during the next few years. 4. ARE THERE MUTATIONAL CHANGES IN G E N E PROMOTER REGIONS THAT UNDERLY CpG ISLAN D HYPE RM ET HY LATlO N ? The aberrant promoter area CpG island methylation changes in tumor suppressor genes occur without concomitant coding region mutations in the involved genes. However, could it be that mutations in the promoter regions

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themselves might be responsible for structural changes in DNA that incite the aberrant methylation patterns? If so, then the entire process would evolve from a genetic change. This is not an easy question to dissect. If acting through long-range chromatin changes, the mutations involved could be located at some distance from the CpG island regions that actually become aberrantly methylated. What can be said is that the CpG island regions that directly harbor the methylation changes in the VHL and p16 tumor suppressor genes contain no mutations in tumor cells (Herman et al., 1994, 1996a). These studies must be expanded before DNA mutational changes can be ruled o u t as a fundamental cause of aberrant methylation in cancer. As mentioned previously, areas of unusual DNA structure appear to be preferentially methylated by DNA-MTase (Christman et al., 1995; Smith et a[., 1991), and this may hold clues for the aberrant patterns seen in cancer. Findings for other diseases stress the importance of such investigations and further suggest that concentration on repeat sequences may prove rewarding. For example, the inherited mental retardation syndrome, fragile X, is caused by abnormal expansion of a promoter region triplet repeat. This event clearly precedes aberrant methylation of the CpG island containing the repeat sequences, and the disease phenotype results from associated loss of expression of the involved gene (Bell et al., 1991; Pieretti et al., 1991). 5. WHAT LOCAL FACTORS ARE ASSOCIATED WITH PROTECTION O F CpC ISLANDS IN NORMAL CELLS, A N D ARE THESE ALTERED IN NEOPLASTIC CELLS?

One of the most intriguing questions in the biology of DNA methylation is, What normally protects CpG islands from methylation? As previously discussed, we have provided experimental evidence that prolonged exposure o f cells to increased DNA-MTase activity can cause initiation and/or spreading of CpG island methylation (Vertino et al., 1996). In the fibroblast model used, however, only selected regions showed this change, and some of the tumor suppressor genes hypermethylated in other cell types, such as VHL. and p16, did not become hypcrmethylated (Vertino et al., 1996). Although the DNA-MTase increases may be permissive for the CpC island events, local factors that normally maintain islands free of methylation could become disrupted, on a cell type- and gene-specific basis, in neoplasia. Elucidation of such factors in normal cells is a priority of ongoing research, although what little is known has been gleaned largely from studies of one gene, APRT, in hamsters and mice. Three laboratories, using gene insertion studies in cell culture and/or transgenic mice, have ascertained that three consensus and one nonconsensus Sp1 binding sites located just 5’ to the transcription start site of the APRT gene are important for protecting a typical CpG island in the region from becoming niethylated (Brandeis et d., 1994; Macleod et d., 1994; Mumma-

Fig. 4 A model for the anatomy of a typical human gene 5’ region CpG island and the methylation status in normal and neoplastic cells. Data represent average structural content and distances compiled from analyses of database sequences for the human VHL, E-cad, APRT, GST (glutathione-S-transferase)-K, and TIMP (tissue inhibitor of metalloproteinase)-2 genes. (A) The typical 5’ C p C island in normal cells is shown as an unmethylated CpC-rich area of about 1.5 kb extending from the immediate 5’ flanking region, through the transcription start site and the first exon, and well into the first intron. The unmethylated island (blue area, labeled protection) is embedded between 5’ and 3’ flanking areas, which are densely methylated (all gene regions outside protected area). Each gene analyzed contained two Alu repeats within 1.0 kb of the 5’ portion of the island, and the VHL gene has an Alu within 0.5 k b of the 3’ end. These Alu repeats are heavily methylated in the VHL and E-cadherin genes, as analyzed by the MSP procedure and by bisulfite genomic sequencing (see text). The average position for clusters of SPl sites toward the borders of the island is depicted. DNA-MTase activity is shown to be excluded from the “protected” island and accessible to the flanking regions. (B) A hypermethylated C p C island is shown, as would be typical for our analyses of the E-cadherin gene in many breast cancers and the VHL gene in certain renal cancers. The entire island is methylated, as shown by absence of the blue central area of protection. DNA-MTase now has access to the island because the depicted relative increase in activity has overidden the 5’ and 3’ borders that normally protect against methylation and/or abnormalities of local features of these borders have led to disruption of the protective function.

