molecular epidemiology of human cancer: contribution of … · [cancer research 58. 4023-4037,...

[CANCER RESEARCH 58. 4023-4037, September 15, 1998]

Perspectives in Cancer Research

Molecular Epidemiology of Human Cancer: Contribution of Mutation Spectra

Studies of Tumor Suppressor Genes

S. Perwez Hussain and Curtis C. Harris1

Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201 IS. P. H.Â¡. and Laboratory of Human Carcinogenesis. National CancerInstitute, N1H, Bethesda, Maryland 20892 Â¡C.C. H.]

Molecular Epidemiology

The identification of individuals at high risk of cancer can offerpromising avenues for the prevention of cancer. The majority ofhuman cancer cases are caused by environmental, occupational, andrecreational exposures to carcinogens (1). These carcinogens canaffect one or multiple stages of carcinogenesis through both geneticand epigenetic mechanisms (2-4). Cancer risk assessment, a highly

visible discipline in the field of public health, has historically relied onclassical epidemiology, chronic animal bioassays of potential carcinogens, and the mathematical modeling of these epidemiological andlaboratory findings (5). Public health regulators are forced to steer aprudent course of conservative risk assessment because of the limitedknowledge of the complex pathobiological processes during carcinogenesis: differences in the metabolism of carcinogens and DNA repaircapacities, variable genomic stability among animal species, andvariation among individuals with inherited cancer predispositionmake a definitive analysis of cancer risk extremely difficult. A molecular epidemiological approach integrates molecular biology, invitro and in vivo laboratory models, and biochemistry and epidemiology to infer individual cancer risk (6-9). Achieving this goal is

challenging both current molecular technologies and the epidemiological designs used to resolve bioethical dilemmas (10, 11).

The two major facets of the molecular epidemiology of humancancer risk are the assessment of carcinogen exposure, includingbiomarkers of effect, and the inherited or acquired host cancer susceptibility factors (reviewed in Refs. 12 and 13); thus, it is thegene-environment interaction that determines an individual's cancer

risk (Fig. 1). Whereas the external environment is a major source ofcarcinogens, the cellular microenvironment also contains endogenouscarcinogens including oxyradicals generated by cancer-prone chronic

inflammatory diseases. Kinzler and Vogelstein (14) have further classified cancer susceptibility genes as gatekeepers and caretakers (discussed below).

The gene-environment paradigm can improve cancer risk assess

ment. When combined with carcinogen bioassay in laboratory animals, laboratory studies of molecular carcinogenesis, and classicalepidemiology, molecular epidemiology can contribute to the fourtraditional aspects of cancer risk assessment: (a) hazard identification;(b) dose-response assessment; (c) exposure assessment; and (d) risk

characterization (Fig. 2). Improved cancer risk assessment has broadpublic health and economic implications (5).

The identification of individuals at high risk of cancer raises complex bioethical issues (Refs. 10 and 15-17; Fig. 2). One can argue that

Received 4/15/98; accepted 7/17/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Laboratory of Human

Carcinogenesis, National Cancer Institute, N1H, Building 37, Room 2C05, Bethesda, MD20892-4255. Phone: (301) 496-2048; Fax: (301) 496-0497; E-mail: Curtis_Hams@

nih.gov.

the knowledge of one's risk can be beneficial. However, more encompassing bioethical issues arise such as an individual's responsi

bility to family members and psychosocial concerns regarding thegenetic testing of children. Therefore, the uncertainty of the currentindividual risk assessments and the limited availability of geneticcounseling services dictate caution and, many argue, the restriction ofgenetic testing to those conditions amenable to either preventative ortherapeutic intervention.

Cancer Susceptibility Genes

An intricately balanced control of cellular proliferation and deathmaintains normal tissue homeostasis and is accomplished by a network of genes. Many of these genes are implicated in the naturalhistory of human cancer because of their consistent alteration in mosttypes of human cancer. The p53 tumor suppressor gene is a remarkable example, because it is mutated in about half of all cancer typesarising from a variety of tissues, and missense mutations occur at ahigh frequency (18-20). Other tumor suppressor genes that are im

portant in human oncology, e.g., APC, WT1, or NF1, seem to have amore limited tissue contribution (Table 1). In addition to the consideration of single genes, recent data indicate the importance of molecular pathways involving cancer susceptibility genes, e.g., pl6'NK4 and

Rb are involved in both the G, checkpoint pathway (21) and themolecular pathogenesis of many types of cancer (22), and APC andÃŸ-catenin are involved in the initiating events in colon carcinogenesis

(23).Germline mutations in genes, e.g., p53, RB, pl6'NK4, and APC,

have been identified in rare cancer-prone families (Table 1). Somatic

mutations in these genes are also frequently found in common sporadic cancers, which attests to the value of studying rare familialcancer syndromes. These cancer susceptibility genes encode proteinsthat perform diverse cellular functions, including transcription, cellcycle control, DNA repair, and apoptosis. The increased cancer risk ofan individual carrying one of these germline mutations can be extraordinarily high, i.e., more than 1000-fold in xeroderma pigmento-sum (complementation group A-G; Fig. 3), where the "at risk" alÃele

is infrequent in the general population. Whereas germline mutations ingenes involved in carcinogen metabolism increase cancer risk onlyseveralfold, an at risk alÃelecan be very common, e.g., GSTM1(occurring in about 50% in the Caucasian population), thus makingthe attributable cancer risk substantial.

Caretaker and Gatekeeper Genes

Recently, the concept of gatekeeper and caretaker genes characterized by their control of net cellular proliferation or maintenance ofgenomic integrity, respectively, has been introduced (14, 24). Examples of gatekeeper genes include APC and ÃŸ-cateninin colon epithe

lial cells, Rb in retinal epithelial cells, NF1 in Schwann cells, and VHLin kidney cells. The most prominent example of a gatekeeper is theAPC gene in colorectal cancer. It is suggested that an alteration in

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MOLECULAR EPIDEMIOLOGY OF HUMAN CANCER

GENEâ€”ENVIRONMENT

/\ /\GatekeeperGenes

CaretakerGenes

ExogenousAgents

EndogenousAgents andMechanisms

Fig. 1. Gene-environment interaction and human cancer risk. Cancer-related genes indifferent human cancers have been classified as gatekeeper (e.g., APC) and caretakergenes (e.g., MSH1 and MLH1). The interaction of these genes with external and internalenvironmental agents can lead to the derangement of regulatory pathways that maintaingenetic stability and cellular proliferation.

APC leads to a derangement of the cellular proliferation pathway thatis important for maintaining a constant cell population. However, thisfunction of APC has a high specificity for colonie epithelial cells butis lacking in most other organs. In murine as well as human coloncancer, APC mutation occurs early on in the process of carcinogenesis(25-27). Although other genes such as K-ras and p53 play important

roles in the later stages of colorectal carcinogenesis, APC mutationand the less common ÃŸ-cateninmutations are essential events in the

initiation of neoplasia (reviewed in Ref. 24). If this concept of agatekeeper pathway holds true for the initiation of neoplasia in general, then the identification of other gatekeeper genes can be anticipated.

