Adenosine Diphosphate Ribosyl Transferase and X-Ray Repair Cross-Complementing 1 Polymorphisms in Gastric Cardia Cancer
2006; Elsevier BV; Volume: 131; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.05.050
ISSN1528-0012
AutoresXiaoping Miao, Xuemei Zhang, Lingqiang Zhang, Yongli Guo, Bingtao Hao, Wen Tan, Fuchu He, Dongxin Lin,
Tópico(s)Cancer therapeutics and mechanisms
ResumoBackground & Aims: Adenosine diphosphate ribosyl transferase (ADPRT) and x-ray repair cross-complementing 1 (XRCC1) are major DNA base excision repair proteins acting interactively in repair processes. This study examined the effects of ADPRT Val762Ala and XRCC1 Arg399Gln polymorphisms on ADPRT-XRCC1 interaction in vitro in cells and their contributions to gastric cardia adenocarcinoma (GCA) risk. Methods: The ADPRT-XRCC1 interaction in cells transfected with ADPRT and XRCC1 variant complementary DNA (cDNA) constructs were examined by immunoprecipitation and immunoblotting analysis. Genotypes were analyzed in 500 patients and 1000 controls, and odds ratios (ORs) were estimated by logistic regression. Results: Interactions between ADPRT-762Val and XRCC1-399Arg or XRCC1-399Gln were robust, but interactions between ADPRT-762Ala and either XRCC1-399Arg or XRCC1-399Gln were very weak. A case-control analysis showed ORs of 2.17 (95% CI, 1.55–3.04) and 1.61 (95% CI, 1.06–2.44) for GCA in the ADPRT Ala/Ala or XRCC1 Gln/Gln genotype carriers, respectively, compared with noncarriers. Gene-gene interaction of ADPRT and XRCC1 polymorphisms increased the OR of GCA in a multiplicative manner (OR for the presence of both ADPRT Ala/Ala and XRCC1 Gln/Gln genotypes, 6.43; 95% CI, 1.80–22.97). A supermultiplicative joint effect between the ADPRT polymorphism and smoking was observed. The ORs (95% CIs) of the Ala/Ala genotype for nonsmokers and smokers who smoked ≤24 or >24 pack-years were 1.44 (0.89–2.32), 2.00 (1.09–3.67), or 3.19 (1.59–6.42), respectively (Ptrend test = .008). Conclusions: The ADPRT and XRCC1 polymorphisms confer host susceptibility to GCA, which might result from reduced ADPRT-XRCC1 interaction and attenuated base excision repair capacity. Background & Aims: Adenosine diphosphate ribosyl transferase (ADPRT) and x-ray repair cross-complementing 1 (XRCC1) are major DNA base excision repair proteins acting interactively in repair processes. This study examined the effects of ADPRT Val762Ala and XRCC1 Arg399Gln polymorphisms on ADPRT-XRCC1 interaction in vitro in cells and their contributions to gastric cardia adenocarcinoma (GCA) risk. Methods: The ADPRT-XRCC1 interaction in cells transfected with ADPRT and XRCC1 variant complementary DNA (cDNA) constructs were examined by immunoprecipitation and immunoblotting analysis. Genotypes were analyzed in 500 patients and 1000 controls, and odds ratios (ORs) were estimated by logistic regression. Results: Interactions between ADPRT-762Val and XRCC1-399Arg or XRCC1-399Gln were robust, but interactions between ADPRT-762Ala and either XRCC1-399Arg or XRCC1-399Gln were very weak. A case-control analysis showed ORs of 2.17 (95% CI, 1.55–3.04) and 1.61 (95% CI, 1.06–2.44) for GCA in the ADPRT Ala/Ala or XRCC1 Gln/Gln genotype carriers, respectively, compared with noncarriers. Gene-gene interaction of ADPRT and XRCC1 polymorphisms increased the OR of GCA in a multiplicative manner (OR for the presence of both ADPRT Ala/Ala and XRCC1 Gln/Gln genotypes, 6.43; 95% CI, 1.80–22.97). A supermultiplicative joint effect between the ADPRT polymorphism and smoking was observed. The ORs (95% CIs) of the Ala/Ala genotype for nonsmokers and smokers who smoked ≤24 or >24 pack-years were 1.44 (0.89–2.32), 2.00 (1.09–3.67), or 3.19 (1.59–6.42), respectively (Ptrend test = .008). Conclusions: The ADPRT and XRCC1 polymorphisms confer host susceptibility to GCA, which might result from reduced ADPRT-XRCC1 interaction and attenuated base excision repair capacity. Human genomic DNA can be damaged by environmental factors such as ultraviolet light, ionizing radiation, chemical carcinogens, and certain products generated by normal cellular metabolism. If not properly repaired, such DNA damage can induce mutations, genome instability, and DNA strand breaks, which in turn may cause cellular malignant transformation and tumorigenesis. Nevertheless, human cells have evolved a set of complex DNA repair systems that safeguard the integrity of genome to minimize the consequences of detrimental damages.1Hoeijmakers J.H.J. Genome maintenance mechanisms for preventing cancer.Nature. 