Fig. 5 A hypothetical model for progression of a generic tumor that takes into account genetic changes and altered DNA methylation. Details are found in the text, and salient features of the model are as follows. (A) A normal epithelial cell system, such as the normal colon, in an older person. Two cells have age-related CpG hypermethylation in the ER gene. ( B ) This change participates in a growth advantage for the involved cells, which leads to their forming a “field effect” for colon cancer predisposition. Exposure of this field to carcinogens and/or chronic injury leads, in some cells, to a relative increase in DNA-MTase activity (green nuclei) and to a transient increase in overall DNA methylation (red circle around the nucleus). (C) In one such cell, an initiating mutation occurs, and the heightened sensitivity of the cell to this event, by virtue of the ER gene change, leads to clonal expansion into the preneoplastic area of dysplasia. In the dysplasia, the cells retain the relative increase in DNA-MTase activity, but now have either normal o r reduced levels of overall DNA methylation (absence of red circle around the nuclei). Heterogenous cells now appear in the dysplastic lesion, which have, individually, hypermethylation of the p16 gene and a mutation of the pS3 gene. ( D )There are phenotypic consequences for these events, however, only when they occur together in a single cell (bottom right of the lesion), and this cell leads to clonal expansion into carcinoma in sztu. (E) This carcinoma develops a heterogenous pattern for hypermethylation and inactivation of the E-cadherin gene (cell at the left, bottom of the carcinoma), and this event results in evolution o f invasion of the basement membrane and metastatic capacity of the carcinoma.

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neni et al., 1995). The CpC-rich Sp-l-binding motif is contained in most CpG islands (Antequera and Bird, 1993a). The APRT gene CpG island is protected from methylation as long as the two most 3’ of the four Sp-1 sites are intact (Mummaneni et al., 1995).Interestingly, although such deletion of the more 5 ’ Sp-1 sites leads to loss of protection from methylation, this does not interfere with transcriptional activity when the unmethylated proximal promoter is used to drive a reporter gene (Dush et al., 1988). Mummaneni and colleagues (1993) have designated a region just 5 ’ to the APRT Sp-1 sites, and to the island in which they reside, as a “de novo methylation center,” which can initiate methylation and from which the nearby island is protected. Their data suggest the presence of czs-acting elements that, when preferentially methylated by the DNA-MTase enzyme, can facilitate spreading of methylation bidirectionally. Deletion studies reveal that the putative methylation center resides in an about 800-base-pair (bp) area that harbors tandem arrays of B-1 elements (Mummaneni et al., 1993), the murine counterpart to human Alu repeat elements (Quentin, 1994). Interestingly, Alu elements, independent of location, appear t o be heavily methylated in all somatic cell types (Hellman-Blumberg et al., 1993; Kochanek et ul., 1990), and these sequences have been proposed to initiate sites of normal DNA methylation in the p53 gene (Magewu and Jones, 1994). Taking all of these findings into account, how does the anatomy of CpG islands of tumor suppressor genes that become methylated in human cancers fit with the APRT model? In this regard, we have compared the VHL and Ecad genes to the human APRT, TIMP2, and GST-ITgenes and derived a strikingly generic anatomy (Fig. 4 and see Graff et al., 1997). Each gene has two Alu repeat elements located within 1.0 k b 5’ of the CpG islands that span the transcription start sites, and the VHL gene has another Alu repeat located within 500 bp 3’ to the CpG island. At the 5 ’ and 3’ ends of each island, within the area where the CpG density is beginning to decline, there are multiple Sp-1 sites. Using a combination of the new MSP PCR technique (Herman et al., 1996a) and bisulfite genomic sequencing (Myohanen et al., 1994), we have mapped, in normal tissues and culture lines of tumors, the methylation status of 80 or more CpG sites located within the CpG islands of the VHL and E-cad genes and in the regions flanking the islands (Graff et a/., 1997 and summarized in Fig. 4). In all instances, even in normal cells, the Alu sequences were heavily methylated, as were adjacent CpG sites, extending up to the Sp-1 sites at both ends of the islands (see Fig. 4). The island regions within the Sp-1 sites were completely free of methylation in the normal tissues and in tumors expressing the genes. In contrast, in cultured breast tumors previously shown to have a nonexpressing and hypermethylated E-cad gene, and in a renal cancer with a similar situation for the VHL gene, all the island CpG sites, as well as the island-flanking regions, were extensively methylated (Graff et al., 1997). We conclude from these studies that the CpG islands of human genes may