Unlike gatekeeper genes, caretaker genes, generally maintaingenomic stability and are not involved directly in the initiation of theneoplastic process. Genetic instability due to mutations in caretakergenes enhances the probability of mutation in other genes, includingthose in the gatekeeper pathway. Mismatch DNA repair genes, e.g.,MSH2 and MLH1, are caretaker genes, and abnormalities in thesegenes enhance genomic instability and increase the risk of humancolon cancer. Animal models based on this knowledge of humancolon carcinogenesis have been developed (28-30). Similar in vivo as

well as in vitro models should be developed for other tissue sites ofcarcinogenesis. Breast cancer susceptibility genes BRCA1 and BRCA2have been recently included in the list of caretaker genes (14). In thesame report, it is suggested that a predisposed individual with aninherited mutated alÃeleof a caretaker gene is at a lower risk of cancerwhen compared with an individual with a mutated gatekeeper alÃele.This difference has been attributed to the finding that three or moreadditional somatic mutations are required to initiate neoplasia in thecaretaker pathway, whereas only one additional somatic mutation isrequired to initiate neoplasia in the gatekeeper pathway. In addition tocancer as an endpoint, the molecular analysis of gatekeeper andcaretaker genes in presumed preneoplastic morphological lesionscould provide short-term and less expensive pathobiological end-

points for hazard identification and molecular epidemiological studies.

Mutator Concept

Multiple mutations are found in cancer cells. The presence of amutator phenotype has been suggested as an important step in tumordevelopment and forms the basis of the mutator hypothesis (Ref. 31,reviewed in Ref. 32). MIS2 arises from either deletions or insertions

in short repetitive sequences in the human genome known as micro-

satellites, and it is used as a measure of genetic instability. Theoccurrence of MIS was initially reported in colorectal cancer, partic-

2 The abbreviations used are: MIS, microsatellite instability; HNPCC, hereditary

nonpolyposis colorectal cancer; BP, benzo(a)pyrene; BPDE. BP-7,8-diol 9,10-epoxide.

ularly HNPCC (33-35). The presence of MIS as a biomarker of a

mutator phenotype has been linked to the defect in mismatch repairgenes, e.g., HMSH2, hMLHl, hPMSl, and HPMS2 (36-39). Tissue ofMSH2-null mice shows hypermutability in a transgenic lacl-reportersystem on exposure to /V-methyl-Ar-nitrosourea (40). A proportion of

HNPCC with MIS does not show a mutation in mismatch repairgenes, which indicates the possible involvement of other mutatorgenes. In a recent study of five Japanese HNPCC families without agermline mutation in MSH2 or MLH1, one family showed MIS and agermline mutation in MSH6/GTP (41). However, tumors in mice withnull mutations in MSH6 do not show MIS (42), which indicatespossible subtle differences in DNA mismatch repair among animalspecies.

Candidate mutator genes are involved in multiple cellular functionsthat are important for maintaining genetic instability, such as DNArepair, DNA replication, chromosomal segregation, cell cycle regulation, and apoptosis. One example is the p53 tumor suppressor gene.Cells lacking expression of wild-type p53 displayed a high frequencyof gene amplification, but the expression of wild-type p53 in Li-

Fraumeni syndrome cells with mutant p53 reduced the frequency ofPALA (a uridine biosynthesis inhibitor)-selected gene amplification

(43, 44). In murine cells, p53 mutations lead to genomic instabilityand the amplification of drug-resistant genes (43). The role of p53 in

DNA repair is less certain. A number of studies have shown p53modulation of nucleotide excision repair in which the loss of p53functions leads to a decrease in DNA repair and thus an increase ingenomic instability (45-51). Suppression of spontaneous homologous

recombination is another suggested function of p53 that contributes tothe maintenance of genetic stability. Isogenic cell pairs from tumorcells or primary fibroblasts showed a 100-fold increase in the recom

bination rate when p53 is inactivated (52). Transfection of normalcells with either the dominant-negative p53ala143 variant expression

vector or the human papilloma virus E6 gene showed a 10-80-fold

higher spontaneous recombination rate (53). p53 is also involved inthe fidelity of centrosoma! duplication, which ensures balanced segregation of the chromosomes during cell division. Mouse embryofibroblasts lacking p53 protein produce multiple copies of centro-

somes (54), resulting in unequal segregation of the chromosomes andgenetic instability. Mutant p53 may increase the sensitivity of cells tomutagens. A 5-fold increase in the mutation frequency, as determinedby the appearance of hypoxanthine guanine phosphoribosyl trans-ferase-deficient (6-thioguanine-resistant) cells (induced by X-rays),was observed in human fibroblast SUSM-I/p53 cells containing amutant p53 gene (codon 273 Arg-His) when compared with theparental SUSM-I cells (55). Human lymphoblastoid cell lines TK6and WTK1 containing wild-type and mutant p53, respectively,showed different levels of (X-ray-induced) mutagenicity and apoptosis (56). A 28-fold increase in the spontaneous mutation frequency atthe heterozygous thymidine locus was observed in WTK1 cells (p53-

mutated) when compared with TK6 (normal) cells (57). TK6 cellswere also more sensitive at the tk locus to the mutagenic effects ofX-rays, ethylmethane sulfonate, methylmethane sulfonate, and mito-

mycin C. Mutant p53 induced an increase in the frequency of chromosomal breaks in dividing LIM12 cells (58). A study of the replication of the pZ402 shuttle vector containing the supF gene inLi-Fraumeni fibroblasts with mutated p53 alÃeleO showed a frequentdeletion of a portion of SV40 T antigen (58). A 20-fold increase in

silent mutations has been observed in the mutated p53 gene in humancancer, which indicates its hypermutability (59). These silent mutations were not found to be selected or represented as genetic polymorphisms in the normal population. However, the fact that thealteration in p53 has been described as a relatively late event incolorectal carcinogenesis argues against its candidacy as a mutator

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Laboratory Animal Studies

1Cancer Epidemiology

Molecular Epidemiology

_L_L

Molecular Dosimetry of CarcinogenExposure

â€” Carcinogen-macromolecular adducts

â€” Cytogenetic endpoints

â€” Mutational spectra and frequency

f

Internal Exposure Assessment

1Inherited Cancer Predisposition

â€” Genetic polymorphism of enzymes involved in

activation and detoxification of carcinogensâ€” Genetic polymorphisms in DMA repair genes, DMA

repair deficiency, and genomic instabilityâ€” Germline mutations or polymorphisms in tumor

suppressor genes

Host Susceptibility Assessment

_LHuman Cancer Risk Assessmentâ€¢Hazard Identificationâ€¢Dose Response Assessmentâ€¢Exposure Assessmentâ€¢Risk Characterization

_LBioethical Issuesâ€¢Autonomyâ€¢Privacyâ€¢Justiceâ€¢Equity

QualitySensitivitySpecificityEffectivenessLimit genetic testing to conditionsthat are correctable by successfulintervention