2001; 411: 366-374Crossref PubMed Scopus (3114) Google Scholar As a result, the normal functions of DNA repair proteins are critical for cells to remove damage and thus prevent carcinogenesis. The base excision repair (BER) pathway, among other DNA repair systems, is basically responsible for rectifying DNA base damage and single-strand breaks.1Hoeijmakers J.H.J. Genome maintenance mechanisms for preventing cancer.Nature. 2001; 411: 366-374Crossref PubMed Scopus (3114) Google Scholar, 2Wood R.D. Mitchell M. Sgouros J. Lindahl T. Human DNA repair genes.Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1098) Google Scholar BER includes 2 major processes, ie, excision of damaged base residues and core BER reaction including strand incision at the abasic site, 1-nucleotide gap-filling reaction, and sealing of the remaining nick.1Hoeijmakers J.H.J. Genome maintenance mechanisms for preventing cancer.Nature. 2001; 411: 366-374Crossref PubMed Scopus (3114) Google Scholar, 2Wood R.D. Mitchell M. Sgouros J. Lindahl T. Human DNA repair genes.Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1098) Google Scholar It is well known that several proteins are involved in these steps, of which adenosine diphosphate ribosyl transferase (ADPRT) and x-ray repair cross-complementing 1 (XRCC1) play key roles. In response to DNA damage, ADPRT specifically binds to DNA strand breaks at which it is autoactivated and recruits the XRCC1-Ligase IIIα complex to stimulate BER, and XRCC1 subsequently interacts with ADPRT to recruit other partner proteins such as DNA polymerase β (Ploβ) to execute and complete BER processes.3Caldecott K.W. XRCC1 and DNA strand break repair.DNA Repair. 2003; 2: 955-969Crossref PubMed Scopus (506) Google Scholar, 4Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage.Mol Cell Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (825) Google ScholarGenetic variants have been identified for both ADPRT and XRCC1 genes.5Lockett K.L. Hall M.C. Xu J. Zheng L.S. Berwick M. Chuang S. Clark P.E. Cramer S.D. Lohman K. Hu J.J. The ADPRT V762A genetic variant contributes to prostate cancer susceptibility and deficient enzyme function.Cancer Res. 2004; 64: 6344-6348Crossref PubMed Scopus (152) Google Scholar, 6Cottet F. Blanche H. Verasdonck P. Le Gall I. Schachter F. Burkel A. Murias M.L. New polymorphisms in the human poly(ADP-ribose) polymerase-1 coding sequence lack of association with longevity or with increased cellular poly(ADP-ribosyl)ation capacity.J Mol Med. 2000; 78: 431-440Crossref PubMed Scopus (51) Google Scholar, 7Shen M.R. Jones I.M. Mohrenweiser H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans.Cancer Res. 1998; 58: 604-608PubMed Google Scholar, 8Hao B. Wang H. Zhou K. Li Y. Chen X. Zhou G. Zhu Y. Miao X. Wen T. Wei Q. Lin D. He F. Identification of genetic variants in base excision repair pathway and their associations with risk of esophageal squamous cell carcinoma.Cancer Res. 2004; 64: 4378-4384Crossref PubMed Scopus (199) Google Scholar There are at least 38 reported variants in ADPRT (http://snp500cancer.nci.nih.gov), one of which is a T to C transition at codon 762 of ADPRT located in the sixth helix of catalytic domain that causes Val-to-Ala amino acid substitution. A recent study has shown that the 762Ala polymorphism is associated with altered ADPRT function, with the Ala allele contributing to significantly low poly(ADP-ribosyl)ation activities in an allele dosage-dependent manner.5Lockett K.L. Hall M.C. Xu J. Zheng L.S. Berwick M. Chuang S. Clark P.E. Cramer S.D. Lohman K. Hu J.J. The ADPRT V762A genetic variant contributes to prostate cancer susceptibility and deficient enzyme function.Cancer Res. 2004; 64: 6344-6348Crossref PubMed Scopus (152) Google Scholar In the XRCC1 gene, at least 17 variants have