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have a generic anatomy characterized by sharp boundaries a t both the 5’and 3’ edges, which normally separate unmethylated sequences within the islands from densely methylated flanking regions often containing Alu repeat sequences, In addition, multiple Sp-1 sites are located at both the 5’ and 3’ boundaries in a strategic position to potentially protect the island from spread of methylation from the flanking regions. Interestingly, in the studies of the APRT gene previously discussed, only the Sp-1 sites at the 5 ’ edge o f the CpC island were tested for protective function. Determining precisely what molecular mechanisms normally allow the unmethylated CpG islands to coexist with nearby methylated areas must be the goal of intensive research during the next years. If this anatomy helps define the protection boundaries for the unmethylated status of a typical C p C island, how does this protection break down during the evolution of a neoplasm? There is little experimental data to address this critical question. Certainly, experimental data indicate that during tumor progression, chronically increased DNA-MTase activity can override the normal protective mechanisms that maintain the unmethylated status of these regions (Vertino et al., 1996). We have looked a t the dynamics of how this process occurs for the E-cad CpG island in our fibroblast model for overexpression of the human DNA-MTase gene. In this setting, E-cad hyper-methylation appears to spread, over 30 to 40 cell passages, inward from both the 5’ and 3’ borders, to involve the transcription start site at the center of the island as the last event (Graff et al., 1997). These data fit well with those of others indicating that de nova methylation of CpC-rich regions evolves by a spreading process that requires multiple cell cycles (Toth et al., 1989). The data also indicate how prolonged exposure to increased DNA-MTase activity during tumor progression could exert constant pressure to disrupt the boundaries that normally protect C p C islands from methylation. As mentioned earlier, defects other than DNA-MTase dysregulation may contribute to CpC island hypermethylation in neoplasia and could well involve the local mechanisms that constitute the C p C island protective borders. One attractive target would be loss of binding proteins like those that may interact with the Sp-1 sites at the island borders. If these were deficient in amount or function, the CpC islands might be rendered vulnerable to methylation. Again, this mechanism could represent a genetic origin to aberrant methylation if such proteins were disrupted by mutational events. Although the proteins involved with protection of C p C islands from methylation are yet to be defined, some may be transcription factors, such as Sp-1. These proteins could help to maintain chromatin structure around promoter areas, so that the access of DNA-MTase to the island is precluded. Proteins known to be essential for chromatin structure, such as histones, are now known to share structural homologies to other proteins that simultaneously

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function to modulate transcription and chromatin structure around gene promoters (Felsenfeld, 1996; Wolffe, 1994). In this regard, one particular variant of histone H1, H l e , has been shown to have binding affinity for CpG-rich DNA and to block DNA-MTase activity in vitro when bound to such sequences (Santoro et al., 1995; Zardo et al., 1996).The authors of this work actually propose that this histone is a candidate factor to explain the protection of C p C islands from methylation. If alterations in transcription factors and/or constituents vital to active transcription could play a primary role in the aberrant C p C island methylation in neoplasia, then this would raise a vital issue regarding the associated loss of gene function, that is, which comes first, the loss of transcription o r the appearance of aberrant C p C island methylation? Another way of phrasing the question would be, does promoter region methylation start the process of gene inactivation or simply reflect it? Of course, this same question has plagued understanding of the role of promoter region methylation in normal cells. The answer to this conundrum in both the normal and cancer cell settings is probably that either scenario can be operative and that the sequence of events may be cell type- and gene-specific. Experimentally, we know, as Antequera and Bird (1993a) have pointed out, that methylation may both respond to chromatin changes and initiate them. Thus, CpC islands methylated in vitro can form condensed chromatin when placed in cells (Keshet et al., 1986). In contrast, inactivation of genes on the X-chromosome of females appears to precede the hypermethylation of C p C islands in the corresponding gene promoters, and the latter event may “lock in” the transcriptionally inactive state (Lee et al., 1996). Likely, this dual role of methylation will emerge for individual gene inactivation events in cancer cells. In some instances, during tumor progression, aberrant methylation may be the initial event, and loss of gene transcription may then ensue. In other situations, a decrement in transcription factors that normally maintain transcriptional activity may precede aberrant methylation of certain genes. We and others have some evidence for this type of transcriptional deficit from studies of the E-cad gene in cultures of human breast and prostate cancer. Transient transfection of reporter constructs for the promoter of this gene into cell lines that have hypermethylation of the endogenous gene consistently yields less activity than in cell lines in which the endogenous gene is unmethylated (Graff et al., 1995; Hennig et al., 1995). Although, in our hands, the degree of reduction in the reporter gene activity does not quantitatively account for the complete lack of native E-cad transcripts in the lines with the hypermethylated endogenous gene, the results reflect a decrement in one o r more trans-acting factors important to E-cad expression. In such settings, definition of the proteins involved, and the mechanisms underlying loss of their transcriptional function may be most revealing for our understanding of aberrant C p C island methylation in tumor cells.