Interventionâ€¢Reduce carcinogen exposureâ€¢Increase medical surveillanceâ€¢Therapeutic strategies including chemopreventionâ€¢Formulation of health policy

Fig. 2. Human cancer risk assessment and bioethical issues associated with molecular epidemiology and human cancer.

gene involved in the initiation of sporadic colorectal carcinoma (60).For example, there was a lack of any association between p53 statusand MIS or loss of heterozygosity in a recent study analyzing 58sporadic colorectal tumors (61). An analysis of 40 primary gastriccarcinomas showed a negative correlation between p53 mutation andMIS and indicates two distinct pathways for their contributions incancer development (62). In the same study, a positive associationwas found between MIS and mutation in the TGFÃŸ-RIIgene. Treatment of wild-type and p53-null mouse embryo fibroblasts with 4-nitroquinoline 1-oxide did not show any difference in accumulation of

point mutations in the lad target gene (63). Also, in the same study,DNA from thymus and thy mie tumors in both p53+/+ and p53â€”/â€”

mice did not show any distinct difference in the mutation frequency inthe target gene. However, the spontaneous mutation frequency of thelad gene was high, i.e., 10~5, so that the modulating effects of p53,

if any, might not be detected. In addition, the rate of nucleotideexcision repair is substantially lower in rodent-derived cells than it isin human cells (64, 65), and the repair of UV damage in the nontran-

scribed strand of the active gene is markedly reduced in rodents whencompared with that of humans (66).

Mutational Spectra of Tumor Suppressor Genes

Several endogenous and exogenous mutagens have been describedas inducing a characteristic pattern of DNA alteration. Mutationalspectra analyses are required to study the type, location, and frequency of DNA changes. Alterations of cancer-related genes found in

tumors not only represent the interaction of a carcinogen with DNAand cellular DNA repair processes but also reflect the selection ofthose mutations that provide premalignant and malignant cells with aclonal growth and survival advantage. Study of the frequency, timing,

and mutational spectra of p53 and other cancer-related genes provides

insight into the etiology and molecular pathogenesis of cancer andgenerates hypotheses for future investigations. These include questions regarding carcinogen-DNA interactions, the function of the

affected gene products, the mechanism of carcinogenesis in specificorgans or tissues, and general cell biological processes such as DNAreplication and repair.

Nonsense mutations, deletions, and insertions are the most frequenttypes of mutations in tumor suppressor genes that produce either anabsentee or a truncated protein product. These mutations are clearlyloss-of-function mutations. In contrast, the p53 tumor suppressor gene

shows an unusual spectrum of mutations when compared with othersuppressor genes, e.g., APC, BRCA1. or ATM, in which the mutationslead to a loss of function (Fig. 4). Missense mutations, in which theencoded protein contains amino acid substitutions, are commonlyfound in the p53 tumor suppressor gene. p53 missense mutations cancause both a loss of tumor suppressor function and a gain of onco-

genic function by changing the repertoire of genes whose expressionis controlled by this transcription factor (67-69). This functional

duality may be one explanation for the high frequency of p53 mutations in human cancer.

p53

The p53 gene is well suited for mutational spectrum analysis forseveral reasons: (a) p53 mutations are common in many humancancers, and a sizeable database of more than 8000 entries hasaccrued, so that the analysis of this large database can yield statistically valid conclusions (70, 71) and can be readily accessed on theWorld Wide Web (http://www.iarc.fr/p53/homepage.html); (b) themodest size of the p53 gene ( 11 exons, 393 amino acids) permits the

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Table 1 Examples of tumor suppressor genes involved in human cancers"

Tumorsuppressorgenep53RBIAPCATMChromosomallocus17pl3.113ql45q21Ilq22Location/proposedfunctionNuclear/transcription

factorNuclear/transcription

modifierCytoplasmic/signal

transductionNuclear/kinase

Ser/Major

typesof somatic

mutationMissenseDeletion

andnonsenseDeletion

andnonsense

DeletionSomatic

mutationsExamples

of neoplasms withsomaticmutationsMost

human cancer typesexamined todateRetinoblastoma,

osteosarcoma,and carcinomas ofthebreast,

prostate, bladder,andlungCarcinoma

of the colon,stomach, andpancreasLeukemiaInherited

mutationsSyndromeLi-FraumeniRetinoblastomaFamilialade

nornalous,polyposis

Ataxia telangiectasiaHÃ©tÃ©rozygotecarrier

rate/IO5births'7~2~2-10

2Typical

neoplasmsCarcinomas

of the breast andadrenal cortex,sarcomas.leukemia,

and braintumorsRetinoblastoma,and

osteosarcomaCarcinomas

of the colon,thyroid, andstomachLeukemia

and lymphoma

WTI

BRCA1

BRCA2

NF1

NF2

pl6INK"

VHL

null p13 Nuclear/transcription

factor17q21 Nuclear/DNA repair

13q12-13 Nuclear/DNA repair

17qll

22q

9p21

3p25

Cytoplasmic/GTPaseactivating protein

Cytoplasmic/cytoskeletal-membrane linkage

Nuclear/cyclin-dependent kinaseinhibitor

Nuclear/adaptor

Missense

Deletionandnonsense

Deletionandnonsense

Deletion

Deletionandnonsense

Deletionandnonsense

Deletion

Wilms' tumor

Breast

Breast

Schwannomas

Schwannomas andmeningiomas

Mesothelioma, pancreasmelanoma, and glioblastoma

Unknown

Wilms' tumor

Familialbreast/ovarian

Familialbreast/ovarian

Neurofibromatosistype 1

Neurofibromatosistype 2

Familial melanoma

von Hippel-Lmdau

-0.5-1

-2-10-1

Wilms' tumor

Carcinoma of the breast andovary

â€”2-10 Carcinoma of the breast andovary

â€”30 Neural tumors

~3 Central Schwannomas andmeningiomas

? Melanoma

-3 Hemangioblastoma and renalcell carcinoma

Â°Reviewed in Ref. 201 (see also Refs. 202-204).

Ref. 205 and A. Knudson, personal communication.

study of the entire coding region, and it is highly conserved invertebrates, allowing the extrapolation of data from animal models(72); and (c) the point mutations that alter p53 function are distributedover a large region of the molecule, especially in the hydrophobicmidportion (Refs. 18, 19, and 73; Fig. 5). These numerous basesubstitutions alter p53 conformation and sequence-specific transacti-

vation activity; thus, correlations between distinct mutants and functional changes are possible. Frameshift and nonsense mutations thattruncate the protein are located outside of these regions (Fig. 5), anddeletions and insertions are most commonly found in exons 2-4 and9-11 (74), so evaluation of the entire DNA sequence yields relevant

data. This situation differs from that of the raÃoncogenes, whosetransforming mutations occur primarily in three codons, a few sequence-specific motifs, and a critical functional domain (75). The

diversity of p53 mutational events permits more extensive inferencesof the mechanism of DNA damage involved.