been identified to date (http://snp500cancer.nci.nih.gov), and, among them, the G to A polymorphism at codon 399 results in Arg-to-Gln amino acid change, which is situated within the BRCT-1 region harboring the ADPRT-binding domain.4Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage.Mol Cell Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (825) Google Scholar, 7Shen M.R. Jones I.M. Mohrenweiser H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans.Cancer Res. 1998; 58: 604-608PubMed Google Scholar An altered DNA repair activity has been suggested to be associated with the XRCC1 399Gln polymorphism.9Lunn R.M. Langlois R.G. Hsieh L.L. Thompson C.L. Bell D.A. XRCC1 polymorphisms effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency.Cancer Res. 1999; 59: 2557-2561PubMed Google Scholar, 10Abdel-Rahman S.Z. El-Zein R.A. The 399Gln polymorphism in the DNA repair gene XRCC1 influences the genotoxic response induced in human lymphocytes by the tobacco-specific nitrosamine NNK.Cancer Lett. 2000; 159: 63-71Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 11Qu T. Morii E. Oboki K. Lu Y. Morimoto K. Micronuclei in EM9 cells expressing polymorphic forms of human XRCC1.Cancer Lett. 2005; 221: 91-95Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar We have recently shown that these functional polymorphisms in ADPRT and XRCC1 have great impact on susceptibility to smoking-related lung cancer and esophageal cancer. Furthermore, a statistically significant interaction between the ADPRT 762Ala and XRCC1 399Gln polymorphisms in risk of the cancer was observed.8Hao B. Wang H. Zhou K. Li Y. Chen X. Zhou G. Zhu Y. Miao X. Wen T. Wei Q. Lin D. He F. Identification of genetic variants in base excision repair pathway and their associations with risk of esophageal squamous cell carcinoma.Cancer Res. 2004; 64: 4378-4384Crossref PubMed Scopus (199) Google Scholar, 12Zhang X. Miao X. Liang G. Hao B. Wang Y. Tan W. Li Y. Guo Y. He F. Wei Q. Lin D. Polymorphisms in DNA base excision repair genes ADPRT and XRCC1 and risk of lung cancer.Cancer Res. 2005; 65: 722-726Crossref PubMed Scopus (34) Google Scholar Our data suggest that these genetic polymorphisms may obstruct the interaction between ADPRT and XRCC1 at protein level in the BER processes and thus abolish or reduce DNA repair capacity in cells carrying the variant alleles. However, this hypothesis has not been examined yet at the molecular level.Gastric cardia adenocarcinoma (GCA) is a common cancer in China as well as in the rest of the world. In China, this cancer is more prevalent in areas of high-risk for esophageal cancer,13Li J.Y. Ershow A.G. Chen Z.J. Wacholder S. Li G.Y. Guo W. Li B. Blot W.J. A case-control study of cancer of the esophagus and gastric cardia in Linxian.Int J Cancer. 1989; 43: 755-761Crossref PubMed Scopus (184) Google Scholar suggesting that it may share common risk factors with esophageal cancer. Although the integrated etiology of esophageal cancer and GCA remains to be fully clarified, several environmental factors such as cigarette smoking, heavy alcohol drinking, micronutrient deficiency, dietary carcinogen exposure, and gastroesophageal reflux disease have been linked to these cancers.14Tran G.D. Sun X.D. Abnet C.C. Fan J.H. Dawsey S.M. Dong Z.W. Mark S.D. Qiao Y.L. Taylor P.R. Prospective study of risk factors for esophageal and gastric cancers in the Linxian general population trial cohort in China.Int J Cancer. 2005; 113: 456-463Crossref PubMed Scopus (534) Google Scholar, 15Engel L.S. Chow W.H. Vaughan T.L. Gammon M.D. Risch H.A. Stanford J.L. Schoenberg J.B. Mayne S.T. Dubrow R. Rotterdam H. West A.B. Blaser M. Blot W.J. Gail M.H. Fraumeni Jr, J.F. Population attributable risks of esophageal and gastric cancers.J Natl Cancer Inst. 2003; 95: 1404-1413Crossref PubMed Scopus (570) Google Scholar, 16Ye W. Chow W.H. Lagergren J. Yin L. Nyren O. Risk of adenocarcinomas of the esophagus and gastric cardia in patients with gastroesophageal reflux diseases and after antireflux surgery.Gastroenterology. 