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6 . SUMMARY The molecular mechanisms underlying aberrant CpG island methylation in neoplasia are complex and may involve multiple simultaneously occurring events. A model can be envisioned (see Fig. 4B)wherein, during tumor progression, a combination of chronic exposure of cells to increased DNAMTase activity, loss of region-specific demethylation activity, and/or loss of binding proteins that help maintain CpG island methylation boundaries lead to promoter region hypermethylation and gene inactivation. Identifying these steps precisely will be an essential target of future research endeavors.

VI. AN OVERVIEW OF TUMOR PROGRESSION THAT INCORPORATES THE ROLES OF ALTERED DNA METHYLATION The data reviewed previously indicate that changes in DNA methylation should be considered a vital aspect of tumor progression. If so, it may be helpful to view the potential contribution of each component of altered DNA methylation in a working model of neoplastic evolution (Fig. 5). We hope that the hypotheses entailed may help to guide future investigations of the methylation changes in neoplasia. One of the cardinal features of our proposed progression model is that the imbalance of DNA methylation begins at the earliest stages of the neoplastic process. For example, in a previously discussed mouse lung tumor model, increased DNA-MTase activity and a transient increase in overall genomic methylation accompany the earliest cellular responses to carcinogen exposure (see Fig. SB).This overall methylation increase could help initiate the promoter region hypermethylation events that influence tumor suppressor gene expression. Despite these initial methylation increases, the steady state for early neoplasms is often one of overall genomic hypomethylation in association with a continued increase in DNA-MTase activity (see Fig. SC). Thus, from the initial neoplastic stages and beyond, the components of DNA methylation imbalance are in place and available to participate in alterations in chromosome structure and gene expression throughout tumor progression. Among the components of the DNA methylation imbalance in neoplasia, a direct role for the regional hypermethylation that targets CpC islands is now, perhaps, best appreciated. In this regard, as outlined earlier, there appear to be two classes of hypermethylated genes that have to be considered. Some genes appear to undergo CpG island hypermethylation only during neoplastic evolution, whereas in others, the change may preexist in normal cells as a function of the aging process. As depicted in Figure 5, both of these