Exogenous mutagenic agents and/or endogenous mutagenic

HIGH GERMLINE MUTATIONSLOW(103-to104

i-fold)CANCER

RISK(2-10-fold),

Rb (Retinoblastoma)p53 (Li-Fraumeni)APC (Familial Polyposis Coll)NF1 (Neurofibromatosis)XPA-G (XerodermaPigmentosum)ATM

(Ataxia Telangiectasia)BRCA1 (Breast-Ovarian Ca)HMLH1 (Hereditary Nonpolyposis

ColorectalCa)CYP1A1"1

CYP2E1 \.CarcinogenNATi Metabolism ,GSTM1 J '(1-5

per 10slivebirths)FREQUENCY

OF AT RISKALLELE(2-50

perlO2livebirths)MODERATE

HIGH

Fig. 3. Examples of the inverse correlation between allelic frequency and cancer riskassociated with inherited cancer susceptibility genes.

mechanisms have been implicated in the induction of mutations.These mutations are archived in the spectrum of p53 mutationsfound in human cancer (18, 19, 73, 76-78). Errors introduced

during DNA replication, RNA splicing, DNA repair, and DNAdeamination are examples of endogenous mutagenic mechanisms.The DNA sequence context is an important factor that determinesthese events. Almost all short deletions and insertions occur atmonotonie runs of two or more identical bases or at repeats of2-8-bp DNA motifs, either in tandem or separated by a short

intervening sequence (74). The mechanism that has been studiedmost is called slipped mispairing, a misalignment of the templateDNA strands during replication that leads to either deletion, if thenucleotides excluded from pairing are on the template strand, orinsertion, if the nucleotides excluded are on the primer strand (79).When direct repeat sequences mispair with a complementary motifnearby, the intervening oligonucleotide sequence may form a loopbetween the two repeat motifs and be deleted (80, 81). Morelengthy runs and sequence repeats are more likely to generateframeshift mutations. The deletions and insertions in the p53 genefound in human tumors also may be biologically selected from thebroad array of such mutations occurring in human cells. Whencompared with the distribution of missense mutations, these typesof mutations occur more frequently in exons 2-4 (54%) and 9-11(77%) than in exons 5-8 (20%; Fig. 5). The NH2 terminus of thep53 protein (encoded by exons 2-4; reviewed in Refs. 82-85) has

an abundance of acidic amino acids that are involved in thetranscriptional function of p53 (86, 87) and binds to transcriptionfactors such as TATA-binding protein in transcription factor IID(82, 88-91). Experimental studies have shown that multiple point

mutations in this domain are required to inactivate its transcriptional transactivation function (92). The COOH terminus (encodedby exons 9-11) of the p53 protein is enriched in basic amino acids

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Fig. 4. Class of mutations in p53, APC, BRCAI,and ATM genes in all human cancers. Missensemutations represent a high proportion of p53 mutations, whereas nonmissense mutations (e.g., frame-

shift, nonsense, and splice site mutations) are common in other tumor suppressor genes. Deletions andinsertions that do not induce frameshifts have beenreferred to as in frame deletions.

p53 (n=6,604)... Nonsense

Frameshift 11% 7o/Splice site 4%

In FrameDel/Ins 2%Silent 2%

Missense74%

ATM (n=130)

Frameshift77%

ARC (n=352)

Frameshift 41%

Splice site 6%

Nonsense24%

Missense16%

Silent 13%

Nonsense15%

Missense6%

In FrameDel/Ins 2%

Frameshift64%

BRCA-1 (n=580)

Nonsense 5%

Missense29%

Splice site2%

that are important in: (a) the oligomerization and nuclear localization ofthe p53 protein (reviewed in Refs. 93-96); (b) the recognition of DNA

damage (97, 98); (c) the negative regulation of p53 binding to promotersequences; (d) the transcription of p53-transactivated genes (99); and (e)

the induction of apoptosis (100). Laboratory studies have shown that at

least two point mutations in the NH2 terminus of p53 are required toinhibit its transcriptional transactivity (92); therefore, deletions and insertions are a more detrimental mutagenic mechanism than single pointmutations for disrupting these NH2-terminal and COOH-terminal func

tional domains.

Transactivation I-Domain

Mutations andSequence Specific

DNA Binding Domain

Tetramerization,Nuclear Localizationand DNA-DamageRecognition Domain

17-29 .97 EVOLUTIONARILY CONSERVED 292, 324 â€” 352

Frameshift27%

Exons 2-4 Exons 5-8Nonsense Frameshift 10% Nonsense

9% Splice site 3%

Exons 9-11

Splice site15%

Silent 8%

Silent 3%Missense

41%

Frameshift23%

n=236

Splice site \'Missense 15%

78% Silent 7%^n=6177 n=129

Nonsense27%

Missense28%

Fig. 5. Schematic of the p53 molecule. The p53 protein consists of 393 amino acids with functional domains, evolutionary conserved domains, and regions designated as mutationalhotspots. Functional domains include the transactivation region (amino acids 20-42. ^), the sequence-specific DNA-binding region (amino acids 100-293), the nuclear localizationsequence (amino acids 316-325, 01), and the oligomerization region (amino acids 319-360, B). Cellular or oncoviral proteins bind to specific areas of the p53 protein. Evolutionaryconserved domains (amino acids 17-29,97-292, and 324-352; black areas) were determined using the Multiple Alignment Construction and Analysis Workbench (MACAW) program.Seven mutational hotspot regions within the large conserved domain are identified [amino acids 130-142, 151-164, 171-181, 193-200, 213-223, 234-258, and 270-286 (checkered

blocks)}. Vertical lines above the schematic, missense mutations.

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Structure-Function Relationship of p53

The p53 mutation spectrum can also provide clues to the criticalfunctional regions of the gene that, when mutated, contribute to thecarcinogenic process. Because about 75% of the missense mutationsare in the sequence-specific DNA-binding midregion of the protein

(Refs. 18, 19, and 73; Fig. 5), investigators have focused on thetranscription transactivator function of p53. However, these missensemutations and the resultant amino acid substitutions can cause aberrant protein conformations (101) that may alter other functional domains, including those in the COOH terminus of the p53 protein. Thispositively charged region contains the putative major nuclear localization signal (amino acids 316-325), the oligomerization domain(amino acids 319-360), and a DNA damage binding domain (aminoacids 318-393; Refs. 102-105). p53 sequence-specific DNA bindingand transcriptional transactivation can also be modulated by posttrans-

lational mechanisms, including serine phosphorylation (103, 106) andthe redox regulation of the cysteine residues responsible for bindingzinc to p53 (107-109). Recently, it has been demonstrated that acety-lation of the p53 COOH-terminal domain at amino acids 373 and 382stimulates its sequence-specific DNA-binding activity (110). Furthermore, in another recent study, O-glycosylation of the p53 COOHterminus has been shown to activate DNA binding (111). In ML-1cells exposed to Adriamycin or cisplatin, a COOH-terminal cleavage

product of endogenous p53 has been shown to correlate with theup-regulation of p21 (112). The function-structure relationship re

vealed by the analysis of the p53 mutation spectrum (18, 73), itsnuclear magnetic resonance and crystallographic three-dimensionalstructure (93, 94, 113), and functional studies of wild-type versus

mutant p53 activity (reviewed in Ref. 83) have generated both hypotheses for further study and strategies for the development ofrational cancer therapy.