2001; 121: 1506-1508Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 17Mayne S.T. Risch H.A. Dubrow R. Chow W.H. Gammon M.D. Vaughan T.L. Farrow D.C. Schoenberg J.B. Stanford J.L. Ahsan H. West A.B. Rotterdam H. Blot W.J. Fraumeni Jr, J.F. Nutrient intake and risk of subtypes of esophageal and gastric cancer.Cancer Epidemiol Biomarkers Prev. 2001; 10: 1055-1062PubMed Google Scholar All these factors can induce or enhance DNA damage mediated by either oxidative stress or DNA-binding electrophiles, which in turn may initiate and/or promote tumorigenesis. However, even in the high-risk areas, only a small fraction of exposed individuals develop GCA in their life span, suggesting that there may be important genetic basis rendering such individuals more susceptible to the cancer. Because DNA repair mechanisms play a pivotal role in preventing tumorigenesis, suboptimal DNA repair capacity throughout lifetime caused by germ-line polymorphisms might be a susceptibility factor for the development of GCA.In this report, we describe a case-control study to test the hypothesis that the genetic polymorphisms of ADPRT and XRCC1 contribute to host susceptibility to GCA. We also examined the effect of these polymorphisms on the interaction between ADPRT and XRCC1 at the protein level in cells to explore further the underlying functional mechanisms.Materials and methodsStudy SubjectsThe case-control study consisted of 500 patients with GCA and 1000 cancer-free controls. All subjects were biologically unrelated ethnic Han Chinese. Patients were consecutively recruited from January 1997 to July 2004 at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing). In this study, GCA was defined as tumors arising at the gastric cardia and/or gastroesophageal junction with or without involvement of other esophageal and/or gastric subsites. Five hundred forty-four patients with histopathologically confirmed GCA, regardless of age, sex, and tumor stage, were enrolled, and the response rate was 92% because of reluctance of 44 patients to participate in the study. Tumors confined entirely to the esophageal or gastric subsites were excluded. Three hundred fifty-six patients were participants in our previous molecular epidemiologic study of GCA.18Miao X. Yu C. Tan W. Xiong P. Liang G. Lu W. Lin D. A functional polymorphism in the matrix metalloproteinase-2 gene promoter (−1306C/T) is associated with risk of development but not metastasis of gastric cardia adenocarcinoma.Cancer Res. 2003; 63: 3987-3990PubMed Google Scholar In the present study, we added 144 patients, for a total of 500 patients with GCA. Control subjects were cancer-free individuals living in the Beijing region, who were selected from a community cancer-screening program for early detection of cancer conducted during the same time period as the patients were collected.19Miao X. Xing D. Tan W. Qi J. Lu W. Lin D. Susceptibility to gastric cardia adenocarcinoma and genetic polymorphisms in methylenetetrahydrofolate reductase in an at-risk Chinese population.Cancer Epidemiol Biomarkers Prev. 2002; 11: 1454-1458PubMed Google Scholar Briefly, the control subjects were selected from a pool of 2800 individuals based on physical examinations. The selection criteria include no individual history of cancer, and controls were frequency matched to GCA patients on age (±5 years) and sex. The characteristics of 789 control subjects were described previously.18Miao X. Yu C. Tan W. Xiong P. Liang G. Lu W. Lin D. A functional polymorphism in the matrix metalloproteinase-2 gene promoter (−1306C/T) is associated with risk of development but not metastasis of gastric cardia adenocarcinoma.Cancer Res. 2003; 63: 3987-3990PubMed Google Scholar In this study, we also selected 211 more control subjects from the same database, for a total of 1000 control subjects, to make a 2:1 match to GCA patients. At recruitment, informed consent was obtained from each subject, and each participant was then interviewed to collect detailed information on demographic characteristics and lifetime history of tobacco use. This study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute and Hospital.Polymorphism AnalysisADPRT Val762Ala and XRCC1 Arg399Gln genotypes were analyzed using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) methods