Chemical and Physical Carcinogenesis: Advances …...CARCINOGENESIS Table 1 Examples of advances in chemical and physical carcinogenesis" A. Cancer etiology I. Life-style factors (tobacco

[CANCER RESEARCH (SUPPL.) 51, 5023s-5044s, September 15. 1991]

Chemical and Physical Carcinogenesis: Advances and Perspectives for the 1990s1

Curtis C. Harris2

Laboratory of Human Carcinogenesis, National Cancer Institute, N1H, Bethesda, Maryland 20892

Abstract

Carcinogenesis is a multistage process driven by carcinogen-induced

genetic and epigenetic damage in susceptible cells that gain a selectivegrowth advantage and undergo clonal expansion as the result of activationof protooncogenes and/or inactivation of tumor suppressor genes. Therefore, the mutational spectra of chemical and physical carcinogens in thesecritical genes are of interest to define endogenous and exogenous mutational mechanisms. The p53 tumor suppressor gene is ideally suited foranalysis of the mutational spectrum. Such an analysis has revealedevidence for both exogenous and endogenous molecular mechanisms ofCarcinogenesis. For example, an informative p53 mutational spectrum offrequent Gâ€”Â»Ttransversions in codon 249 is found in hepatocellularcarcinomas from either Qidong, People's Republic of China, or southern

Africa. This observation links exposure to aflatoxin B,, a known cancerrisk factor in these geographic regions, with a specific mutation in acancer-related gene. Other studies indicate that abnormalities in genes

controlling the cell cycle may cause genomic instability and increase theprobability of neoplastic transformation. Finally, mechanistic understanding of Carcinogenesis is leading to improved cancer risk assessment andto the identification of individuals at high cancer risk.

Introduction

Carcinogenesis is a mature multidisciplinary field of cancerresearch that has a rich history of scientific accomplishmentsachieved by epidemiolÃ³gica! observations (1-3); developmentand exploitation of both animal models relevant to humancancer (4) and in vitro cell and tissue models including the useof human tissues (5, 6); and most recently, advances in molecular genetics (7-II).3 Examples of conceptual advances and

seminal observations in the areas of chemical and physicalCarcinogenesis are listed in Table 1; advances in viral Carcinogenesis are reviewed by Howley in this issue (12).

Since the research accomplishments of Carcinogenesis havebeen extensively reviewed (13-25), this commentary will focuson selected topics that are especially relevant to current understanding of human Carcinogenesis and, in particular, to themolecular epidemiology of human cancer, an emerging field incancer research.

Multistage Carcinogenesis

Carcinogenesis is a multistage process driven by carcinogen-induced genetic and epigenetic damage in susceptible cells thatgain a selective growth advantage and undergo clonal expansionas the result of activation of protooncogenes and/or inactivationof tumor suppressor genes. The traditional view of carcinogen-esis (Fig. 1) is derived primarily from studies of animal models.The first stage of the carcinogenic process, tumor initiation,involves exposure of normal cells to chemical, physical, ormicrobial carcinogens that cause a genetic change(s) providing

1Presented at the Symposium "Discoveries and Opportunities in CancerResearch: A Celebration of the 50th Anniversary of the Journal Cancer Research."

May 15, 1991, during the 82nd Annual Meeting of the American Association forCancer Research, Houston, TX.

2To whom requests for reprints should be addressed, at Laboratory of Human

Carcinogenesis, National Cancer Institute, N1H, Building 37, Room 2C05, Bethesda, MD 20892.

3 Due to space limitations and the breadth of this topic, comprehensive reviewswill be frequently cited and are the source of additional citations.

the initiated cells with both an altered responsiveness to theirmicroenvironment and moreover exerts a selective clonal expansion advantage when compared to the surrounding normalcells (26-28). The initiated cells may have decreased responsiveness to the inter- and intracellular signals that maintainnormal tissue architecture and regulate the homeostatic growthand maturation of cells. For example, initiated cells may be lessresponsive to negative growth factors, inducers of terminal celldifferentiation and/or programmed cell death (4, 26, 29-35).Perturbation of the normal microenvironment by (a) physicalmeans, e.g., wounding of the mouse skin or partial hepatectomyin rodents, (b) chemical agents, e.g., exposure of the mouseskin to certain phorbol esters or exposure of the rat liver tophÃ©nobarbital, (c) the inflammatory process, or (d) microbialagents, e.g., influenza virus enhancement of rodent lung carci-nogenesis or hepatitis viruses in human liver Carcinogenesis,may also foster such selective clonal expansion of the initiatedcells (4, 17, 36, 37).

Tumor promotion results in proliferation and/or survival ofthe initiated cells to a greater extent than normal cells andenhances the probability of additional genetic damage includingendogenous mutations accumulating in the expanding population of these cells. The probability of a subpopulation of initiated cells converting to malignancy can be substantially increased by their further exposure to DNA-damaging agents(38-40) that may activate protooncogenes (41) and/or inactivate tumor suppressor genes. Malignant cells continue to exhibit progressive phenotypic changes during tumor progression(42) and may exhibit intrinsic genomic instability that is manifested by the abnormal number and structure of chromosomes,gene amplification, and altered gene expression (43-45).

This classical view of two stage Carcinogenesis involving amutation, tumor initiation, and an epigenetic change, tumorpromotion, has been conceptually important but is also considered to be simplistic in that the number of independent geneticand epigenetic events may be 6 or more in certain types ofcancer (46-48). Furthermore, chemical carcinogens may: (a)not be considered mutagens, termed nongenotoxic carcinogens(49); (b) have both genetic and epigenetic effects such as theability to induce mutations and the property of decreasing DNAmethylation, respectively (50) or increase cellular proliferation;and (c) have a linear or non-linear dose-response relationshipwith respect to tumor formation (51, 52). These and otherobservations have generated an active debate concerning boththe contribution of endogenous mutagenic mechanisms, e.g.,oxy-radical DNA damage, DNA depurination, DNA polymer-ase infidelity, and deamination of 5-methylcytosine, versus exogenous environmental mutagens and the value of animal bioas-says to identify carcinogens and to aid in human cancer riskassessment (53-63). This debate is not merely an academic onein that societal and regulatory decisions critical to public healthare at issue.

Carcinogen Metabolism and DNA Damage

Many chemical carcinogens require metabolic activation generally to high energy electrophiles to exert their carcinogenic

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CARCINOGENESIS

Table 1 Examples of advances in chemical and physical carcinogenesis"

A. Cancer etiologyI. Life-style factors (tobacco snuff or smoke) can cause human cancer.

2. Occupational agents and factors can cause human cancer.a. Nun (childlessness)b. Coal sootc. Sunlightd. Aromatic aminese. Benzenef. Ionizing irradiation

g. Asbestos

3. Mixtures of chemicals (coal tar) can cause cancer in experimental animals.

4. Specific chemicals, e.g., dibenzanthracene or 2-naphthylamine, are carcinogens in experimental animals.

5. Solid "inert" substances such as plastic can cause cancer when implanted s.c. in experimen

tal animals.

6. Naturally occurring compounds isolated from plants, e.g., croton oil, or marine organisms,e.g.. teleocidin B and okadaic acid, have cocarcinogenic and/or tumor-promoting activities inexperimental animals.

7. Naturally occurring chemicals such as alkaloids from plants, Â¡-.K--bracken fern (Pleritliumaquilinum). cycad. and Senecio, and fungal toxins, e.g.. aflatoxin B,. can be carcinogens inexperimental animals.

8. yV-Nitrosamines can be carcinogenic in laboratory animals.

9. Cooking of red meat, fish, or specific amino acids can generate polynuclear aromatic hydrocarbons and, in some cases, heterocyclic amines that are carcinogenic in animal models.

10. Tobacco smoking and alcoholic beverages are human cancer risk factors and have multiplicative effects.

11. Exposure of the human fetus to ionizing radiation increases risk of leukemia.

12. Tobacco smoking and occupational exposure to asbestos increases risk of human broncho-genie carcinoma.

13. Dietary exposure to aflatoxin B, correlates with prevalence of human hepatocellular carcinoma in Africa.

14. Animal bioassays can identify chemical carcinogens, e.g., chloromethyl ethers and vinylchloride, prior to epidemiological evidence.

15. Carcinogen-macromolecular adducts can be used as molecular dosimeters including exposureto life-style and occupational carcinogens.