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gene classes may contribute to the earliest steps in neoplasia. For the age-related changes, for example, evidence points to the possibility that colon tumors may initiate in cells that harbor hypermethylation of the ER gene. In Figure 5A and B, such cells are hypothesized to be particularly sensitive to initial carcinogen effects and to respond with a selective growth advantage that provides a field effect for tumor potential. In contrast to genes hypermethylated during aging, the classic tumor suppressor genes examined appear to acquire this change only after the neoplastic process initiates. What is the timing of these events during tumor progression? This is an important question for which information is only beginning to emerge. Studies suggest, however, that this CpG island methylation often begins early and with a pattern of cellular heterogeneity (see Fig. 5C through SE), which is critical to our progression model. Evidence for such heterogeneity emerged early in our studies of the calcitonin gene on chromosome 1Ip, where we found both hypermethylated and normal alleles in leukemic cells proven to be a clonal population by molecular analyses (Baylin et al., 1987). This same phenomenon was later recognized for the p l 5 gene in these same neoplasms (Herman et al., 1996b). More recently, in ongoing studies of breast cancer progression, hypermethylated alleles for the ER and E-cad genes can be clearly detected by the new MSP technique in 25 to 35% of ductal carcinoma in situ (DCIS) lesions (Graff et al., unpublished results). This incidence increases in the next stage of invasive tumors. Similarly, in lung cancer progression, hypermethylated alleles of the p l 6 gene have been detected in preinvasive lesions in patients who simultaneously harbored established lung carcinomas with this same change (Herman et d.,unpublished results). This situation for human lung cancers is paralleled by findings in a rat model for lung carcinogenesis, in which hypermethylated p16 alleles were detected at a stage of epithelial hyperplasia that preceded frank adenoma and subsequent adenocarcinoma development (Belinsky et al., unpublished results). These early methylation changes can subsequently progress during tumorigenesis in several ways. First, the density of methylation within the involved gene can increase over time, as observed for the candidate tumor suppressor gene, HIC1, between the stages of benign adenomatous colon polyps and frank carcinomas (Makos et al., 1992). Second, cells with the early methylation changes may be selected for with time as the increasing methylation density becomes associated with loss of key gene function. This selection may be facilitated, especially if these gene expression changes coincide in the same cell with other genetic and/or epigenetic events that favor clonal expansion (see Fig. 5B through E). For example, in breast tumor progression, we are now finding that concordancy for ER and E-cad gene hypermethylation may track with the invasive and/or metastatic phenotype (Graff et al., unpublished data).

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The concept for progressive involvement of CpG island hypermethylation during tumor progression is similar to that for accrual of classic gene mutations (Kinder and Vogelstein, 1996).The role of cellular heterogeneity, however, so accepted for DNA changes such as chromosome alterations (Nowell, 1976), is less often considered for specific gene mutations because we often think of these latter events as leading to immediate selective advantage, which triggers rapid clonal expansion. In fact, much evidence suggests that key gene mutations reside in cell subpopulations a t stages of tumor progression. For example, p53 gene mutations exist in subpopulations of cells early in progression of tumors such as brain cancer (Sidransky et a/., 1992) and in other, more recently studied tumor types (Ziegler et al., 1994). Similarly, as previously discussed, homozygous deletions of the p16 gene can be heterogeneous within tumor cell populations. Thus, both promoter region hypermethylation and gene mutations may exist in subclones of tumor cells and not result in a dominant progression event until these changes act cooperatively to initiate clonal expansion. Each individual molecular change, however, is still critical because the involved cell population is rendered particularly sensitive to expansion when additional events are superimposed. The model in Figure 5 summarizes this discussion by depicting a cooperative interaction between CpC island hypermethylation events and classic mutational changes that drives tumor progression through clonal expansion of heterogeneous cell populations. Note that after an initiating mutation occurs in a cell harboring an age-related hypermethylation change (see Fig. SB), clonal expansion ensues to produce a dyspastic or preneoplastic lesion in which all cells carry the molecular alterations (i.e., ER gene hypermethylation and the first mutation) that have occurred to this point (see Fig. SC). At this dysplastic state, however, two types of cellular heterogeneity arise. Subpopulations of cells have developed a pS3 mutation, and a separate set have evolved hypermethylation of the p16 gene promoter region (see Fig. 5C). There are no selective advantages from these molecular changes, however, until both coincide in the same cell. Such cooperation for inactivation of these two genes has been suggested in a recent study (Kinoshita et al., 1996). Allelic inactivation of the .opposite copy of the mutated o r hypermethylated genes must, of course, also occur. The cell now harboring two mutational changes (the initiating mutation, plus a pS3 mutation) and two hypermethylation changes (in the ER and p l 6 genes) clonally expands to produce carcinoma in situ (see Fig. 5C and D). In the next critical stage of tumor progression (see Fig. 5D and E), a carcinoma with metastatic potential evolves, again through expansion of a cell subpopulation. A cell within the carcinoma in situ develops a reduction in E-cad gene expression in conjunction with promoter region hypermethylation (see Fig. SD), and the progeny exhibit decreased homotypic cell-to-cell adhesiveness and capacity to invade (see Fig. 5E).