Missense mutations in the p53 gene domains encoding loop 2 or 3have been associated with more aggressive breast cancer (114) thatwas less responsive to chemotherapy with doxorubicin (115). Unlikemutations affecting interaction at the interface between the p53 protein and its consensus sequences in DNA, loop 2 or 3 mutants are lesslikely to be temperature sensitive. Additional studies are warranted todetermine the importance of somatic p53 mutation in specific structural and functional domains and the success of cancer therapy. Thestructural and functional consequences of specific germline p53 mutations in Li-Fraumeni syndrome families should also be investigated.

Mutation spectra studies of DNA repair genes such as XPD haveshown dramatic gene phenotype effects (116). Germline mutations inXPD can exhibit either a phenotype of trichodystrophy without anincreased risk of cancer or xeroderma pigmentosum with a > 1000increased risk of sunlight-induced skin cancer. Therefore, similar genephenotype effects are likely to be found in Li-Fraumeni families.

DNA Methylation and Mutational Hot Spots

Methylated CpG sites of p53 harbor a strikingly high proportion ofmutations that constitute major hotspots in human cancer (reviewed inRef. 73). Deamination of 5-methylcytosine at CpG sites is considered

to be one of the major endogenous mechanisms for the induction ofthese mutations (117). Deamination of 5-methylcytosine forms thy-midine and generates a G-T mismatch which, if not repaired, produces

a C to T transition. Deamination of cytosine can also generate a C toT transition if uracil glycosylase and a G-T mismatch repair are

inefficient. The presence of a high frequency of C to T transitions atCpG dinucleotides in colon carcinomas (18) suggests the involvementof an endogenous deamination mechanism. However, a considerablevariation also has been reported in mutational type at CpG sitesamong different types of cancer. Compared to colon carcinoma, low

frequencies of transition mutations are observed in liver and lungcancer at CpG dinucleotide sites, and, in contrast, high frequencies ofG to T transversions are reported in these cancer sites (73). A distinctpattern of mutation at CpG sites in different tissue types indicateseither the possible involvement of exogenous agents or the selectionof p53 mutants that vary among tissues in their pathobiologicalactivities (118).

Recently, a role for cytosine methylation at CpG sites has beensuggested in determining the preferential site of DNA damage byexogenous carcinogen. Interestingly, treatment of HeLa cells andbronchial epithelial cells with BPDE selectively induced guanineadduci formation at CpG sites inp53 codons 157, 248, and 273 (119).In contrast to nonmethylated cloned p53 gene sequences containingnonmethylated cytosines, treatment of methylated sequences withBPDE preferentially produced BPDE-guanine adducts at CpG sites

(120). In the same study, a similar trend also is observed in the PGK1gene, with different methylation status, derived from an active orinactive X chromosome. BPDE, aflatoxin B,, 8,9-epoxide, /V-acetoxy-2-acetylaminofluorene, and benzo(g)chrysene diol epoxide, which

interact with different moieties within guanine, showed more bindingat methylated CpG sites than at nonmethylated CpG sites in the hot-

spot codons of p53 (121). The precise role by which methylationresults in the preferential interaction of BPDE is not clear; however,its influence in the creation of an intercalation site has been suggested(120). Rates of DNA repair may vary among p53 exons (122).

DNA Strand Bias of p53 Mutation

The transcribed strand of an active gene is usually repaired at amore rapid rate than the nontranscribed (DNA coding) strand (123).For example, DNA damage on the transcribed strand of the p53 geneis repaired faster compared with that of the nontranscribing or codingstrand (124, 125). This preferential DNA repair is suggested as animportant factor in determining the mutation pattern leading to strandbias (123, 126). The positive correlation between cigarette smokingand G:C to T:A transversions on the nontranscribed strand of p53 inlung cancer (127) supports the above-mentioned hypothesis. Labora

tory studies have also shown that the mutations induced by exogenouschemical carcinogens occur preferentially on the nontranscribed coding strand of p53 (128-130). C to T transitions at CpG sites are a

common mutation produced by endogenous mechanisms such asdeamination of 5-methylcytosine (117, 131, 132). It can be hypothe

sized that C:G to T:A transitions arising from the deamination of5-methylcytosine at CpG dinucleotide sites in the p53 gene would not

display a DNA strand bias, whereas C:G to T:A transitions arisingfrom bulky chemical DNA adducts would occur primarily on thenontranscribed strand, due to the efficient repair of the transcribedstrand. Alternatively, the reported association between transcription-

coupled repair and mismatch repair (133) implies a possible strandbias for mismatch repair. Analysis of the p53 mutation database (70)for C to T transitions at CpG sites at mutational hotspot codons on thenontranscribed strand showed either bias for both transcribed andnontranscribed strands or no bias, depending on the specific codonand cancer type (Table 2). Although the total number of mutations issmall, mutations at codon 175 (nucleotide residue 13,202) show asubstantial bias for the transcribed strand in breast, colon, esophageal,head and neck, and lung cancers. In contrast, mutations at codon 282(nucleotide residue 14,513) show a strong bias for the nontranscribedstrand in brain, breast, colon, esophageal, head and neck, and lungcancers. Mutations at codon 248 (nucleotide residue 14,068) show nostrand bias in breast, colon, and head and neck cancers, whereasesophageal and lung cancers show a bias for the nontranscribedstrand. Mutations at codon 248 (nucleotide residue 14,068) only show

4028




Table 2 DNA strand bias at somatic and germline mutation hotspots in the p53 tumor suppressor gene

AB.Cancer

type.

Somaticmutation:BrainBreastColonEsophagealHead

andneckLiverLung%

codon157%

codonn175C

to T at CpG sites(nontranscribedNANANANANANANA<5<5<5<5<5<5<5strand)0NA0300NA0n<521732713<5S%

codon24832424586423864n2833101726814%

codon27324654336179248n17606214121221%codon28288100100100100NA100n784086<513G

lo T at CpG siles (nonlranscribedstrand)0BrainBreastColonEsophagealHead

andneckLiverLungC.

GermlinemutationGermlineNA100NANA100100100:C to T atCpGNA<55<5<571123NANA80NANANANA<5<55<5<5<5<5NANANA100NANA100<5<5<56<5<512NANANANA1008388<5<5<5<57524NANANANANANANA<5<5<5<5<5<5<5sites(nontranscribedstrand)"'6<5NA<5205164369

" The number of mutations less than five (<5) was not assessed (NA; Ref. 50).b The germline data do not include multiple mutations from the same family.

a bias for the nontranscribed strand in brain and liver cancers. Mutations at codon 273 (nucleotide residue 2,896) show a transcribedstrand bias in brain, esophageal, and head and neck cancers but showa nontranscribed strand bias in breast and liver cancers. No strand biaswas observed at this codon in colon and lung cancers. One possibilityof the occurrence of strand bias for C to T transitions at CpG sitescould be due to the different rate or the fidelity of G-T mismatch

repair, depending on either the surrounding DNA sequences of themutational hotspot sites or differences in the etiological agents amongcancer types. These findings are consistent with the hypothesis thatchemical carcinogens found in the environment, diet, or tobaccosmoke are responsible for this subset of p53 mutations.