as described previously.12Zhang X. Miao X. Liang G. Hao B. Wang Y. Tan W. Li Y. Guo Y. He F. Wei Q. Lin D. Polymorphisms in DNA base excision repair genes ADPRT and XRCC1 and risk of lung cancer.Cancer Res. 2005; 65: 722-726Crossref PubMed Scopus (34) Google Scholar All subjects were successfully genotyped. To ensure quality control, genotyping was performed without knowledge of case/control status of the subjects, and a 15% random sample of patients and control subjects was genotyped twice by different persons; the reproducibility was 100%.Plasmid ConstructionThe complementary DNA (cDNA) encoding wild-type ADPRT (762Val) was generated by PCR from the human fetal liver cDNA library (Clontech, Mountain View, CA), with the forward primer 5′-gaagatcttcggatggcggagtcttcg-3′ and reverse primer 5′-ccgctcgagcgggtcgacttaccacagggaggt-3′, which contain 5′-BglII and 3′-SalI sites (underlined sequences). To ensure high-fidelity amplification, LA Taq polymerase (TaKaRa, Dalian, China) was used in the PCR. The PCR product was subsequently used as a template to generate mutant ADPRT (762Ala) cDNA by using site-specific mutagenesis with aforementioned PCR primers and primers of 5′-caggccaaggccgaaatgcttgacaac-3′ and 5′-catttcggccttggcctgcacact-3′ (underlined sequences encode 762Ala). Both ADPRT 762Val and 762Ala cDNA were ligated into pFlag-CMV-2 vector (Sigma Chemical Company, St. Louis, MO), and the resulting constructs were designated as pFlag-CMV-2-ADPRT-762Val or pFlag-CMV-2-ADPRT-762Ala. For XRCC1 constructs, 2 XRCC1 cDNA clones with 399Arg or 399Gln genotype, kindly provided by Prof. Keith W. Caldecott of University of Sussex, United Kingdom, were used as template in PCR amplification. The PCR primers used were 5′-ccggaattcacatgccggagatccgc-3′ and 5′-gaagatcttcaggcttgcggcaccaccccata-3′, which consist of 5′-EcoRI and 3′-BglII sites (underlined sequences). Both XRCC1 399Arg and XRCC1 399Gln cDNA were ligated into pCMV-Myc vector (Clontech), respectively, and designated as pCMV-Myc-XRCC1-399Arg or pCMV-Myc-XRCC1-399Gln. All constructs used in this study were restriction mapped and sequenced to confirm their authenticity.Cell Culture and TransfectionHuman breast cancer cell line MCF7 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen, Carlsbad, CA) in a humidified, 5% CO2 incubator at 37°C. Human gastric cancer cell line MGC-803 cells and Barrett’s esophageal adenocarcinoma cell line SEG-1 cells were maintained in RPMI 1640 containing 10% fetal bovine serum. The MCF7 and MGC-803 cells were cotransfected with pFlag-CMV-2-ADPRT-762Val or pFlag-CMV-2-ADPRT-762Ala and pCMV-Myc-XRCC1-399Arg or pCMV-Myc-XRCC1-399Gln plasmids using Lipofectamine 2000 (Invitrogen), and SEG-1 cells were transfected using FuGene 6 reagent (Roche Diagnostics, Shanghai, China) according to the manufacturer’s protocol.Immunoprecipitation and ImmunoblottingImmunoprecipitation (IP) and immunoblotting (IB) were performed as previously described20Zhang L. Xing G. Tie Y. Tang Y. Tian C. Li L. Sun L. Wei H. Zhu Y. He F. Role for the pleckstrin homology domain-containing protein CKIP-1 in AP-1 regulation and apoptosis.EMBO J. 2005; 24: 766-778Crossref PubMed Scopus (57) Google Scholar with minor modifications. Briefly, the cells, harvested 48 hours after transfection, were washed twice with cooled phosphate-buffered saline, and cell lysates were prepared in lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Tween 20, 0.2% Nonidet P-40, 10% glycerol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mmol/L NaF and 1 mmol/L Na3VO4) by sonication and subsequent centrifugation at 15,000g for 10 minutes. The supernatant was incubated with mouse control IgG (Santa Cruz Biochemical Co, Santa Cruz, CA) at 4°C for 1 hour and then were incubated with protein A/G-agarose for another 1 hour. After a brief centrifugation, the pellet was washed 3 times with lysis buffer (designated IP-IgG), and the supernatant was divided equally into 2 parts, and each was incubated with anti-Myc antibody or anti-Flag antibody and protein A/G-agarose (Santa Cruz) at 4°C to immunoprecipitate Myc-tagged XRCC1 proteins and Flag-tagged ADPRT