DNA Repair Gene XRCC 3 Codon 241 Polymorphism , Its Interaction with Smoking and XRCC 1 Polymorphisms , and Bladder Cancer Risk

DNA repair efficiency varies among individuals, with reduced repair capacity as a risk factor for various cancers. This variability could be partly explained by allelic variants for different DNA repair genes. We examined the role of a common polymorphism in the XRCC3 gene (codon 241: threonine to methionine change) and bladder cancer risk. This gene plays a role in the homologous recombination pathway, which repairs double-strand breaks. The functional consequences of the XRCC3 codon 241 polymorphism are still unknown. We hypothesized that this polymorphism could affect repair of smoking-associated DNA damage and could thereby affect bladder cancer risk. We genotyped 233 bladder cancer cases and 209 controls who had been frequency matched to cases on age, sex, and ethnicity. We observed little evidence of a positive association between subjects who carried at least one copy of the codon 241 Met allele and bladder cancer (odds ratio: 1.3; 95% confidence interval: 0.9–1.9). Among heavy smokers, individuals with the Met allele had about twice the risk of those without it; however, a test of interaction was not statistically significant (P 0.26). Previously, we observed in these subjects an association between bladder cancer risk and allelic variants of the XRCC1 gene, which is involved in the repair of base damage and single-strand breaks. In this study, we found some evidence for a genegene interaction between the XRCC1 codon 194 and XRCC3 codon 241 polymorphisms (P 0.09) and some support for a possible gene-gene-smoking three-way interaction (P 0.08). Introduction DNA repair plays a key role in carcinogenesis through the removal of DNA damage induced by endogenous and environmental sources. We are interested in polymorphic genes involved in the repair of smoking-induced DNA damage and in their association with bladder cancer risk. The main risk factor for bladder cancer is cigarette smoking, which can increase risk up to four times (1, 2). Cigarette smoke is a rich source of chemical carcinogens and ROS. Chemical carcinogens form bulky adducts on DNA, whereas ROS can induce base damage, SSBs, and DSBs (3). Bulky adducts are repaired through the nucleotide excision repair pathway (4). ROS-induced base damage and SSBs are repaired through the base excision repair pathway (5), whereas DSBs can be repaired by either HRR or nonhomologous end joining (6). We have previously reported an association between bladder cancer risk and allelic variations in the XRCC1 gene, which is involved in BER (7). In this study, we focused on the XRCC3 gene, which is required for efficient repair of DSBs through the HRR pathway (8), for repair of DNA cross-linking (9), and for chromosomal segregation (10). During HRR, the XRCC3 protein interacts with the Rad51 protein, enabling Rad51 protein multimers to assemble at the site of damage (9, 11). Furthermore, Rad51 has also been found to colocalize with the XRCC1 protein after base damage, suggesting coordination between XRCC1dependent SSB repair and recombination events during DNA replication (12). A common polymorphism in the XRCC3 gene in codon 241 results in a Thr to Met substitution (13). Using a bladder cancer case-control study, we analyzed a possible association between the XRCC3 codon 241 polymorphism and bladder cancer and a possible interaction with cigarette smoking as a measure of ROS exposure. We also tested for an XRCC1XRCC3 interaction and a possible effect modification of this interaction on the effect of cigarette smoking. Materials and Methods Subjects. Bladder cancer patients (n 235) and control individuals (n 213) were enrolled from the urology clinics at Duke University Medical Center and the University of North Carolina Hospitals, as described previously (2, 7). Briefly, cases had histologically confirmed transitional cell carcinoma. Controls were urology patients without a history of cancer from the same clinics, the most frequent diagnoses being benign prostate hypertrophy, impotence, and urinary incontinence. Controls were frequency matched to cases based on ethnicity Received 3/30/01; revised 6/3/02; accepted 6/5/02. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Present address: Department of Preventive Medicine, University of Southern California, Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, CA 90089. 2 Present address: Toxicology Operations Branch, National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709. 3 To whom correspondence should be addressed, at National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709. Phone (919) 451-4631; E-mail: taylor@niehs.nih. gov. 4 The abbreviations used are: ROS, reactive oxygen species; SSB, single-strand break; DSB, double-strand break; HRR, homologous recombination repair; OR, odds ratio; CI, confidence interval. 939 Vol. 11, 939–943, September 2002 Cancer Epidemiology, Biomarkers & Prevention Research. on September 20, 2017. © 2002 American Association for Cancer cebp.aacrjournals. org Downloaded from (black, white), sex, and age at interview (10-year interval). After giving written informed consent, all individuals were administered a questionnaire that detailed their smoking exposure history and provided blood samples collected under protocols approved by the institutional review boards of each participating institution. Genotype Analysis by PCR-RFLP. DNA was extracted from peripheral blood lymphocytes by standard methods. The DNA segment surrounding codon 241 was amplified by PCR using 50 ng of DNA in a final volume of 15 l containing 1 PCR buffer, 0.2 mM dNTPs, 2 mM MgCl2, 0.8 M forward primer (at 17,724 bp: 5 -TTGGGGCCTCTTTGAGA-3 ), 0.8 M reverse primer (at 18,258 bp: 5 -AACGGCTGAGGGTCTTCT-3 ), and 0.5 units of AmpliTaq Gold (Perkin-Elmer, Foster City, CA). PCR reactions were carried out in a Perkin-Elmer 9700 thermocycler with an initial denaturation step of 8 min at 94°C, followed by 30 cycles at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. PCR products were digested with NlaIII restriction enzyme (New England Biolabs, Beverly, MA), resolved in 3% NuSieve 3:1 agarose gels (FMC Bioproducts, Rockland, ME), and stained with ethidium bromide. NlaIII recognizes an invariant restriction site around bp 17,963, which serves as an internal control for complete enzyme digestion. The XRCC3 codon 241 Met-allele creates a NlaIII restriction site at bp 18,067, a site that is not present in the codon 241 Thr-allele. Statistical Analysis. We used standard methods for 2 k contingency tables, including Fisher’s exact test, as appropriate, to analyze categorical variables without adjustment for covariates. We checked among controls for differences between the observed genotypic frequencies and those expected under the Hardy-Weinberg law using estimates of the disequilibrium coefficient (14). When adjusting for age, sex, or ethnicity, and when examining interactions between the polymorphism and smoking, we used standard logistic regression methods (15). We tested for interaction on a multiplicative scale. Given the small number of blacks in our study, data from blacks are included with whites in combined OR estimates after including ethnicity as a covariate. For gene-environment interaction analyses, we used categorized versions of pack-years as measures of smoking exposure and years of smoking as a continuous variable as described previously (2). We examined the combined effects of the continuous smoking variable and the XRCC3 genotype and the combined effects of smoking with the XRCC3 and XRCC1 genotypes using an approach that we have used previously (2, 7). This approach fits a series of logistic regression models and compares them, testing relevant hypotheses with likelihood ratio tests. Within a model, differences Table 1 Genotypic frequencies and XRCC3 codon 241 polymorphism and smoking combined analysis Smoking XRCC3 codon 241 Whites and blacks Cases n 233 Controls n 209 ORadj b 95% CI Test for interaction P Nonsmokers and smokers combined Thr/Thr 90 (39%) 94 (45%) 1 Thr/Met 110 (47%) 91 (44%) 1.2 0.8–1.9 NA Met/Met 33 (14%) 24 (11%) 1.5 0.8–2.7 NA