The frequency of G to T transversions at CpG sites is found to berelatively low and exhibits a strong bias for the nontranscribed strandat a number of mutational hotspots. All G to T transversions at CpGsites at codon 157 (nucleotide residue 13,147) occur on the nontranscribed strand in breast, head and neck, liver, and lung cancers.Similarly, at codon 248 (nucleotide residue 14,069), a strong bias forthe nontranscribed strand exists in esophageal and lung cancers. G toT transversions at codon 175 (nucleotide residue 13,203) in coloncancer and codon 282 (nucleotide residue 14,514) in lung, liver, andhead and neck cancers also show a substantial bias for the nontran

scribed strand. The presence of strong bias for the nontranscribedstrand in G to T trans versions at CpG sites of hotspot codons suggeststhe involvement of exogenous chemical carcinogens that form bulkyDNA adducts, rather than the endogenous mechanism such as thedeamination of 5-methylcytosine.

Interestingly, analysis of germline C to T transitions at CpG sites ofhotspot codons in Li-Fraumeni families shows a considerable bias for

the transcribed strand (Table 2). G to T germline mutations at CpGsites are not reported in these families.

pl6'NK4

pl6'NK4, which encodes a cell cycle-inhibitory protein, has been

found to be commonly mutated in a variety of human cancers (134-140). Epigenetic control of pl6INK4 expression by DNA methylation

may also inactivate this cancer susceptibility gene (141, 142). Germ-line mutations in p!6INK4 have been associated with familial cancers

including melanoma, pancreatic cancer, and head and neck cancer(143). Because of the common occurrence of germline mutations inmelanoma families, pl6'NK4 has been considered a melanoma suscep

tibility gene; however, half of the chromosome 9p-linked melanomafamilies do not show pl6'NK4 germline mutations. In vitro studies

Germline(n=19)

Somatic(n=96)

Del. + ins.5%

G:C->C:G

A:T->T:A16%

CC:GG->TT:AA 4%

Del. + ins33%

G:C->A:Tat Non-CpG

26%

G:C->C:G16%

G:C->T:A21%

A:T->C:G 2%G:C->A:T A:T->G:C 3%at CpG 5% A:T->T:A 2%

G:C->T:A23%

Polymorphism(n=37)

G:C->C:GA:T->G:C 8% 3% Q:C->T:AA:T->T:A

3%

G:C->A:Tat Non-CpG

27%

G:C->A:Tat CpG 21%

G:C->A:Tat Non-CpG 11%

G:C->A:Tat CpG

48%

Fig. 6. Mutation spectra of pI6INK4 in human cancers. Germline and somatic pl6INK4 mutations show distinct patterns. Deletions and insertions constitute 33% of the somatic

mutations, whereas only 5% of the germline mutations represent this class of mutation.

4029




Fig. 7. Germline mutational spectra in BRCA1and BRCA2. Deletions and insertions represent themajor types of mutations in both BRCA1 andBRCA2. Among the less common missense mutations, G:C to A:T and A:T to G:C transitions represent 17 and 12%, respectively, of the total mutations in BRCA1. G:C to A:T and A:T to G:Ctransitions represent only 6 and 5%, respectively, ofthe total mutations in BRCA2.

BRCA1 (n=889)G:C->C:G

Del. + ins59%

G:C->T:A 6%G:C->A:T

at CpG 5%G:C->A:T at

non-CpG 12%A:T->T:A 1%

A:T->G:C 12%

A:T->C:G 4%

BRCA2 (n=168)G:C->C:G 2%

Del. + ins80%

G:C->T:A 3%G:C->A:TatCpG4%

G:C->A:Tatnon-CpG 2%A:T->T:A 2%A:T->G:C 5%

A:T->C:G 2%

have shown that germline melanoma-predisposing mutants are unableto inhibit the catalytic activity of cyclin D1/CDK4 and cyclin Dl/CDK6 complexes (144). The absence of pl6'NK4 alteration in a largeportion of the melanoma-prone families linked to chromosome 9p21supports the possible existence of additional melanoma susceptibilitygenes. There are some interesting distinctions between the germlineand somatic pl6'NK4 mutational spectra (Fig. 6). Deletions and insertions, which constitute 33% of the somatic pl6INK4 mutation, are

represented by only 5% of germline mutations. CC to TT tandemdouble mutations, which are characteristically induced by UV radiation, constitute 4% of somatic p!6INK4mutations but are never found

as a germline mutation. Although several germline polymorphismsalso have been reported for pl6'NK4, their functional and prognostic

significance is not yet known.

BRCA1 and BRCA2

BRCA1 on chromosome 17ql2-21 and BRCA2 on chromosome13ql2-13 have been recently identified as cancer susceptibility genesthat are involved in the familial predisposition to breast cancer (145-151). In addition to breast cancer, germline mutations in BRCA1 alsopredispose individuals to ovarian cancer (152-154), whereas BRCA2

mutations are implicated in male breast and pancreatic cancers (155-157). Mutations in these genes are considered to be responsible forabout 80% of familial breast cancer cases. In contrast to other cancersusceptibility genes, somatic mutations in BRCA1 or BRCA2 areinfrequent in breast and ovarian cancers (158-162). Mutation spectrastudies of BRCA1 and BRCA2 have revealed that the majority of themutations lead to a loss of function and include deletions and insertions leading to frameshift and nonsense mutations. Deletion andinsertion mutations constitute about 60% of all mutations in BRCA1and 80% of all mutations in BRCA2(Fig. 7). However, a considerablenumber of missense mutations have been reported previously (163).The major missense mutations reported in BRCA1 are G:C to A:Ttransitions (17%, with 12% at non-CpG sites) and A:T to G:C transitions (12%), and in BRCA2,only 6% of the total mutations are G:Cto A:T transversions (with 2% at non-CpG sites), and 5% are A:T toG:C transitions. Although there are 100 different mutations reportedthroughout the BRCA1gene, two mutations, i.e., 185delAG and 5382insC, occur with considerably high frequencies and constitute about20% of the total mutations reported so far. Interestingly, one of thesemutations, 185delAG, has an ethnic commonality and segregates withearly-onset breast cancers among 20% of Ashkenazi Jews (164). In a

CODONI 57

Colon19%

CODON 175 CODON 179

Head &Neck 11%

Lung 7%Liver 2HeadiNeck5%

CODON 248

Colon17%

Head &Neck 27%

Breast20%

(Ã®olon66%

CODON 249

Skin Breast3% 6% Colon

3%Head&

Neck5%

Liver69%

Breast19%

Colon15%

Head&Neck

Liver 5%4%

CODON 278

Breast

Skin59%

Colon5%Head&

Neck16%

Liver2%

Lung9%

Fig. 8. p53 mutational hotspots in human cancers. Most types of human cancers show the domination of specific p53 mutations at particular mutational hotspots. The characteristicpatterns hypothesize molecular linkage between a particular cancer and a specific exogenous or endogenous carcinogen.