proteins, respectively. The lysates and immunoprecipitates were immunoblotted with the anti-Myc (Clontech; 1:1000) or anti-Flag M2 (Sigma Chemical Company; 1:1000) antibodies after gel electrophoresis and electrotransfer of separated proteins on nitrocellulose sheets. After incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz; 1/2000 dilution), bands were visualized using SuperSignal chemiluminescence kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.Statistical AnalysisThe χ2 test was used to examine differences in demographic variables, smoking, and distribution of genotypes between patients and control subjects. The associations between genotype and risk of gastric cardia cancer were estimated by calculating odds ratios (ORs) and their 95% confidence intervals (CIs) with unconditional logistic regression models. The ORs were adjusted for age, sex, and smoking status. The number of pack-years smoked was determined as an indicator of the cumulative cigarette dose level (pack-years = cigarettes per day/20 × years smoked). Light and heavy smokers were categorized by using the 50th percentile pack-year value of the control subjects as the cut-off points (ie, ≤24 and >24 pack-years). We tested the null hypotheses of multiplicative gene-gene and gene-smoking joint effect evaluated departures from multiplicative joint effect models by including main effect variables and their product terms in the logistic regression model. A more than multiplicative joint effect21Brennan P. Gene-environment interaction and etiology of cancer what does it mean and how we measure it?.Carcinogenesis. 2002; 23: 381-387Crossref PubMed Scopus (217) Google Scholar was suggested when OR11 > OR10 × OR01. The homogeneity test was done to compare the difference between smoking-related ORs among different genotypes or between the product of related ORs and the joint effect OR. All analyses were performed with the computer programs of Statistical Analysis System (version 6.12; SAS Institute, Cary, NC.ResultsSubject Characteristics and Genotype DistributionsBaseline characteristics of patients and control subjects are summarized in Table 1. The sex and age distributions were fairly identical among patients and control subjects, indicating that the matching of controls to cases was adequate. There were no statistically significant differences between patients and control subjects in terms of smoking status and levels of pack-year smoked. Genotyping results (Table 2) showed that the allele frequencies for the ADPRT 762Ala and XRCC1 399Gln variants were 0.36 and 0.27 in control subjects and 0.44 and 0.32 in patients, respectively. The frequencies of the 3 ADPRT genotypes, ie, 762Val/Val, -Val/Ala, and -Ala/Ala, were 39.6%, 49.2%, and 11.2%, respectively, among control subjects, which conformed to the Hardy–Weinberg equilibrium. The respective ADPRT genotype frequencies among GCA patients were 30.0%, 51.4%, and 18.6%, which differed significantly from those among control subjects (χ2 = 22.12, P < .0001), with the Ala/Ala genotype being significantly more prevalent in patients than in control subjects. The frequencies of XRCC1 399Arg/Arg, -Arg/Gln, and -Gln/Gln genotypes among control subjects were 53.2%, 40.2%, and 6.6%, which conformed to the Hardy–Weinberg equilibrium but significantly differed from those among patients (44.2% Arg/Arg, 46.8% Arg/Gln, and 9.0% Gln/Gln, respectively) (χ2 = 11.40, P = .003).Table 1Frequency Distribution of Select Characteristics by Case Control StatusVariablePatients (n = 500)Control subjects (n = 1000)P valueaTwo-sided χ2 test.No.(%)No.(%)Sex1.00 Male430(86.0)860(86.0) Female70(14.0)140(14.0)Age (yrs)1.00 ≤4014(2.8)28(2.8) 41–5062(12.4)124(12.4) 51–60165(33.0)330(33.0) >60259(51.8)518(51.8)Smoking statusbNonsmokers were defined as subjects who smoked 24135(51.3)281(49.6)a Two-sided χ2 test.b Nonsmokers were defined as subjects who smoked <10 cigarettes lifetime. Open table in a new tab Table 2Genotype Frequencies of ADPRT and XRCC1 Among Cases and Control Subjects and Their Association With Gastric Cardia AdenocarcinomaGenotypeControl subjects (n = 1000)Patients (n = 500)PtrendNo.(%)No.