16. Transplacental exposure to diethylstilbestrol increases risk of human vaginal carcinoma.

17. Fiber dimension and length is correlated with carcinogenicity in animal models

Hill. 1759(329)Soemmerring, 1795 (330)Doll and Hill. 1950(331)Wynder and Graham, 1950(332)

Ramazzini. 1713(333)Pott, 1775(334)Thiersch, 1875(335)Rehn. 1895(337)DeLore and Borgman, 1928 (340)Harting and Hesse, 1879 (336)Trieben, 1902 (338)Clune!, 1910(339)Martland and Humphries, 1929 (341)Ministry of Labour and National Serv

ice, 1949(342)Wagner et al., 1960(343)

Yamagiwa and Ichikawa. 1918 (344)

Kennaway and Hieger, 1930 (345)Hueper et al.. 1938(346)

Turner. 1941 (347)

Berenblum, 1941 (348)Fujiki et al.. 1979(349)

Cook el al., 1950(355)Schoentalf/a/.. 1954(356)Laquer, 1964(357)Evans and Mason, 1965 (358)Carnaghan, 1965(359)Wogan, 1966(360)Wogan and Newberne. 1967 (361)

Magee and Barnes. 1956 (362)

Kuratsume, 1956(363)Lijinsky and Shubik, 1964 (364)Sugimura et al.. 1977(365)Kasai el al.. 1980(366)

Wynder el al.. 1957(367)Keller. 1967(368)Rothman and Keller, 1972 (369)

Stewart Ã©tal.,1958(370)

Selikofff/a/., 1968(371)

Alpert et al.. 1968(372)

Van Duuren el al.. 1968 (373)Viola el al., I97I (374)

Ehrenberg el al.. 1974(296)Tornqvist et al.. 1986 (298)Farmer elal.. 1986(300)

Herbst et al., 1977(375)

Stanton el al., 1981 (376)

B. Multistage carcinogenesis

1. Chemicals can cause cancer at sites distant from the point of application in experimentalanimals.

2. Metabolites of chemical carcinogens are predicted to be the active carcinogenic agent.

3. Carcinogenesis is a multifactorial process involving tumor initiation and promotion in animal models.

Sasaki and Yoshida. 1935 (377)

Boyland. 1935(378)

Rousand Kidd. 1941 (379)Berenblum. 1941 (348)Mottram. 1944(380)Berenblum and Shubik. 1949 (381)

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Table 1 Continued

4. Metabolic activation of some chemical carcinogens is essential for their carcinogenic activity.

5. Tumor promoters can inhibit cell-cell communication of mammalian cells in vitro.

6. A tumor promoter in experimental animals can specifically bind and activate a specificcellular receptor.

7. Oncogenes cooperate in neoplastic transformation of primary rodent cells.

8. Some tumor promoters act via the protein kinase C pathway for signal transduction.

9. Cooperation between oncogenes is demonstrated in transgenic animal models.

10. Overexpression of protein kinase C leads to morphological transformation of rodent cells.

11. Multiple genetic events in protooncogenes and tumor suppressor genes are associated withhuman carcinogenesis.

Millerand Miller. 1947(382)Miller et al., 1964(383)

Murray and Fitzgerald, 1979 (384)\ott\etal., 1979(385)

Driedgerand Blumberg, 1980 (386)Castagna et a!., 1982 (387)

Land et al., 1983(388)Ruley, 1983(389)

Davis and Czech. 1985 (390)Arroleo and Weinstein. 1985 (391)Ebeling et al., 1985(392)

Sinn Ã©tal.,1987(153)

Housey et al., 1988(393)Personsera/., 1988(394)

Volgelsteinera/., 1988(395)

C. Genetic and epigenetic mechanisms of carcinogenesis

1. Human cancer can be inherited.

2. Chromosomal abnormalities are predicted to be involved in cancer.

3. Carcinogenic X-rays are also mutagens.

4. Inbred mouse strains differ in their cancer incidence.

5. Cancer may arise by inactivation of both alÃelesof a tumor suppressor gene.

6. Chemical carcinogens can be mutagens.

7. Chemical carcinogens may bind to DNA.

8. A specific chromosomal abnormality can be associated with human leukemia.

9. UV light causes a specific type of DNA damage, i.e., thymidine dimers (cyclobutane).

10. Carcinogenic potency of some chemical carcinogens correlates with their binding level toDNA in experimental animals.

11. Defective DNA repair may predispose to UV-induced human skin cancer.

12. Interindividual variation is observed in carcinogen metabolism and/or formation of carcino-gen-DNA adducts in human cells and tissues.

13. Normal cells are dominant in normal-tumor murine cell hybrids.

14. In vitro bacterial mutagenesis can be used to screen putative carcinogens.

15. RNA viral oncogenes have a normal cellular counterpart (protooncogene).

16. Data consistent with the autocrine growth factor theory are presented.

17. DNA from chemically transformed rodent cells contain an oncogene(s).

18. Specific chromosomal loss is associated with tumorigenic revenants of normal-carcinomahuman cell hybrids.

19. Chromosomal translocation can activate protooncogenes in human cells.

20. Oncogenes are cloned from human cancers.

21. raj oncogene contains a base substitution when compared to protooncogene.

Watkins, 1904(396)

Boveri, 1914(397)

Muller. 1927(398)

Forth et al., 1933(399)Strong, 1935(400)

Charles and Luce-Clausen, 1942 (401)Knudson, 1971 (95)Comings, 1973(402)CaveneeÂ«a/.. 1983(96)

Auerbach et al., 1947 (403)Boyland and Horning, 1949 (404)

Boyland, 1952(405)Wheeler and Skipper. 1957 (406)Brookes and Lawley. 1960 (407)Farber and Magee. 1960 (408)

Nowell and Hungerford. 1960 (409)

Beukers and Berends, 1960 (410)

Brooks and Lawley, 1964 (411)

Cleaver, 1968(412)

Kuntzman et al., 1968 (413)Welch et al., 1968(414)Huberman and Sachs, 1973 (71)Harrisera/., 1976(415)

Harris et al.. 1969(416)

Ã•mese/a/., 1973(417)McCann ff a/., 1975(418)

Stehelin et al., 1976(419)

DeLarco and Todaro. 1978 (420)

Shihetal., 1979(421)

Stanbridge et al., 1981 (422)

Dalia-Pavera et al., 1982(423)deKlein et al., 1982(424)Shen-Ong et al.. 1982 (425)TaubÂ« al.. 1982(426)

Goldfarb et al., 1982(427)Pulciani et al.. 1982(428)ShÃ¬hetal.. 1982(429)

Ready et al., 1982(430)Takinera/., 1982 (431)Taparowsky et al., 1982(432)

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CARCINOGENESIS

Table 1 Continued

22. Chemical carcinogens can activate ras protooncogenes in animal models.

23. Some mutant growth factor receptors are oncogenes in rodent cells.

24. Site-specific mutagenesis analysis of carcinogen-DNA adducts confirms predications of nu-cleotide misincorporation opposite specific adducts.

25. Transferred oncogenes can cause neoplastic transformation of human epithelial cells.

26. The human rctinoblastoma susceptibility (Kin gene is cloned.

27. Nuclear oncogenes can act as transcription factors.

28. Direct evidence of the tumor suppressor activity of the Rb gene in human cells.

29. pS3 is frequently mutated in diverse types of human cancers.

30. p53 functions as a tumor suppressor gene in rodent and human cells.

31. A candidate tumor suppressor gene on chromosome 18q may be involved in human coloncarcinogenesis

32. NF1. a GTPase-activating protein, may be a negative regulator of ras p2l and a tumorsuppressor gene

33. A candidate Wilm's tumor suppressor gene is cloned.

34. Germline />.\Ã¬mutations are found in certain cancer-prone families.

35. Candidate tumor suppressor genes (MCC and APC) may be involved in familial adenoma-tous polyposis and colorectal carcinoma.

36. An environmental chemical carcinogen (aflatoxin B,) is linked with a specific mutation inthe p53 tumor suppressor gene in human hepatocellular carcinogenesis.

37. A candidate tumor suppressor gene (protein tyrosine phosphatase -y)on chromosome 3pmay be involved in human lung and renal carcinogenesis.

Balmain and Pragnell. 1983 (433)Sukumar et al., 1983 (434)

Downward et al., 1984 (435)

Loechler et al.. 1984(116)Preston et al.. 1986(436)

Yoakum et al., 1985(437)Kliim Â«-iÂ«/..1985(438)

Friend et al., 1986(439)

Makiera/., 1987(440)Vogtefa/., 1987(441)HÃ¶hnumn et al., 1987 (442)

Huang Ã©tal..1988(147)

Baker et al., 1989(443)Nigroero/., 1989(127)Takahashi et al.. 1989(128)

Eliyahu et al., 1989(444)Finlay Â«a/.. 1989(445)Baker et al., 1989(443)

Fearonera/., 1990(446)

Cawthon et al., 1990 (447)Xuetal., 1990(448)Wallace Ã©tal..1990(449)

Callera/.. 1990(450)Gessler, 1990(451)

M:ilkiii <â€¢/Â«/..1990(141)Srivastavaera/., 1990(142)Kinzler et al., 1991 (452)Kinzler et al., 1991 (453)Nishisho et al., 1991 (454)Graden et al., 1991 (455)

Hsufia/., 1991 (137)Bressac et al.. 1991 (138)

LaForgia et al., 1991 (456)

Â°These historical reviews have been useful references and are a source of additional examples of advances in carcinogenesis (350-354).

Initiation Promotion Conversion Progression

Fig. I. Carcinogenesis is a multistage process involving multiple genetic and epigeneticevents in protooncogenes. tumor suppressorgenes, and anti-metastasis genes.