4030




LUNG (64%)

BREAST (15%)

HEAD & NECK (20%)

CodonsFig. 9. p53 mutational hotspots of G to T Iransversions in human breast, head and neck, and lung cancers. Codon 157 is one of the mut alumal hotspots in lung cancer. A transversion

of G to T at codon 157 in lung cancer is found more frequently among smokers than never-smokers and is a candidate early marker for identifying individuals at higher cancer risk.

recent study involving 5318 Ashkenazi Jews, the 185delAG and5382insC mutations in BRCA1 and the 6174delT mutation in BRCA2were analyzed. Among 120 carriers oÃBRCA1 and BRCA2 mutations,the estimated risk at the age of 70 years was reported to be 56% forbreast cancer and 16% for both ovarian cancer and prostate cancer(165). Similar to BRCA1, a large percentage of known BRCA2 germ-

line mutations (33%) is represented by two deletion mutations,6174delT and 997del5. The 6174delT is found in about 8% of early-

onset breast cancer cases in the Ashkenazi Jewish population (166),whereas 997del5 has been commonly reported in early-onset familial

breast cancer cases from Iceland (153). Another interesting observation is the apparent occurrence of genotype-phenotype correlation

with respect to a specific BRCA1 or BRCA2 mutation and the risk ofbreast and ovarian cancers. The presence of a truncating mutation inthe first two-thirds of the BRCA1 gene instead of in the last third of the

gene may significantly increase the risk of ovarian cancer in comparison with that of breast cancer (153). Likewise, the analysis of 25families with multiple cases of breast and ovarian cancers withBRCA2 mutations also showed a significant genotype-phenotype cor

relation. Mutations leading to the truncation of BRCA2 in familieswith the highest risk of ovarian cancer were all found to be clusteredin a region of approximately 3.3 kb in exon 11 (154).

Although the normal functions of BRCA1 and BRCA2 are notknown, recent studies are providing interesting clues. For example,the transcriptional activation function of BRCAl and BRCA2 has beenreported, and the COOH-terminal region of BRCAl (amino acids1528-1863) showed significant transcriptional activation when fusedto the GAL4 DNA-binding domain; this transactivation function waslost when a mutation was introduced in the COOH-terminal region

(167). BRCA2 exon 3 has been found to have a sequence homologyfor the activation domain of c-Jun and showed transcriptional activation in yeast when linked to the lex-A DNA-binding domain as well

as in two different mammalian cell lines, U20S and NMuMG, whenlinked to the GAL4 DNA-binding domain (168). BRCAl and BRCA2

have been suggested to play a role in embryonic cellular proliferationand development (169, 170). Mutations in BRCAl or BRCA2 causelethality at different stages of development in mouse embryos. Interestingly, BACA5"6 mutants (deletion of the fifth and sixth exons) with

a homozygous null p53 or p21 background showed an enhancedsurvival of embryos (171), suggesting a functional interaction between BRCAl, p53, and p21. An increased expression of p21 hasbeen observed in the BRCA5"6 mutants (169). Furthermore, the

formation of a complex between BRCA1/BRCA2 and Rad 51 andhypersensitivity of BRCA2 mutant mouse embryos to y radiation hasindicated a role for BRCAl and BRCA2 genes in DNA repair pathways (170, 172, 173). These functions of BRCA] and BRCA2, whichhave been suggested to play a role in maintaining genomic stability,give BRCAl and BRCA2 an important place in the cancer susceptibility gene family. The finding of inherited mutations in the BRCAland BRCA2 genes in families with early-onset breast and ovarian

cancers raises the significance of predictive testing or early diagnosiswhile prompting the debate of several technical and bioethical concerns (reviewed in Ref. 151).

Considering the variety of hereditary cancers and alleile deletions,one can predict the discovery of additional tumor suppressor genes,some of which may have a conspicuous role in carcinogenesis. Thefrequency of these cancer susceptibility genes and their attributablecancer risk are important considerations in the development of a

4031




Table 3 Assessment of causation by the Bradford-Hill criteria

Hypothesis: Dietary AFB," exposure can cause 249"' (AGG â€”>ACT) p53 mutations during human liver carcinogenesis

Strength of associationâ€¢Consistency

â€¢Positive dose-response correlation between estimated dietary AFB!exposure and the frequency of 249*" p53 mutations in three different

ethnic populations on three continents (179, 180, 206)â€¢Specificity

â€¢249s*' p53 mutant cells are observed in nontumorous liver in high HCC

incidence geographic areas (177)

Biological plausibilityâ€¢AFB ! is a potent mutagen and carcinogen in laboratory studies (207-209)â€¢AFB, is enzymatically activated by human hepatocytes (210, 211), and the

8,9-AFB, oxide binds to the third base (G) in codon 249 (212)â€¢AFB, exposure to human liver cells (178, 184) in vitro produces codon 249"'

p53 mutationsâ€¢249s" p53 expression inhibits apoptosis (213) and p53-mcdialcd transcription

(118) and enhances liver cell growth in vitro (214)'AFB,, aflatoxin B,.' HCC, hepatocellular carcinoma.

public health policy for genetic screening of the general population.Different public health and bioethical considerations apply to thegenetic screening of family members of individuals carrying a highcancer risk alÃelein their germline (16).

Molecular Linkage between Carcinogen Exposure and Cancer

A number of specific p53 mutational hotspots have been recognizedin different types of human cancer (Fig. 8). The occurrence of anidentical mutation that is experimentally induced by a carcinogensupports a causative role of a specific environmental carcinogen incertain tumor types. Molecular linkage between exposure to carcinogens and cancer is best exemplified by the p53 mutational spectra ofhepatocellular carcinoma, skin cancer, and lung cancer. The mostcommon techniques used for p53 mutation analysis include PCR-based assays like single-strand conformational polymorphism, dena

turing gradient get electrophoresis, and DNA sequencing. Recently,another method based on yeast functional assays was developed todetect p53 mutations (174-176). In this assay, loss of DNA binding

and transcriptional transactivation function in mutant p53 is detectedby the colony color of the yeast. Measuring p53 mutation load or thefrequency of mutated alÃeles in nontumorous tissue may indicateprevious carcinogen exposure and identify individuals at increasedcancer risk. However, the detection of rare cells with mutations in aproto-oncogene or tumor suppressor gene in normal-appearing human

tissue represents a challenging task. The average spontaneous mutation per base pair in human cells is estimated to be in the range of10~8 to 10~'Â°, and these frequencies increase only 10- to 1000-fold

on exposure to a mutagen. Therefore, methods that allow the detectionof few altered DNA sequences from IO5 to IO10 copies of the corre

sponding wild-type sequences in the presence of large quantities of

cellular DNA are required. The development of a highly sensitivegenotypic assay is allowing the detection of low-frequency mutationsin normal-appearing human tissues (177, 178). The detection of aparticular mutation in normal-appearing tissue provides further sup

port for the involvement of a specific carcinogen in a particular humancancer and may help identify individuals at increased cancer risk.

