NER Pathway Research Articles

Aging and photo-aging DNA repair phenotype of skin cells—Evidence toward an effect of chronic sun-exposure

Several studies have demonstrated the deleterious effect of aging on the capacity of cells to repair their DNA. However, current existing assays aimed at measuring DNA repair address only a specific repair step dedicated to the correction of a specific DNA lesion type. Consequently they provide no information regarding the repair pathways that handle other types of lesions. In addition to aging, consequences of photo-exposure on these repair processes remain elusive. In this study we evaluated the consequence of aging and of chronic and/or acute photo-exposure on DNA repair in human skin fibroblasts using a multiplexed approach, which provided detailed information on several repair pathways at the same time. The resulting data were analyzed with adapted statistics/bioinformatics tools. We showed that, irrespective of the repair pathway considered, excision/synthesis was less efficient in non-exposed cells from elderly compared to cells from young adults and that photo-exposure disrupted this very clear pattern. Moreover, it was evidenced that chronic sun-exposure induced changes in DNA repair properties. Finally, the identification of a specific signature at the level of the NER pathway in cells repeatedly exposed to sun revealed a cumulative effect of UVB exposure and chronic sun irradiation. The uses of bioinformatics tools in this study was essential to fully take advantage of the large sum of data obtained with our multiplexed DNA repair assay and unravel the effects of environmental exposure on DNA repair pathways.

The C/A polymorphism in intron 11 of the XPC gene plays a crucial role in the modulation of an individual’s susceptibility to sporadic colorectal cancer

Epidemiological data show that colorectal cancer (CRC) is the second most frequent malignancy worldwide. The involvement of "minor impact genes" such as XME and DNA-repair genes in the etiology of sporadic cancer has been postulated by other authors. We focused on analyzing polymorphisms in DNA-repair genes in CRC. We considered the following genes involved in DNA-repair pathways: base excision repair (OGG1 Ser326Cys, XRCC1 Trp194Arg and Arg399Gln); nucleotide excision repair [XPA (-4)G/A, XPC C/A (i11) and A33512C (Lys939Gln), XPD Asp312Asn and A18911C (Lys751Gln), XPF Arg415Gln, XPG Asp1104His, ERCC1 C118T]; homologous recombination repair [NBS1 Glu185Gln, Rad51 135G/C, XRCC3 C18067 (Thr241Met)]. The study group consisted of 133 patients diagnosed with sporadic CRC, while the control group was composed of 100 age-matched non-cancer volunteers. Genotyping was performed by PCR and PCR-RFLP. Fisher's exact test with a Bonferroni correction for multiple testing was used. We found that: (i) XPC C/A (i11) heterozygous variant is associated with increased risk of CRC [OR is 2.07 (95% CI 1.1391, 3.7782) P=0.038], (ii) XPD A18911C (Lys751Gln) is associated with decreased risk of CRC [OR=0.4497, (95% CI 0.2215, 0.9131) P=0.031] for an individual with at least one A allele at this locus. (1) The XPC C/A (i11) genotype is associated with an increased risk of sporadic colorectal cancer. (2) The NER pathway has been highlighted in our study, as a most important in modulation of individual susceptibility to sCRC.

Abstract 4199: Genetic variation in the in vitro genotoxic response to glycidamide and gene expression of DNA repair genes

Abstract Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuff upon heating. Glycidamide (GA), formed via epoxidation presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite that plays a central role in AA genotoxicity. The high levels of AA that are orally consumed could be an additional factor for global cancer risk. However, cancer risk estimation in population is still problematic and the mechanisms of DNA repair triggered by GA are still poorly understood. The aim of this work was to evaluate the role of DNA repair genes in the genotoxicity induced by GA in human cells, using complementary methodologies. For this purpose, the effect of GA (250 µM) in cultured human lymphocytes from healthy non-smoker individuals (n=13) was assessed using the sensitive sister-chromatid exchange assay (SCE, 46 h in vitro exposure). GA was found to be genotoxic, with a large inter-individual variation. Therefore we studied the possible influence of individual genetic polymorphisms of key genes codifying for DNA repair enzymes related to the BER (XRCC1, OGG1, PARP1, PARP4, APEX1 and MUTYH), NER (RAD23B, ERCC1, ERCC2, ERCC4, ERCC5, ERCC6 and XPC) and HRR (RAD51, NBS, XRCC3 and XRCC2) pathways. No relevant associations between SCE/cell levels and the individual genetic polymorphisms were found. However, the only individual with the ERCC1 Q504K polymorphism revealed a significant increase in GA-induced levels of SCE/cell (p<0.03). We further studied the effect of the same concentration of GA in peripheral white blood cells from healthy individuals (n=25) using the comet assay (CA, 1 hour in vitro incubation). GA also showed to be genotoxic. The % DNA in Tail for all individuals was analysed considering the abovementioned polymorphisms. In this assay, the MUTYH H335Q was found to influence the repair capacity of GA, being the variant genotype associated with an increased damage in the comet assay (6.1± 1.7 heterozygous versus 11.9 ± 1.8 homozygous variant; p<0.001). Finally, a different strategy focusing on the role of DNA repair enzymes upon GA treatment was used. The non-malignant mammary cell line MCF10A was exposed to GA (10 µM) for 24 h and gene expression was studied using RT2 ProfilerTM PCR Array: Human DNA Damage Signalling Pathway by SABiosciencesTM. Concerning gene expression results, GA at this lower concentration level did not influence the expression of the genes considered when compared to respective non-treated controls. Further studies should be conducted in order to test other exposure periods and use other human cell types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4199. doi:10.1158/1538-7445.AM2011-4199