â€¢Defects in Terminal Differentiation

â€¢Defects in Growth Control

â€¢Resistance to Cytotoxicity

GeneticChange

inhiortion

NORMAL CELL

v â€”/ rÃ¯Ã¯t$<'INITIATED

PRENEOPLASTICCELL LESIONMALIGNANT TUMORP*

'CLINICAL

CANCERâ€¢

Activation of Proto-Oncogenes

â€¢Inactivation of Tumor Suppressor Genes

â€¢Inactivation of Antimetastasis Genes

effects (64). Although interspecies differences have long beenknown in the metabolism of xenobiotics (65-69), the metabolicpathways of activation and the resultant carcinogen-DNA adducts are generally qualitatively similar among various animalspecies, including humans (6). These observations support the

qualitative extrapolation of carcinogenesis data from laboratoryanimals to the human situation. Quantitative and qualitativedifferences in cytochrome P-450 enzymes in various tissues (70)may be partly responsible for the divergent sites of cancerinduction among animal species; e.g., 4-aminobiphenyl causes

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HypothesisGeneration

ClinicalObservation

CARCINOGENESIS

In Vivo Studies In Vitro Studies

Fig. 2. Strategy for studies of molecularcarcinogenesis and molecular epidemiology toimprove interspecies extrapolation of carcinogenesis data and to identify high cancer riskindividuals. Epidemiology

Clinical Investigations

Molecular Epidemiology

Explant vCulture

CellCulture

(epithelial)

Molecules

Molecular Dosimetryof Carcinogen Exposure

Inherited Cancer Predisposition

MolecularCarcinogenesis

liver cancer in rodents and bladder cancer in dogs and humans(65) and may confound tissue site-specific extrapolation ofcarcinogenesis data among animal species. Quantitative differences in carcinogen metabolism and in the formation of carcin-ogen-DNA adducts are, however, found among different celltypes (71, 72), tissue types (6, 70, 73), and outbred individualsof the same species (74, 75). As discussed (see below) in thesection "Molecular Epidemiology of Human Cancer," these

quantitative differences may influence tissue site of cancer andan individual's cancer risk.

The influence of carcinogen metabolism on interspecies extrapolation of carcinogenesis data is becoming more well defined by the cloning of genes encoding enzymes involved inactivation and detoxication of chemical carcinogens. Investigators have recently cloned more than 150 CYP4 genes (76)

and other genes involved in carcinogen metabolism, such ashuman /V-acetyltransferase (77), human epoxide hydrolase (78,79), human NAD(P)H:menadione oxidoreductase (80), humanglutathione 5-transferases (81), and pig flavin-containing mon-ooxygenase (82). As discussed by Gonzalez et al. (70), interspecies differences in carcinogen metabolism may, in part, be dueto: (a) qualitative differences in CYP genes due to an absenceof a CYP expression in an animal species; (b) quantitative andqualitative variations in regulation of CYP genes among animalspecies; and (c) differences in CYP substrate or product specificity. Since examples of interspecies differences in each of the3 types noted above have been described, in vitro model systemsincluding those using human cells and tissues (6) or geneticallyengineered cells or rodents containing single or combinationsof human genes directing the expression of specific enzymesresponsible for carcinogen metabolism (70, 83) will serve as abridge between in vivo studies in animal models and the humansituation (Fig. 2).

The molecular and atomic analyses of the physical interactions between chemical carcinogens and DNA represent a majorachievement of cancer researchers in the last two decades (84).The information gleaned from these studies of carcinogen-DNA

4The abbreviations used are: CYP, cytochrome P-450; CHO, Chinese hamsterovary; BPDE, benzo(a)pyrene diol-epoxide; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; AHH, aryl hydrocarbon hydroxylase; GST, glutathione S-transferase; PAH, polycyclic aromatic hydrocarbon; HPLC, highperformance liquid chromatography; PTPase y, protein-tyrosine phosphatase y.

adducts has provided a mechanistic explanation of the muta-genicity of many carcinogens, including their mutational spectra, and a rationale for improved carcinogen exposure assessment in molecular epidemiological studies of human cancer.

Critical DNA Targets: Protooncogenes and TumorSuppressor Genes

Protooncogenes are normal cellular genes that, when inappropriately activated as oncogenes, cause dysregulation ofgrowth and differentiation pathways and enhance the probability of neoplastic transformation. Carcinogens can cause thegenetic changes that can lead to activation of protooncogenes,including base substitution mutations (84), chromosomal trans-locations (85), and gene amplification (45, 86). For example,ras protooncogenes are activated primarily at codons 12, 13,and 61 by base substitution mutations caused by chemical andphysical carcinogens in both cultured mammalian cells andanimal models (87-90) including transplacental exposure tochemical carcinogens (91). Mutation of the Ha-ras protooncogene is an early event in rodent models of skin and mammarycarcinogenesis (87, 92). In addition, the v-Ha-ras transgene cansubstitute for the initiation step in mouse skin carcinogenesisin transgenic mice (93, 94). This animal model should be usefulfor investigating the molecular mechanisms of tumor promotersand antipromoters.

In contrast to protooncogenes, tumor suppressor genes arenormal cellular genes that, when inappropriately inactivated,cause dysregulation of growth and differentiation pathways andenhance the probability of neoplastic transformation. Based on

Table 2 Examples of functions of putative tumor suppressor genes

Induce terminal differentiationMaintain genomic stabilityTrigger senescenceInduce programmed cell deathRegulate cell growth

Signal transducers of negative growth factorsRegulators, e.g., PTPase7. of tyrosine kinases

Inhibit proteasesAlter DNA methylase activityModulate histocompatibility antigensRegulate angiogenesisFacilitate cell-cell communication

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CARCINOGENESIS

the multifunctional classes of tumor suppressor genes (Table 2)their number may equal that of the oncogenes. Loss of functionof the products of both alÃelesis the basis of the 2-hit hypothesisand the recessive nature of the genetic process (95). In additionto inactivation of both alÃelesby mutational mechanisms (basesubstitution mutations, deletions, chromosomal nondisjunc-tion, and recombination) (96), inactivation of a normal alÃeleby genomic imprinting (97) and its gene product by an increasedrate of proteolytic digestion (98) or dominant negative mechanisms (99) have been proposed. Based on target size theory, theinactivation of a single alÃeleof a tumor suppressor gene shouldhave an intrinsically higher probability than activation of aprotooncogene, e.g., one of the ras protooncogenes in whichmutations in only a few specific codons will lead to activation.The requirement for inactivation of both alÃelesof a tumorsuppressor gene counterbalances this probability except in thosecases in which an inactivated alÃeleis inherited or the mechanism of inactivation is via a dominant negative mechanism, asproposed for the p53 tumor suppressor gene (99, 100).

Ionizing irradiation and carcinogenic hormones, metals, aldehydes, or fibers such as asbestos are generally inefficient inthe production of point mutations at the gene level but aremore efficient at causing chromosomal abnormalities and losses(62). Therefore, one would speculate that tumor suppressorgenes would be targeted by these carcinogens more readily thanprotooncogenes. Whereas many electrophilic metabolites ofchemical carcinogens form carcinogen-DNA adducts and causepoint mutations, chemical carcinogens also cause chromosomalabnormalities and micronuclei. Thus, both cytogenetic andmutagenesis assays have been developed to assess these mutational changes (44, 101-103).

Mutational Spectrum

Since mutations are largely responsible for activating protooncogenes and inactivating tumor suppressor genes, the mutational spectra of chemical and physical carcinogens are ofinterest to define endogenous and exogenous mutational mechanisms. Studies using prokaryotic and simple eukaryotic assaysand site-specific mutagenesis assays (104, 105) have shown thateach carcinogenic agent produces a "fingerprint" (106) linking

specific types and locations of DNA adducts with the mutational spectrum of the agent. Both exogenous genes, which areintroduced into the appropriate host cell by a shuttle vector(105, 107-110), and endogenous genes (106) such as adeninephosphoribosyltransferase (aprt), dihydrofolate reducÃase(dhfr), and hypoxanthine-guanine phosphoribosyl transferase(hprt) have been utilized in the eukaryotic studies. These assayswill underestimate the mutational frequency due to deletions,chromosomal nondisjunction, and frame-shift mutations because other genes essential for cell survival may be adjacent tothe hemizygous nonessential detector gene. In addition, certainmutations may cause either clonal expansion or inhibition ofthe mutant cell and further complicate the interpretation of themutational spectrum.

In both exogenous and endogenous types of target genes, thecarcinogen-induced mutational spectra can be compared to thespontaneous mutational spectrum. The majority of spontaneous mutations analyzed in these assays are single base substitutions. However, interesting differences have been observedin the spontaneous mutational spectrum in the endogenous aprtof CHO cells compared to bacterial cells and exogenous genescarried by shuttle vectors in mammalian cells (111). Transitionsof G:Câ€”Â»A:Tare frequent in CHO cells suggesting deaminationof 5-methylcytosine and/or DNA replication errors, whereasdeletions and insertions are more frequent in the other assaysystems. A comparison of the spontaneous mutational spectrumat the aprt locus in CHO cells with the spectra induced byionizing radiation, UV radiation or benzo(a)pyrene diol epox-ide is shown in Table 3. The mutational spectrum of UV lightis consistent with the mutagenesis models of the promutageniccyclobutane and pyrimidine:pyrimidone (6:4) photoproductsinduced by UV light. As noted previously, ionizing radiation ismost efficient in causing deletions. BPDE, which binds to the2-amino group of G, produces the predicted G:Câ€”Â»T:Atrans-versions; a similar mutational spectrum for BPDE has beenobserved in human cell assays (112). Polycyclic aromatic hydrocarbons can also cause mutations at regions of the genesencoding pre-mRNA consensus splice sites, resulting in mRNAsplicing defects. For example, mutations affecting splicing inthe CHO dhfr gene were preferentially induced by 3Â«,4ÃŸ-dihy-droxy-1 a,2Â«-epoxy-1,2,3,4-tetrahydrobenzo(c)phenanthrene(113). The hprt locus is of particular interest because of thepotential to compare mutational spectra obtained from studiesusing cultured cells experimentally exposed to carcinogens withthose observed in human lymphocytes obtained from donorsexposed in vivo to environmental carcinogens (114).