(%)ADPRT Val762Ala Val/Val396(39.6)150(30.0) Val/Ala492(49.2)257(51.4) Ala/Ala112(11.2)93(18.6) One Ala, OR (95% CI)aData were calculated by unconditional logistic regression and adjusted for sex, age, and smoking status.1.35 (1.06–1.72) Two Ala, OR (95% CI)aData were calculated by unconditional logistic regression and adjusted for sex, age, and smoking status.2.17 (1.55–3.04)<.0001bTests for trend of odds were 2-sided and based on likelihood ratio tests assuming a multiplicative model.XRCC1 Arg399Gln Arg/Arg532(53.2)221(44.2) Arg/Gln402(40.2)234(46.8) Gln/Gln66(6.6)45(9.0) One Gln, OR (95% CI)aData were calculated by unconditional logistic regression and adjusted for sex, age, and smoking status.1.40 (1.12–1.75) Two Gln, OR (95% CI)aData were calculated by unconditional logistic regression and adjusted for sex, age, and smoking status.1.61 (1.06–2.44).0009bTests for trend of odds were 2-sided and based on likelihood ratio tests assuming a multiplicative model.a Data were calculated by unconditional logistic regression and adjusted for sex, age, and smoking status.b Tests for trend of odds were 2-sided and based on likelihood ratio tests assuming a multiplicative model. Open table in a new tab Association Between Individual Polymorphism and GCA RiskThe unconditional logistic regression model was used to estimate the association between genotypes and risk of GCA (Table 2). It was observed that the odds of having the ADPRT 762Ala/Ala genotype in the GCA group was 2.17 (95% CI: 1.55–3.04; P < .0001) compared with the 762Val/Val genotype. A significantly increased OR was also associated with the 762Val/Ala heterozygous genotype (OR, 1.35; 95% CI: 1.06–1.72; P = .009), suggesting an allele-dose relationship (trend test, P < .0001). Similarly, the XRCC1 399Gln allele was shown to be a risk allele. Subjects carrying the 399Gln/Gln or 399Arg/Gln genotype had an odds of 1.61 (95% CI: 1.06–2.44; P = .017) or 1.40 (95% CI: 1.12–1.75; P = .003) for developing GCA, respectively, compared with subjects carrying the 399Arg/Arg genotype (trend test, P < .001), suggesting a dominant effect of this polymorphism in GCA. In the stratification analysis, age and smoking had no effect on the risk of GCA related to both ADPRT and XRCC1 genotypes (data not shown).Effects of Polymorphisms on ADPRT and XRCC1 InteractionIt has been well known that ADPRT and XRCC1 act interactively at the protein level in the BER process.3Caldecott K.W. XRCC1 and DNA strand break repair.DNA Repair. 2003; 2: 955-969Crossref PubMed Scopus (506) Google Scholar, 4Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage.Mol Cell Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (825) Google Scholar We therefore examined whether the investigated polymorphisms obstruct the interaction between ADPRT and XRCC1 at the protein level. The expression plasmids consisted of Myc-XRCC1-399Arg (designed as R), Myc-XRCC1-399Gln (designated as Q), Flag-ADPRT-762Val (designed as V), or Flag-ADPRT-762Ala (designed as A) and were constructed and cotransfected into human cancer cells (designed as RV, QV, RA, and QA). Equal cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibody in reciprocal coimmunoprecipitation assays (Co-IP). The Co-IP experiments were performed in MCF-7 cells (Figure 1A), MGC-803 cells (Figure 1B), and SEG-1 cells (Figure 1C). Western blot analysis of whole cell lysates showed that, under our experimental conditions, the constructed plasmids expressed respective proteins, and the expression levels of each plasmid in cells were similar (Figure 1I), indicating that our experimental model is valid. It was found that a similar amount of XRCC1-399Arg and XRCC1-399Gln protein was specifically immunoprecipitated by anti-Myc antibody (Figure 1II, upper panels, lanes 2, 4, 6, and 8) but not by control IgG (Figure 1II, upper panels, lanes 1, 3, 5, and 7). Strikingly, interactions between XRCC1-399Arg or -399Gln and ADPRT-762Val were robust, and the levels of the 2 XRCC1 isoforms bound to ADPRT-762Val were quite similar in the 3 cell lines (Figure 1AII, 1BII, and 1CII, bottom panel, lanes 2 and 4), suggesting that the XRCC1 399Arg→Gln change had li
Referência(s)