In liver tumors from persons living in geographic areas in whichaflatoxin B, and hepatitis B virus are cancer risk factors, most p53mutations are at the third nucleotide pair of codon 249 (179-182). A

dose-dependent relationship between dietary aflatoxin B, intake andcodon 249scr/>53 mutations is observed in hepatocellular carcinoma from

Asia, Africa, and North America (reviewed in Refs. 8 and 183). Themutation load of 249"" mutant cells in nontumorous liver also is posi

tively correlated with dietary aflatoxin B, exposure (177). Exposure ofaflatoxin B, to human liver cells in vitro produces 249"" (AGG to AGT)

p53 mutants (178, 184). These results indicate that expression of the249scr mutant p53 protein provides a specific growth and/or survival

advantage to liver cells (185) and are consistent with the hypothesis thatp53 mutations can occur early in liver carcinogenesis.

Sunlight exposure is a well-known risk factor for skin cancer.Tandem CC to TT transition mutations are frequently found in squa-

mous and basal cell skin carcinoma (186), whereas they are rarelyreported in other types of cancers (73). In vitro studies have shown theinduction of the characteristic CC to TT mutations by UV exposure(187-191). Sunlight-exposed normal skin and precancerous skin con

tain CC to TT tandem mutations (192,193). These results indicate thatCC to TT mutations induced by sunlight exposure may play a role inthe occurrence of skin cancer.

Cigarette smoking has been established as a major risk factor for theincidence of lung cancer. Codons 157,248, and 273 of \hep53 gene havebeen designated as mutational hotspots in lung cancer. The majority ofmutations found at these codons are G to T transversions. Furthermore, inaddition to lung cancer, codon 157 also constitutes one of the hotspots forG to T transversions in breast and head and neck cancers (Fig. 9). Insmoking-associated lung cancer, the occurrence of G to T transversions

has been linked to the presence of BP in cigarette smoke. Interestingly,codon 157 (GTC to TTC) mutations are not found in lung cancer fromnever-smokers (73, 194). A dose-dependent increase in p53 G to T

transversion mutations with cigarette smoking has been reported in lungcancer (127). Recently, it has been shown that BPDE, the metabolicallyactivated form of BP, binds to guanosine residues in codons 157,248, and273, which are mutational hotspots in lung cancer (119). Cigarette smokecondensate or BP also neoplastically transforms human bronchial epithelial cells in vitro (195).

Assessment of Causation by the Bradford-Hill Criteria

Results obtained from molecular epidemiological studies can beused for the assessment of causation. Using the "weight of the


Hypothesis: The chemical carcinogen BP in tobacco smoke can cause p53 hotspot mutations at codons 157, 248, and 273 in human lung carcinogenesis


â€¢Cigarette smoking is associated with a dose-response increase in p53mutations (G to T transversions) in human lung cancer (127)

â€¢Specificity

â€¢Codon 157 (GTC -> TTC) mutations are uncommon in other types of

cancer, including lung cancer in never smokers (73)â€¢Temporality

â€¢p53 mutations can be found in bronchial dysplasia (215-222)

Biological plausibilityâ€¢Tobacco smoke and BP are mutagens (223-225)â€¢BP is metabolically activated and forms BPDE DNA adducts in human

bronchus in vitro (75-fold interindividual variation; Refs. 226 and 227)â€¢BP diol epoxide binds to Gs in codons 157, 248, and 273, which are p53

mutationalâ€¢BP exposure to human cells in vitro produces codon 248 (COG â€”>CTG) p53

mutations (228)â€¢Cigarette smoke condensÃ¢tes or BP can neoplastically transform human

bronchial epithelial cells in the laboratory (195, 229)

4032





Hypothesis: Sunlight exposure can cause a characteristic CC to TT tandem double mutation in p53 in human skin cancer


â€¢Commonality of CC to TT tandem double mutation in squamous and basalcell skin carcinoma (186, 193, 230)

â€¢Specificityâ€¢Tandem dipyrimidine CC to TT mutations are uncommon in other types of

cancer(73)â€¢Temporality

â€¢CC to TT mutations are found in normal and precancerous skin (193, 231)

Biological plausibilityâ€¢UV is a mutagen and carcinogen in laboratory studies (232)â€¢Cylobulane pyrimidine dimers and pyrimidine photoproducts are the two major adducts

produced by UV-irradiation and induce C to T at non-CpG sites and CC to TTmutations (189, 191)

â€¢Skin cancer in xeroderma pigmentosum patients with defective nucleotide excisionrepair contains a high frequency of CC to TT mutations (233)

â€¢UV exposure produces CC to TT mutations in studies using phage, bacteria, and mice(187-190)

evidence" principle, Bradford-Hill (196) proposed criteria in the

assessment of cancer causation including strength of association(consistency, specificity, and temporality) and biological plausibility. Three examples of weight of the evidence consistent withthe hypotheses include the following: (a) dietary aflatoxin B,exposure that can cause codon 249ser (AGG to AGT) p53 muta

tions during human liver carcinogenesis (Table 3); (b) the chemicalcarcinogen BP in tobacco smoke that can cause p53 hotspot mutations in human lung carcinogenesis (Table 4); and (c) sunlightexposure that can cause a characteristic CC to TT double mutationin p53 in human skin cancer (Table 5).

Furthermore, the influence of geographic location, race, and genderneeds to be taken into account when determining an individual's

susceptibility or risk for cancer (13). The p53 mutation spectra inbreast cancer from Japanese women show a different pattern whencompared with that of European and American women (197, 198).Even among African-American and Caucasian-American women, adifferent p53 mutational pattern in breast cancer is observed (199).The difference in mutation spectra and cancer incidence within apopulation or race could be due to either the effect of a regionalenvironmental agent or the polymorphic variation in genes responsible for carcinogen metabolism and DNA repair.

Comprehensive databases of tumor suppressor genes with mutations can be used as an important tool in molecular epidemiology.Examples of databases available on the Internet include the following:(a) for p53, http://www.iarc.fr/p53/home page.html; (b) for germlinemutations in Li-Fraumeni syndrome, http:cuni.cz/win/projects/readme.htm (71, 200); (c) for BRCA1 and BRCA2, http://www.nhgri.nih.gov/Intramural research/Lab transfer/Bic/; and (d) for APC,http://perso.curie.fr/Thierry.Soussi/p53database.html. We have previously discussed the limitations and advantages of the methodologiesto detect mutations and the need for improved epidemiolÃ³gica!designand quality control (73). Further improvement of these databases byincluding information on geographical location, race, ethnicity, mutagen exposure, clinical course, genetic polymorphism, and so forthwill enhance the analyses of the mutation spectra of these genes.

Acknowledgments

We thank Mohammed Khan and Karman Raja for help with the mutationdatabase analysis and Dorothea Dudek for editorial and graphic assistance.

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1998;58:4023-4037. Cancer Res S. Perwez Hussain and Curtis C. Harris Mutation Spectra Studies of Tumor Suppressor GenesMolecular Epidemiology of Human Cancer: Contribution of

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