The molecular analysis of mutationally activated ras protooncogenes in animal models of carcinogenesis generally supportsthe concept of mutational spectrum reflecting the DNA adduciformation and mutagenic activiiies elicited by specific chemicalcarcinogens. For example, the mulalions found in activated rasprotooncogenes associated with tumors of rodents exposed loIhe melhylaling AAnilroso compounds are predominancyG:Câ€”Â»A:Tbase subslitutions (Table 4); these are likely due lomelhylalion of deoxyguanine al ihe Oh posilion followed bymispairing wilh ihymine during DNA synthesis (92, 110, 115-117). Although there are several deoxyguanine residues in rascodons lhal would generale a transforming protein if substi-luled wilh adenine, Ihe animal experimenls have revealed lhatthe mutalions occur overwhelmingly al only one of the "possible" mutation sites. However, preferential mutalion siles are

within the admittedly narrow range of possibilities for aclivalingras genes. Unexplained mutational specificities have been observed in other experimental systems (118-120) and may reflectthe differences in the spectrum of promulagenic carcinogen-DNA adducls, specificily of DNA repair enzymes (121), and/or Ihe resullanl amino acid changes are eilher silent or lethal.

Allhough the mutalional speclrum of ras prolooncogenes inrodent tumors caused by methylaling carcinogens is consistent

Table 3 Percentage distribution of mutations in the endogenous aprt locus, including multiple events in Chinese hamster ovary-cells"

TransitionsMutagenSpontaneous

UV radiationIonizing radiationBenzo(a)pyrene diol epoxideG:C-Â»A:T71615A:T-Â«:C0

260G:Câ€”T:A13

S662TransversionsG:Câ€”

C:G3

10614A:Tâ€”

C:G0

5190A:T-Â»T:A7

12139Deletions6

2315Insertions0

205

'Adapted from Ref. 106.

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CARCINOGENESIS

Table 4 Examples of predominant base substitution mutations in ras protooncogenes in rodent tumors

ConditionSpontaneousMethylating

carcinogen''Tetranitromethane

Benzo(a)pyreneSkin

(H-ras)!G35â€”

Â»A'(NMU,

MNNG)MouseLiver

Lung Mammary Skin(H-ras) (K-ras) (H-ras)(H-ras)C'Â»-.A

G3!-Â»TG3'^A/

*

(NNK, DMN)Liver

Lung(K-ras)(K-ras)G3'-^A*RatMammary(H-ras)G35â€”A*(NMU)Kidney(H-ras)(DMN-OMe)Esophagus(H-ras)G3'-Â»AJ(MB)

7,12-DimethylbenzanthraceneAflatoxin B,

Benzidine and congeners

Urethan AiÂ«_TÂ» An

" Ref. 457.* Ref. 458.' Ref. 459.''NMU, jV-methyl-A"-nitrosourea; MNNG, /V-methyl-iV'-nitrosoguanidine; NNK, 4-(methylnitrosamine)-l-(3-pyridyl-l-butanone), DMN, A'-nitrosodimethyla-

mine; DMN-OMe, methyl(methoxymethyl)nitrosamine: and MB, jV-nitrosomethylbenzylamine.' Ref. 87./Ref. 460.'Ref. 461.* Ref. 117.1Ref. 462.' Ref. 467.* Ref. 463.'Ref. 150." Ref. 464."Ref. 139.Â°Ref. 465.' Ref. 466.* R. W. Wiseman, personal communication.'Ref. 139.

Table 5 Examples of human cancers with base substitution mutations in the pS3 tumor suppressor gene"

V\orldwidecancerburden(rank)*234781112CancerLungBreastColonEsophagusLiverBladderLeukemia

andlymphomasSarcomasBrainNo.

ofmutationsdetectedin tumor

celllinesandtumors^43"31393719'15531220Mutational

'hotspots"(codon)273175.248273,

282249273Mutations

atA:T65712131903G:C372632251712341217CpG-TpG74267052579G:C-A:T121331163730815G:C-T:A19709142221G:C-C:G661013221

Â°Hollstein et al. (132).* From Muir (468).' Only mutations in exons 5, 6, 7. and 8 are included in this analysis. Tumor cell line mutations are included in the analysis with the understanding that some of

these may have arisen in vitro.d Chiba et al. (136) reported a higher p53 mutation frequency in lung squamous cell carcinoma than in other non-small cell lung carcinomas.' Frequency and mutational spectrum may vary considerably with geographic origin of tumor.

with the formation of the promutagenic 06-melhyldeoxyguano-sine and mutational spectrum caused by A/-nitroso-/V'-methyl-

urea in the aprt gene of CHO cells (106), an alternative hypothesis is that the mutant ras genes preexist in certain cells andthat exposure to the methylating agent facilitates clonal expansion of these mutant cells. This latter hypothesis has been testedby investigating the induction of rodent tumors by chemicalcarcinogens that form DNA adducts with differing chemicalspecificity. As seen in Table 4, the mutational spectra werehighly correlated with each chemical carcinogen and reflectedthe predicted base substitution (84), i.e., G:Câ€”Â»T:Atransversionfor benzo(a)pyrene which forms predominantly the /V2-BPDE-deoxyguanosine adduci (122) and A:Tâ€”>T:Atransversion for7,12-dimethylbenzanthracene which forms predominantly theA*-7,12-dimethyIbenzanthracene diol-epoxide-deoxyadenosine

adduci (123).

The mutalional speclra of aclivaled ras prolooncogenes inlumors and preneoplaslic lesions caused by chemical carcinogens in animal models (Table 4) may prove lo be inslructive inthe inlerprelalion of mulalions in prolooncogenes and tumorsuppressor genes found in human cancers (Table 5). The spectraof Ki-ras protooncogene mutations in human adenocarcinomasvary according lo lissue sile; e.g., G:Câ€”Â»T:Atransversions occurin lung tumors, whereas G:Câ€”Â»A:Ttransit ions predominale incolonie lumors. The inlerprelalion of Ihese results, however,may be confounded by sponlaneous mulalions engendered byeilher endogenous mulagens or DNA polymerase errors. Astwo examples, 8-hydroxyguanine, caused by oxy radical damage(124) and DNA depurination, can produce G:Câ€”Â»T:Airansver-sions, and DNA polymerase infidelity will also produce G:Câ€”Â»T:A transversions as well as olher changes (125).

The p53 lumor suppressor gene is ideally suiled for analysis5029s



CARCINOGENESIS

of mutational spectrum: (a) p53 is well conserved in evolution(126) and 5 domains are >90% homologous in DNA sequenceamong humans and rodents that are frequently used in animalmodels of carcinogenesis; (b)p53 is mutated in diverse types ofhuman cancer (127-131) and, where examined, cancers inlaboratory animals5"7; (c) a wide spectrum of mutational types

and codon sites have been observed that presumably defineregions of the p53 protein likely to be essential for its biologicalactivity in cell cycle control, in tumor suppression, and for itsinteraction with other cellular and viral proteins. A recentreview (132) has concluded that the majority of the mutationsin human tumors occur in the evolutionarily highly conserveddomains in exons 5-8 of the p53 gene, and the missensemutations are predominantly transitions at G:C base pairs andare almost all (>95%) at amino acids that are entirely conservedin mouse, rat, monkey, and human. Mutational spectra alsovary among cancer types (Table 5). The G:Câ€”Â»A:Ttransitionsare the most frequent in colon tumors and 68% of the mutationsoccur at CpG dinucleotides. These findings are consistent withthe endogenous mutational mechanism due to deamination of5-methylcytosine residues found at CpG dinucleotides in themammalian genome. More than one-half of the p53 mutationsin colon tumors are at "hot spots," codons 175, 248, 273, or

282, each of which is a CpG dinucleotide.In contrast to colon tumors, CpG dinucleotides are less

frequently sites of mutations in most other types of humancancers. Whereas G:Câ€”>T:Amutations are rare in colon tumors, these transversions are significantly more common inbreast and lung cancers. The G:Câ€”>T:Amutations in Ki-rojprotooncogene are also found in human lung adenocarcinomas( 133-135) and in rodent lung cancers caused by benzo(a)pyrene(Table 4). Interestingly, the p53 mutational spectra differ insmall cell lung cancer and non-small cell lung cancer; G:Câ€”Â»

T:A transversions are common in the latter group of cancers(136). Whereas CpGâ€”Â»TpGmutations occur with intermediatefrequency in esophageal cancers, 36% of the mutations are atA:T pairs which may be due in part to DNA depurination and/or exposure to chemical carcinogens such as urethan (Table 4),a contaminant of certain alcoholic beverages.