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Thus, this hypothetical model embodies the classic concepts of cellular heterogeneity in tumor progression (Nowell, 1976) and stresses that, from the earliest time points, cooperative interaction between genetic and epigenetic events drives critical stages. Although aspects of this model must be continually verified or refuted by experimental approach, we think the dynamics depicted fit nicely with existing data. It is important to note that cellular heterogeneity for any molecular change can appear at virtually every stage of tumor evolution. In the model, the timing for the DNA changes shown is only one possibility and is not meant to imply an absolute order. In fact, even for a single tumor type, the model predicts that, for example, cells harboring changes favoring invasive and/or metastatic potential could arise a t virtually any progression stage shown. The timing for resultant phenotypic consequences would, again, depend on the number of changes simultaneously affecting a given call.

VII. CLINICAL IMPLICATIONS OF ALTERED DNA METHYLATION IN CANCER In addition to the discussed biologic consequences of altered DNA methylation for tumorigenesis, the advances in understanding of these processes justify considering how the data may prove useful to the diagnosis and treatment of cancer. Particularly, the newer findings for inactivation of specific genes in association with CpG island methylation and the potential direct role of increased DNA-MTase activity in tumor progression bear comment. The fact that the CpC island methylation events for many involved genes may represent a process specific to neoplastic cells makes this change attractive to consider for development of molecularly based biomarkers. With the advent of PCR procedures, such as MSP (Herman et al., 1996a),that can sensitively detect the methylation status of specific CpG sites in rare alleles, DNA from small clinical specimens, processed at the time of routine pathology examination, can be analyzed for methylation changes. Aberrantly hypermethylated alleles could potentially detect stages of early tumor evolution, or such analyses of lymph nodes or margin tissues removed a t the time of initial surgery could provide a sensitive index of micrometastases. Finally, PCR analyses of posttreatment samples, such as bone marrow from patients with leukemia, could provide a sensitive means for assessing residual tumor or predicting disease recurrence. Tests of these hypotheses should be forthcoming in the next several years. The findings from the Jaenisch group that lowered levels of DNA-MTase activity reduce the incidence of adenoma formation in mice with APC gene mutations (Laird et al., 1995), combined with the evidence that increased

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DNA-MTase activity may be characteristic of multiple types of carcinogenesis, have legitimized consideration of targeting inhibition of this enzyme as an anticancer strategy. In fact, the demethylating agent 5-AZA-cytidine and its congener, 5-deoxy-AZA-cytidine, have been tried clinically. Some response rates have been seen in diseases such as leukemia (Gattei et al., 1993; Mandelli, 1993; Petri et al., 1993; Silverman et al., 1993; Willemze et al., 1993; Zagonel et al., 1993). Because these drugs incorporate into DNA and may have effects other than inducing demethylation, the mechanisms underlying antitumor effects are not known. It is now possible to test whether reactivation of specific genes through relief of CpG island hyperrnethylation might correlate with any observed therapeutic effects. Also, new and more specific ways to inhibit the enzyme are being studied. Promising cell-differentiating and tumor cell-inhibiting activity, bo:h in vitro and in vivo, has been reported with use of antisense approaches (MacLeod and Szyf,l995; Ramchandan et al., 1997; Szyf et al., 1992). The next several years should see testing of multiple approaches aimed at the possibility that a reduction of DNA-MTase activity may be useful for tumor treatment. Preclinical testing can take advantage of several excellent murine and rodent carcinogenesis models to study tumor responses, to outline the mechanisms involved, and to delineate the most sensitive stages of tumor progression. Such investigations should help determine whether decreasing DNA-Mtase activity is most useful for prevention, early intervention, and/or treatment of established tumors and should justify the building interest in the alterations of DNA methylation that appear to be a fundamental hallmark of the neoplastic state.

ACKNOWLEDGMENTS We gratefully acknowledge all of the colleagues over the years who have contributed to studies i n our own laboratories and the many workers in the DNA methylatioii field whose work is cited in this review. Of course, space limitations preclude referencing many other papers that have provided important data, and we apologize for those exclusions. We thank Tammy M. Means for invaluable secretarial services. Portions of work performed in the authors’ laboratories were supported by National Cancer Institute grants #CA43318, #CA.54396, and #SPOCA58184 and b y a grant from the Tobacco Research Council (#1987B). J.G.H. is recipient of a Valvano Fouindation Research Award and J.P.I. of a Kiminel Foundation Award.

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