The most striking p53 mutational spectrum is found in hep-atocellular carcinomas from either Qidong, People's Republic

of China (137) or southern Africa (138). Eleven of 12 basesubstitution mutations in 26 tumors were at a single site, the3rd base position of codon 249, and all but 1 were G:Câ€”>T:Atransversions. One additional mutation at codon 157 was alsoa G:Câ€”>T:Atransversion. These tumors were from patientswho live in geographic areas where aflatoxin B, and hepatitisB virus are major risk factors. Aflatoxin B, is a naturallyoccurring hepatocarcinogen that forms /V7-deoxyguanosine pro-

mutagenic adducts and induces primarily G:Câ€”Â»T:Atransversions and G:Câ€”Â»A:Ttransitions in experimental systems (139)(Table 4). Analysis of liver tumors from geographical areaswhere aflatoxin BI is considered not to be a significant riskfactor will be instructive in determining whether exposure tothis fungal carcinogen may be responsible for these p53mutations.

An analysis of mutational spectra in human tumors frompopulations with differing risk factors may provide insight in amanner similar to those of the studies in experimental animalsdescribed above (Table 4). In addition to further studies of liver

'' A. Balmain, personal communication.' A. Klein-Szanto. personal communication.7G. Wogan, personal communication.

cancer, the mutational spectra of Ki-ras and/or the p53 genecould be compared to bronchogenic cancers, including the useof paraffin-embedded, fixed archival tissue, from cigarettesmokers versus nonsmokers, and from workers in high riskoccupations, e.g., uranium mining, mustard gas production,asbestos mining, and chloromethyl ether production.

Selective Clonal Expansion

The majority of human cancers and those induced by chemical and physical carcinogens in animal models are unicellularin origin (43, 140). The clonal nature of neoplasia is thecornerstone in the concept of multistage carcinogenesis andprovides the basis for current strategies designated to elucidatethe component genetic changes. If all of the clonally derivedcells in a tumor, although heterogeneous in other aspects (discussed below), have in common the critical genetic lesion(s)that triggers and/or drives their selective clonal growth thenthe identification of this lesion(s) is a realistic goal.

Recent data suggest that alterations in thep53 and Rb tumorsuppressor genes represent such critical genetic lesions. Forexample, germline mutations in the Rb gene predispose toretinoblastoma (95, 96) and similar mutations in the p53 geneconfer an inherited susceptibility to cancers of the breast, softtissues, brain, and other tissues (141, 142). In vitro transfectionof wild type p53 into neoplastic colon (143), bladder (144),brain (145), and bone ( 146) cell lines containing an endogenousmutant p53 gene suppresses cellular growth and/or tumori-genicity. Similar transfection studies using putative wild typeRb have also suppressed, to varying degrees, cell growth (147,148). Although the mechanism(s) of growth retardation of thesecell lines by p53 or Rb is uncertain, the inhibition of the cellcycle progression into S phase following induction of wild typep53 is accompanied by selective down-regulation of mRNA andprotein expression of proliferating cell nuclear antigen, a cofac-tor of DNA polymerase Â¿,an important component of DNAreplication (149).

Studies of animal models suggest that certain activated pro-tooncogenes may also be candidate critical genes. Mutations inthe Ha-ras protooncogene are early genetic events in mouseskin carcinogenesis (150) and rat mammary carcinogenesis(151, 152). Certain oncogenes impart, as transgenes, an "initiated" state (93, 94), and cooperation among oncogenes has

been shown to increase cancer risk in transgenic mouse models(153). Whereas germline mutations occur in tumor suppressorgenes that increase cancer risk in the affected individuals (141,142), germline mutations that activate protooncogenes in humans have yet to be described. The success of transgenic mousestudies argues against the possibility that such mutations wouldlead to fetal death and thus not be observed in the humanpopulation.

Activated protooncogenes can modulate the responsivenessof mammalian cells to their microenvironment and their cellular neighbors. For example, rodent cells transformed by certaintransfected oncogenes have defective intercellular communication (154) and the EIA oncogene induces resistance to thegrowth-inhibitory effects of transforming growth factor ÃŸ,(155). When Ha-ras, Ki-ras, or the combination of c-myc andc-ra/are transferred into SV40 T "immortalized" human bron

chial epithelial cells (BEAS-2B), the cells had a reduced responsiveness to growth inhibition by either transforming growthfactor type ÃŸor serum (156-158)* and thus have potentially

* Unpublished results.

5030s



CARCINOGENESIS

gained a selective clonal expansion advantage. Antisense oli-gonucleotides or expression vectors producing antisense DNAfragments of the mRNAs of these oncogenes should be usefulin assessing the direct role of raÃ,myc, or ra/in the modulationof growth and differentiation in this in vitro model system ofhuman bronchial epithelial carcinogenesis.

The molecular mechanisms responsible for selective growthadvantage of initiated cells are likely to be numerous and involvecell-cell interactions (154), cell-matrix interactions (159), cell-

growth factor interactions (34, 160) and intrinsic aberrationsin regulation of the cell cycle (161), terminal differentiation(41), and programmed cell death (162). Deregulated expressionof Bcl-2, a novel inner mitochondrial membrane protein, extends the survival of lymphoid cells presumably by blockingprogrammed cell death, apoptosis, and, as a hybrid minigenewith an immunoglobulin gene, increases the probability oflymphoma in transgenic mice (163). Epstein-Barr viral lategene products may also extend the survival of B-lymphocytesby blocking their death by apoptosis (164). An example of astudy that has identified candidate mutant genes and/or reversed the neoplastic state by transfecting the wild type gene isthe transfection of Â«5and /3Â¡integrin complementary DNA intomalignant CHO cells with a much decreased intrinsic level ofthese cognate integrins leads to suppression of tumorigenicity(165). Indirect proof of mechanisms leading to abnormal cell-growth factor interactions include interruption of signal trans-duction pathways of positive growth factors by use of neutralizing antibodies to either growth factors, e.g., gastrin-releasingpeptide (166), or their cell membrane receptors, e.g., epidermalgrowth factor receptor (167), resulting in decreased tumorigenicity of the human carcinoma cells injected into athymicnude mice. Transfection of antisense oligonucleotides or vectorsexpressing antisense oligonucleotides of certain oncogenes maylead to a reversal of certain in vitro phenotypes, such as softagar growth or transformed morphology, and in some casescause a decrease in tumorigenicity (168-172).

Protein kinase and protein phosphatases are responsible forthe reversible phosphorylation of cellular proteins that controlgrowth and differentiation of mammalian cells (173,456). PKCis a multigene family with at least 8 members that have differential expression in specific tissue types and functions in signaltransduction (28, 174). Exposure of certain cells to the tumorpromoter TPA causes PKC to translocate to cellular membranes and the resultant activation of PKC isoforms is associated with rapid induction of expression of nuclear protoonco-genes c-jun and c-fos (175). Since exposure to TPA produces

many phenotypic responses that are cell type dependent (176),and activated PKC modulates cytosolic calcium concentrationand phosphorylates multiple protein substrates, the signaltransduction pathways involving PKC are difficult to delineate(28). One hypothesis of the mechanistic role of PKC in tumorpromotion in mouse skin carcinogenesis is that certain proteins,when phosphorylated at specific sites directly or indirectly byPKC, activate signal transduction pathway(s) positive for cellgrowth. Assuming that the phosphorylated protein(s) is in thehypothetical active state, dephosphorylation at specific siteswould lead to inactivation. Therefore, inhibitors of the responsible protein phosphatase(s) would increase the steady statelevels of the critical phosphoprotein(s) in the activated stateand would be candidate tumor promoters. Although the phos-phoprotein(s) critical in tumor promotion has not as yet beenidentified, this latter hypothesis is supported by the recentdiscovery of non-TPA-like tumor promoters (177) such as

okadaic acid, a potent inhibitor of protein phosphatases I andIIA (178, 179). The interpretation of the above studies isrestricted because of the intrinsic limitations of experimentaldesigns using agonists and/or inhibitors that frequently havemultiple nonspecific effects. Since members of the PKC andthe protein phosphatase multigene families are being cloned,more powerful genetic strategies are becoming available.

Genomic Instability

Normal human somatic cells maintain a diploid karyotypeand have a mutation frequency of about 10~8 for hemoglobinvariants (point mutations), about 10~7 for hprt variants (meas

ures many classes of mutations, but the assay does not detectmitotic recombination), and about 10~5 for glycophorin A or

HLA variants (measures most classes of mutations includingsomatic recombination, gene conversion, or chromosomal non-disjunction) (114, 180, 181).

The potential sources for these observed mutation frequenciesinclude exposure to "background" levels of environmental

chemical and physical agents and endogenous mechanisms (55)such as: (a) the chemical instability of DNA, e.g., depurination[one can calculate that each cell undergoes IO4 depurinations/day (125)] or deamination of 5-methylcytosine to yield thymi-dine (182, 183); (b) free radicals of oxygen, e.g., hydroxyl ions[from measurements of 8-hydroxyguanine and thymine glycolin urine, about IO4 oxy radical-induced DNA lesions occur in

each human cell per day (54)]; and (c) DNA replication errors.Imbalances in deoxynucleoside triphosphate pools (184) andmutations in DNA polymerase Â«(185) are examples of mechanisms leading to infidelity of DNA synthesis. Inactivation ofRAD18, a putative DNA repair gene in Saccharomyces cerevis-iae, leads to an increase in the frequency of G:Câ€”>T:Atrans-versions ( 186). Considering the multiplicity of proteins involvedin DNA synthesis, DNA repair, mitosis, and the cell cycle, thenumber of potential gene targets in somatic cells is large andcould result in the mutator phenotype (43, 55, 187) (Table 6)that could increase the probability of both neoplastic transformation and the generation of more malignant subclones duringtumor progression (42, 188). If such mutations occur in germcells and are nonlethal, they could lead to congenital abnormalities and increased cancer risk in an inherited manner. Thehypothesis that genomic instability may have an inherited basisand be associated with increased cancer risk is supported bystudies of Bloom's syndrome, where an increase in cytogenetic

end points is observed (189), and of colonie carcinomas, wherea significant increase in allelic deletions are found in tumorsfrom patients who have first degree relatives with cancer (190).

Cytogenetic studies (191, 192) have provided the bulk of theindirect evidence supporting the hypothesis that genomic instability is a potential mechanism driving tumor progression. Theevidence is less compelling (193) when the frequencies of eitherspontaneous point mutations (194-201) or drug-induced geneamplification (202-206) are compared in normal and neoplasticcells.

Recombinase infidelity (207), telomere reduction (208), andDNA hypomethylation (47, 209) are examples of additionalmechanisms of genomic instability. The frequency of somaticrecombinational events at certain loci, e.g., Â¡mmunoglobulingenes, is relatively high, but specific DNA sequences at theseloci are recognized by the responsible V-(D)-J recombinases.Although the degree of infidelity of wild type and mutantrecombinases is not known, a possible example of "illegitimate"

5031s



CARCINOGENESIS

Table 6 Examples of gene abnormalities associated with genomic instability

Genomic inslabilily

GeneRAD

9RAD18p}4Â«"Topoisomerase

IInodDNA

polymeraseÂ«p53R.4C-IseidUnknownSpeciesS.

cererisiaeS.cerevisiae.9.pombeS.pombeDrosophilaHumanHuman

(Li-Fraumenisyndrome)HumanMurineHuman

(Bloom's syndrome)ActivationMutationMutationMutationMutationMutationMutationMutationMutation"MutationUnknownTypeChromosomal

lossBasesubstitution (G:Câ€”>T:Atransversion)AneuploidyAbnormal

chromosomalsegregationAbnormalchromosomalsegregationBase

substitutionAneuploidyChromosomal

rearrangements anddeletionsAberrantt'(D)J recombination anddouble-strandbreak

DNArepairHyperrecombination

' In vitro experimental studies.

V-(D)-J recombinase activity leading to interstitial deletion andrearrangements in a growth-regulatory gene has been described(210). In addition, the seid (severe combined immunodeficiency)mutation in mice is due to a defect in the site-specific V(D)Jrecombination pathway and has been recently linked to a deficiency in double-strand break DNA repair. This linkage suggests that the seid gene product performs a similar function inboth pathways.

Hastie et al. (208) have shown that telomere reduction iscommon in both colorectal adenomas and carcinomas and havespeculated that telomere reduction may be involved as an earlyevent in carcinogenesis. Interestingly, studies of yeast (211) andmaize (212) have shown that chromosomes without terminalrepeats or telomeres are prone to rearrangements or loss. Studies in yeast have also identified genes involved in cell cyclecontrol and mitosis, e.g., RAD9 and cdc-2, that, when mutated,influence the fidelity of mitosis (213, 214) (Table 6). Forexample, certain mutations in RAD9 of 5. cerevisiae increasethe rate of chromosomal loss by 7-21-fold (215) and topoisom-erase II mutants in Saccharomyces pombe have abnormal segregation of the intertwined daughter chromosomes at the endof DNA replication (216) and abnormal chromosome condensation (217). Since human homologues of such genes are beingidentified, similar investigations could be conducted using cultured human cells, and human tumors could be examined formutations in these genes.

Analogous to the yeast paradigm of a mutator phenotypeassociated with dysregulation of cell cycle control (213), certainactivated protooncogenes or inactivated tumor suppressorgenes could lead to genomic instability. The rapid appearanceof aneuploidy and "spontaneous" immortalization of cultured

human fibroblasts from donors with the Li-Fraumeni syndrome, who have an inherited mutant p53 alÃele(218), isconsistent with this hypothesis. The genomic instability causedby certain DNA viruses such as SV40, adenovirus, and papil-lomavirus could also be due to the inactivation of p53 and/orRb by complexing with the viral proteins.

As argued by Loeb (55), the impression of a high degree ofgenomic stability may be misleading when one considers thatthe human body contains about IO14cells and that, within theaverage human life span, the cells undergo a total of IO'6

divisions. Therefore, spontaneous mutagenesis may significantly contribute to the prevalence of certain cancer types thathave no apparent risk factors and minimal variations in incidence occur among different geographic areas. However, epidemiolÃ³gica! studies have: (a) identified significant cancer riskfactors, e.g., tobacco smoke and hepatitis B virus; (b) foundsubstantial geographic variation in the incidence of the mostcommon types of human cancer; and (c) found that migrant

populations frequently acquire the risk of cancer prevailing intheir new geographic location of residence (1, 2, 219). Basedon the above and other considerations ( 1), it has been estimatedthat about 80% of all cancer in the United States is due toenvironmental factors. Finally, one can speculate that the ratesof spontaneous mutations, e.g., deamination of 5-methylcyto-sine, may be altered by environmental chemicals directly and/or indirectly via generation of endogenous agents such as oxyradicals and the intracellular signal transducer, nitric oxide.

Molecular Epidemiology of Human Cancer

Combined laboratory-epidemiological studies of human cancer have a long history and have been named metabolic (220),biochemical (221), and molecular epidemiology (222). Whereasclassical epidemiology has been successful in identifying highcancer risk populations, e.g., cigarette smokers, the primarygoal of molecular epidemiology is to identify individuals inthese populations who are at the highest cancer risk. Thecurrent strategy involves improved exposure assessment bymeasuring biologically effective doses of carcinogens and secondly, analysis of host susceptibility factors within the framework of well-designed epidemiological studies (223).

Exposure Assessment

Carcinogen-macromolecular adducts, mutations, and cyto-genetic changes can be measured in the target epithelial cellpopulation and in surrogate blood cells as shown in Fig. 3. Thisparadigm is now feasible to explore experimentally because ofrecent advances in methods to measure carcinogen-macromo-lecular adducts (224), somatic gene mutations (181, 225), andcytogenetic endpoints (103). In addition, polymerase chainreaction technology and DNA sequencing allows a rapid assessment of mutations in protooncogenes, tumor suppressorgenes, and antimetastasis genes in the epithelial cells. Since theras protooncogene family is occasionally and the p53 tumorsuppressor gene is frequently mutated in human cancers, asdiscussed above, these genes are suitable candidates for assessing the genetic consequences of carcinogen-DNA adducts andendogenous mutagens. The mutational spectra in ras and p53genes can be measured by current technology due to the clonalexpansion of the cells driven by these mutant genes. Althoughsmall clonal populations of a few hundred cells are sufficientfor analysis of mutational spectra by polymerase chain reactionand direct DNA sequencing, additional advances in methodology will be required to determine the mutational spectra insingle cells that have not clonally expanded and are diluted ina much larger population of normal cells.

5032s



CARCINOGENESIS

Host Susceptibility Factors

Both inherited and acquired host factors that predispose anindividual to cancer caused by environmental agents have beenidentified (37, 226). Inheritance of a germline mutation in thetumor suppressor genes, p53 (141, 142) or, perhaps, Rb (227),predisposes to the development of adult types of cancer (Table7). Other inherited host susceptibility factors include certainDNA repair deficiencies (228), genetic polymorphism in certainxenobiotic metabolizing enzymes (70), and, perhaps, the proox-idant state in Bloom's syndrome (229).

Interindividual Variation in Human Carcinogenesis

No one supposes that all the individuals of the same species arecast in the very same mould. These individual differences arehighly important for us. ...

The Origin of SpeciesCharles Darwin, 1859

Scientists have repeatedly recognized person-to-person differences in behavior, morphology, and risk of disease. Suchinterindividual differences may be either inherited or acquired.For example, inherited differences in susceptibility to physicalor chemical carcinogens have been observed among individuals,including an increased risk of sunlight-induced skin cancer inpeople with xeroderma pigmentosum (228), bladder cancer indye stuff workers with a poor acetylator phenotype (230), andbronchogenic carcinoma in tobacco smokers who have an extensive debrisoquine hydroxylator phenotype (231, 232).

Because most chemical carcinogens require metabolic activation to exert their oncogenic effects and the amount ofultimate carcinogen produced results from the action of competing activation and detoxication pathways, interindividualvariation in carcinogen metabolism is considered to be animportant determinant of cancer susceptibility (75). DNA ad-ducts are one form of genetic damage caused by chemicalcarcinogens and may lead to mutations that activate protoon-

â€¢Environmental Exposure

J

Table 7 Examples of inherited predisposition of human cancer

1â€¢Bio1

rbcsei

ir

odIâ€¢um

wbcr

*â€¢AdductsG.A.â€¢

CytogeneticEndpointsâ€¢Â«

â€¢^CellClonal

Expansionâ€¢

Mutationalspectra, e.g.,HPRT, HLA-*nâ€¢

Targetf Tissue(cell)Â»â€¢

â€¢Adducts1^

â€¢CytogeneticEndpointsCellDonai

Expansionâ€¢

Mutationalâ€” *â€¢ spectra, e.g.,

ras, p53

â€¢Exposure Assessment

Fig. Ã•.Molecular dosimetry, mutational spectra, and exposure assessment.Paradigm for improvement of carcinogen exposure assessment that involves themeasurement of carcinogen-macromolecular adducts, cytogenetic end points, andmutational spectra in protooncogenes, e.g., ras. or tumor suppressor genes, e.g.,p53, found in target tissues and in monitoring genes, e.g., hprt or histocompati-bility genes (HLA), in blood lymphocytes or glycophorin A (G.A.) protein in redblood cells (rbc).

CandidategenesP53RbMCCCYP2D6XPACConditionLi-Fraumeni

syndromeFamilial

retinoblastomaFamilialpolyposiscoliExtensive

debrisoquine phenotypeXeroderma

pigmentosum AExamples

ofcancertypeCarcinomas

ofthebreastand blad

der;sarcomasRetinoblastomaColonLungSkin

cogenes and inactivate tumor suppressor genes in replicatingcells. The steady state levels of these adducts depend on boththe amount of ultimate carcinogen available to bind and therate of removal from DNA by enzymatic repair processes. Thegenomic distribution of adduct formation and repair is nonran-dom and is influenced by both DNA sequence and chromatinstructure (121, 233-235).

Carcinogen Metabolism and DNA Adduct Formation. Chemical carcinogens are metabolized by a wide variety of solubleand membrane-bound enzymes. Multiple forms of human cy-tochrome P-450 are involved in the oxidative metabolism ofchemical carcinogens, e.g., polycyclic aromatic hydrocarbons(70). Several thousand-fold interindividual variation has beenobserved in placental AHH activity which is catalyzed by theCYPIA1; some of this variability is under direct genetic control,but variations are also the result of an enzyme induction processdue to maternal exposure to environmental carcinogens, e.g.,tobacco smoke, or dietary factors (236-238). However, theinduction process itself may have a genetic component (239)and inducible AHH activity is higher in cultured lymphocytesfrom lung cancer cases when compared to controls (240). Trelland coworkers (241) are currently conducting a prospectivestudy to determine if high inducibility of AHH is a risk factorfor the development of cancer. Increased binding ofbenzo(a)pyrene metabolites to DNA in lymphocytes of patientswith lung cancer versus noncancerous controls has also beenobserved-(242). A highly inducible allelic variant CYPIA1 hasbeen identified in humans (243).9 This variant is associatedwith increased risk of smoking-associated lung cancer in aJapanese population (244). This association has not been foundin a U.S. population10.

Cytochrome P-450 (CYP2D6) activity is polymorphic andhas also been linked to lung cancer risk (231, 232). CYP2D6hydroxylates xenobiotics such as debrisoquine, an antihyperten-sive drug, and a tobacco-specific A'-nitrosamine (70), and anindividual's polymorphic phenotype is inherited in an autoso-

mal recessive manner. The rate of 4-hydroxylation of debrisoquine varies several thousand-fold among people, and lung,liver, or advanced bladder cancer patients are more likely tohave the extensive hydroxylator phenotype when compared tononcancer controls (231, 245, 246). In a case-control study oflung cancer in the United States, the extensive hydroxylatorphenotype had an 8-fold increased cancer risk when comparedto the poor hydroxylator phenotype and the increased risk wasprimarily for histolÃ³gica! types other than adenocarcinoma ofthe lung (232). In addition, analysis of data derived from aBritish population (231) has revealed that individuals with theextensive hydroxylator phenotype who are occupationally ex-

9T. L. McLemore, S. Adelberg, and M. C. Liu. Cytochrome P450IA1 geneexpression in lung cancer patients: evidence for cigarette smoke-induced expression in normal lung and altered gene regulation in primary pulmonary carcinomas,submitted for publication.

IOP.Shields, A. Weston. and C.C. Harris, unpublished results.

5033s



CARCINOGENESIS

posed to high amounts of either asbestos or polycyclic aromatichydrocarbons have relative lung cancer risks of 18- and 35-fold,respectively (247). CYP2D6 may activate a chemical carcino-gen(s) found in tobacco smoke, e.g., certain jY-nitrosamines,and/or, a more speculative possibility, inactivate nicotine, theaddictive pleasure component of tobacco smoke that woulddecrease its steadystate level and increase the drive to smokemore cigarettes. Thereby, the individual with the extensivemetabolizer phenotype would be at greater cancer risk. Anotherhypothesis is that an alÃeleof the CYP2D6 gene is in linkagedisequilibrium with another gene that influences cancersusceptibility.

The Â¿Y-acetylationpolymorphism is controlled by two auto-somal alÃelesat a single locus in which rapid acetylation is thedominant trait and slow acetylation is recessive. Acetylation ofcarcinogenic aromatic amines has been proposed as a cancerrisk factor (248). The slow acetylator phenotype has been linkedto occupationally induced bladder cancer in dye workers exposed to large amounts of A'-substituted aryl compounds (230).

Interestingly, the rapid acetylator phenotype is commonlyfound in colon cancer cases (249). Whether or not this association is due to metabolism of a carcinogenic aromatic amine inthe colonie epithelium is not known.

Wide interindividual differences in detoxifying enzymes ofcarcinogens are also found. For example, at each step in themetabolic pathway of benzo(a)pyrene activation to electrophilicdiol epoxides, competing detoxifying enzymes are found (250).Confirming previous observations (251), a recent study of several of the enzymes involved in benzo(a)pyrene metabolismshowed a more than 10-fold person-to-person variation in theiractivities (252) and presented indirect evidence that tobaccosmoke induced many of these enzymes. Genetic control of thepresumed detoxication of benzo(a)pyrene by conversion towater-soluble metabolites has been reported (239).

GSTs are multifunctional proteins (253) that catalyze theconjugation of glutathione to electrophiles, including the ultimate carcinogenic metabolite of benzo(a)pyrene (254), and areconsidered to be a detoxification pathway of carcinogenic poly-cyclic aromatic hydrocarbons (255). The three isoenzymes ofGST (Â«-<,n and w) vary in their substrate specificity, tissuedistribution, and activities among individuals (256). Expressionof GST-ji is inherited as an autosomal dominant trait (257) andindividuals with low GST-^ activity may be at greater risk oflung cancer caused by cigarette smoking (258, 259). Furtherfunctional and genetic studies of acquired and inherited deficiencies in the activities of enzymes involved in the detoxification of chemical carcinogens are warranted.

Metabolism of carcinogens from several chemical classes,including /V-nitrosamines, polycyclic aromatic hydrocarbons,hydrazines, mycotoxins, and aromatic amines, has been studiedusing human tissues, cells, and microsomes (73, 260-267). Theenzymes responsible for the activation and deactivation ofprocarcinogens, the metabolites produced, and the carcinogen-DNA adducts formed by cultured human tissues and cells, ingeneral, are qualitatively similar among donors and tissue types.The DNA adducts and carcinogen metabolites are also similarto those found in most laboratory animals, an observation thatgenerally supports the qualitative extrapolation of carcinogen-esis data from the laboratory animal to the human (73). However, some notable differences among animal species have beenreported, including metabolism of aromatic amines in theguinea pig (65), benzo(a)pyrene in the rat and rabbit (66, 268),and aflatoxin B, in the Syrian golden hamster (67).

Although the major DNA adducts are qualitatively similarfor the chemical carcinogens thus far studied in these in vitromodels, quantitative differences have been found among individuals and their various tissue types (73, 260, 269-271). Thedifferences in formation of DNA adducts range from approximately 10-150-fold among humans, the interindividual distribution is generally unimodal, and the variation is similar inmagnitude to that found in pharmacogenetic studies of drugmetabolism (75).

DNA Repair Rates. DNA repair enzymes modify DNA damage caused by carcinogens in reactions that generally result inthe removal of DNA adducts. Studies of cells from donors withxeroderma pigmentosum have been particularly important inexpanding our understanding of DNA excision repair and itspossible relationship to risk of cancer (228). The rate but notthe fidelity of DNA repair can be determined by measuringunscheduled DNA synthesis and removal of DNA adducts, andsubstantial interindividual variations in DNA repair rates havebeen observed (272). As discussed above, the fidelity of DNArepair could also vary among individuals, and recent advancesin the identification of mammalian DNA repair genes and theirmolecular mechanisms (273, 274) should provide an opportunity to investigate the fidelity of excision DNA repair in thenear future. In addition to finding excision repair rates severelydepressed in xeroderma pigmentosum cells (e.g., complementation group A), an approximately 5-fold variation amongindividuals in unscheduled DNA synthesis induced by UVexposure of lymphocytes in vitro has been found in the generalpopulation (272). A significant reduction in unscheduled DNAsynthesis induced in vitro by yV-acetoxy-2-acetylaminofluorenehas been observed in mononuclear leukocytes from individualswith a history of cancer in first degree relatives when comparedto those without a family history (275, 276).

Interindividual variation has been noted in the activity of O6-alkyldeoxyguanine-DNA alkyltransferase; this enzyme repairsalkylation damage to O6-deoxyguanine (277-280). In additionto these person-to-person differences of about 40-fold, widevariations in this DNA repair activity have been observed indifferent types of tissues (277, 278, 281, 282), fetal tissuesexhibit 2- to 5-fold less activity than the corresponding adulttissues (283), and cells may have lower repair rates after terminal differentiation (284). The activity of this DNA repairenzyme is inhibited by certain aldehydes (281) and alkylatingcancer chemotherapeutic agents (285). A decrease in this DNArepair activity has been observed in fibroblasts from patientswith lung cancer when compared to donors with either melanoma or noncancer controls (286). Therefore, acquired and/orinherited deficiency in 06-alkyldeoxyguanine-DNA alkyltrans

ferase may be a cancer risk factor in tobacco smokers.A unimodal distribution of repair rates of benzo(a)pyrene

diol-epoxide-DNA adducts has been observed using humanlymphocytes in vitro (287). The interindividual variation wassubstantially greater than the intraindividual variation whichsuggests a role of inherited factors. The influence of thesevariations in DNA repair rates in determining tissue site andrisk of cancer in the general population remains to bedetermined.

Tumor Promotion. Because cancer is the result of complexinteraction between multiple environmental factors and bothacquired and inherited host factors, one should consider carcin-ogen-DNA adducts as only one piece in the puzzle. Other piecesinclude determinants of tumor promotion. In the skin carcino-genesis studies, wide variations in susceptibility to tumor-pro-

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CARCINOGENFSIS

moling agents have been observed among animal species andamong different inbred strains of a single species (288-291).Epidemiological studies suggest that tumor promotion mayinfluence both tumor probability and latency period in humans(1), and mathematical models of carcinogenesis indicate thatcell proliferation is an important determinant of cancer risk(292). Methods to identify human tumor promoters and topredict responses to tumor promoters among different humansneed to be developed.

Interindividual Variation in Carcinogen-Macromolecular Ad-ducts in People Exposed to Chemical Carcinogens. Results fromin vivo and in vitro studies discussed above serve as a basis forinvestigations in biochemical and molecular epidemiology. Forexample, the observation that the carcinogen-DNA adductsformed in cultured human tissues are generally the same asthose found in experimental animals in which these chemicalsinduce cancer has encouraged investigators to search- for DNA

adducts in biological specimens obtained from people exposedto either specific carcinogens such as benzo(a)pyrene or chem-

otherapeutic agents.The development of highly sensitive methods to detect car-

cinogen-macromolecular adducts has made it possible to measure adducts in blood and tissue samples (293-295). Althoughhemoglobin and albumin are not considered targets for thepathobiological effects of carcinogens, these macromoleculeshave certain advantages for the molecular dosimetry of exposure to carcinogens. For example, RBC have a lifetime of about120 days in humans, so the levels of carcinogen-hemoglobinadducts may be an integrative measure of exposure during aperiod of nearly 4 months. In addition, these protein adducts,unlike DNA adducts, are not considered to be repaired in thecirculating RBC, and mg quantities of hemoglobin can beobtained from a few ml of blood. Ehrenberg et al. (296) pioneered this area of research by measuring alkylating agentssuch as ethylene oxide bound to hemoglobin. The amounts ofethylene oxide-hemoglobin and 4-aminobiphenyl-hemoglobinadducts are substantially increased in tobacco smokers compared to nonsmokers (297, 298), and the 4-aminobiphenyl-hemoglobin adducts levels decrease upon smoking cessation(299). The kinetics of adduci loss mirrors the 120-day half-lifeof the erythrocytes. Occupational exposure lo elhylene oxidehas also been monilored (300). Dietary exposure to chemicalcarcinogens can be monitored by measuring carcinogen-albumin adducts. Wild et al. (301) have found thai exposure toaflaloxin B, in coniaminaled grain products is widespread inThe Gambia and Senegal, >95% of the subjects had deteclableaflatoxin-albumin adducts, and the interindividual variationeven within a single village can be 100-fold.

Carcinogen-DNA adducts have also been measured in workers exposed to chemical carcinogens usually in complex mixtures such as coke oven exhaust. For example, PAH-DNAadducts in peripheral blood lymphocytes have been measuredby enzyme immunoassays in coke oven workers, foundry workers, roofers, and fire fighters (302-307). In the occupationallyexposed individuals, enzyme immunoassays detecled 1 to 1200adducts (BPDE-DNA aniigenic equivalent) per IO8 unmodified nucleolides in 25 to 100% of the subjects (303-305, 307-309). These adducts have also been detected by complementarymethods in placenta! DNA samples in levels that ranged between 10 and 500 in 10s nucleotides. In this case, synchronous

scanning fluorimetry and gas chromalography-mass spectros-copy have given consistent results (310-315).

A comparison between deteclion methods and end poinls has

also been performed for coke oven workers in ihe United Statesand Scandinavian countries (308, 316). However, levels ofPAH-DNA adducts reported using the 12P-postlabeling assay

for detection are lower than those reported using enzyme im-munoassay for deteclion. These dala can in pari be explainedby ihe relalive subslrate specificities of each assay which havenot yet been fully slandardized or rigorously compared. Anli-PAH-DNA anlibodies that presumably indicate genotoxic exposure to metabolically aclivaled PAHs have also been detectedin human serum of both occupalionally exposed and nonex-posed populations (coke oven workers; smokers and non-smokers) (303, 304, 309).

Alkyl-DNA adducts have also been detecled in human samples. The receÃ±Ãuse of HPLC or Â¡mmunoaffinily chromatog-raphy in combination with the 32P-postlabeling assay has revealed Ihe presence of 06-methyldeoxyguanosine, O6-ethyl-deoxyguanosine, O4-ethylthymidine, and 7V7-melhyldeoxy-

guanosine as well as other types of alkyl adducls in ihe humanpopulation, including cancer patients (317-322). The combination of HPLC and the "P-posllabeling assay has been usedto detect O6mdG in lung cancer cases (320, 323) and immu-

noaffmily chromalography and HPLC have been used lo deleclIhese alkyl adducts in placenta! DNA (321, 324). The power ofHPLC combined with the "P-posllabeling assay is evidenlbecause O6-elhyldeoxyguanosine and /V7-methyldeoxyguano-

sine adducts can be detected with a high degree of sensitivityand specificily in lung lissues al the level of 1 adduci in IO7

nucleotides (325, 326).The wide range in the amounts of carcinogen-macromolecu-

lar adducls measured in ihe sludies discussed above is in pari areflection of occupational, environmenl, or life-style exposurelo chemical carcinogens. Inlerindividual differences in ihe balance belween metabolic activation and deactivation of chemicalcarcinogens and in DNA repair rates are also likely to conlrib-ute to the variation.

Although methods to measure carcinogen-macromolecularadducts are an importanl conlribulion to improved carcinogenexposure assessment, carcinogen-macromolecular adducls willnoi be a precise quanlitative predictor of cancer risk because ofthe multistage and complex nalure of human carcinogenesis.

In biology, one rarely deals with classes of identical entities, butnearly always studies populations consisting of unique individuals.This is true for every level of hierarchy, from cells to ecosystems.

The Growth of Biological ThoughtErnst Mayr, 1982

Cancer Risk Assessment Perspective

Quantitative risk assessment is currenlly more mathematicalextrapolation and scientific inluition than a rigorous science.However, the stalus of cancer risk assessmenl should improveduring the 1990s. This optimism is based largely on the recentadvances that have increased our understanding of the molecular mechanisms of human disease palhogenesis. The discoveryof multiple genetic and epigenelic changes during carcinogenesis is an example lhal provides opporlunilies for bolh earlydiagnosis of preneoplaslic lesions and novel interventive methods to inhibit or reverse carcinogenesis. These advances aredriven in pari by improved biotechnology. For example, polym-erase chain reaction coupled wilh direcl DNA sequencing allows the detection of mutalions in formalin-fixed, paraffin-embedded tissue sections and the "retrospective epidemiologi-

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CARCINOGENESIS

cal" study of archival material (327).

Quantitative methods to measure human exposure to biologically effective doses of chemical and physical carcinogenscontinue to improve and their current limitations and advantages have been extensively discussed (224,293). If developmentand validation continue at the present pace, methodology forcarcinogen exposure assessment should be satisfactory by theend of this decade.

The identification of inherited or acquired host susceptibilityfactors is also becoming an important factor in the risk assessment equation (223) (Table 7). Analogous to the confoundmentof exposure assessment by complex mixtures, time course variables, etc., generic problems concerning the use of data on hostsusceptibility factors in risk assessment are evident: (a) rankorder of "potency or weight" of various host susceptibility

factors influencing the cancer risk in individuals exposed tocarcinogens; (b) "potency" impact of the degree of the interin

dividual variation in a specific host susceptibility factor; and (c)interactive effects among various host susceptibility factors. Inaddition, societal and ethical issues come to the forefront whenever individuals at high disease risk are identified. These issuesare already being actively discussed (328). Finally, the conductof well-designed and multifaceted molecular epidemiolÃ³gica!studies will require the development of a new cadre of scientistswho have knowledge and skills in both laboratory science andanalytical epidemiology.

By the end of the 1990s, molecular epidemiology of humancancer risk is predicted to have a substantial impact on thequality of quantitative risk assessment and should contribute tothe identification of individuals who will most benefit by cancerprevention strategies including intervention by chemopreven-tive therapy during the multistage process of carcinogenesis.

Acknowledgments

The editorial aid of Robert Julia is appreciated. The comments ofMarshal Anderson, Allan Conney, John Essigmann, Frank Gonzalez,John Higginson, Lawrence Loeb, James Miller, Janardan Reddy, Phillip Shubik, Gary Stoner, Benjamin Trump, Bert Vogelstein, ElizabethWeisburger, Ainsley Weston, Gerald Wogan, and Stuart Yuspa areappreciated.

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