DNA repair gene polymorphisms and micronucleus frequencies in Chinese workers exposed to vinyl chloride monomer

Vinyl chloride monomer (VCM) is genotoxic and cancerogen agent. Individual variations in response to the exposure have been noticed and the variations may be due to genetic differences in the removal of VCM-DNA adducts, such as polymorphism in genes NER pathway and BER pathway. This study explores the relationship between genetic polymorphism of seven genes within the NER pathway (XPA, XPD, XPC, XPG, XPF, and ERCC1) and BER pathway (APE1), and susceptibility to chromosomal damage after exposure to VCM. One hundred and eighty workers occupationally exposed to VCM and 43 unexposed controls were investigated. Chromosome damage in peripheral lymphocytes was measured by the cytokinesis-blocked micronucleus assay. PCR-RFLP technique was applied to detect polymorphisms of the seven genes. The influence of genotype, age, gender, cumulative exposure dose, alcohol consumption, and smoking status on the frequencies of MN was determined using univariate and multiple Poisson regression analyses. We found XPA A23G variant workers had significantly higher MN frequencies than those from the wild-type homozygous counterparts (P = 0.01). Those with the variant XPD Lys751Gln genotype had marginally higher MN frequencies (P = 0.07). On the other hand, XPC PAT and XPF 5'-UTR T2063A variant workers had significantly lower MN frequencies compared with those from their wild-type homozygous counterparts (P = 0.04 and P = 0.004, respectively). Our findings suggest that XPC PAT, XPD Lys751Gln, XPA A23G and XPF 5'-UTR T2063A contribute to the level of chromosome damage in occupational exposure to VCM in Chinese population.

Divergent effects of oxidatively induced modification to the C8 of 2′‐deoxyadenosine on transcription factor binding: 8,5′(S)‐cyclo‐2′‐deoxyadenosine inhibits the binding of multiple sequence specific transcription factors, while 8‐oxo‐2′‐deoxyadenosine increases binding of CREB and NF‐kappa B to DNA

DNA is exposed to endogenous and environmental factors that can form stable lesions. If not repaired, these lesions can lead to transcription/replication blocking or mutagenic bypass. Our previous work has focused on 8,5'-cyclopurine 2'-deoxyribonucleosides, a unique class of oxidatively induced DNA lesions that are specifically repaired by the NER pathway (see Brooks PJ [2008]: DNA Repair 7:1168-1179). Here we used EMSA to monitor the ability of sequence-specific transcription factors, HSF1, CREB, and NF-kappaB and "architectural" transcription factor, HMGA, to bind to their target sequences when 8, 5'(S)-cyclo-2'-deoxyadenosine (cyclo-dAdo) is present within their recognition sequences. For comparison, we also tested the effect of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dAdo) in the same recognition sequences. The presence of a cyclo-dAdo lesion in the target sequence essentially eliminated the binding activity of HSF1, CREB, and NF-kappa B whereas HMGA retained some of its binding activity. In contrast, 8-oxo-dAdo had no obvious effect on the binding activity of HSF1 and HMGA in comparison to lesion-free DNA. Notably, though, CREB and NFκB binding increased when an 8-oxo-dAdo lesion was present in their target sequence. Competition EMSA showed about 2-3-fold increased affinity of both proteins for the 8-oxo-dAdo containing target sequence compared to lesion-free DNA. Molecular modeling of the lesions in the NF-kappaB sequence indicated that 8-oxo-dAdo may form an additional hydrogen bond with the protein, thereby strengthening the binding of NF-kappa B to its DNA target. The cyclo-dAdo lesion, in contrast, distorted the DNA structure, providing an explanation for the inhibition of NF-kappaB binding.

Identification of Novel Small Molecule Inhibitors of the XPA Protein Using in Silico Based Screening

The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding, and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.

850 The role of DNA repair gene polymorphisms in the development of radiation-induced late toxicity in prostate cancer patients

Effective DNA repair machinery ensures maintenance of genomic integrity. Environmental insults, ageing and replication errors necessitate the need for proper DNA repair systems. Any alteration in DNA repair efficacy would play a dominant role in progression of squamous cell carcinoma of head and neck (SCCHN).Genotypes of XRCC1 gene–Arg194Trp, Arg280His, Arg399Gln and XPD Lys751Gln, by PCR-RFLP were studied in 278 SCCHN patients and an equal number of matched healthy controls residing in north India.In XRCC1 polymorphisms, Arg194Trp and Arg399Gln variants showed a reduced risk, whereas, XPD Lys751Gln variants exhibited ∼2-fold increase in SCCHN risk. With XRCC1-Arg280His variants, there was no association with SCCHN risk. Arg399Gln of XRCC1 appears to have a protective role in people those consume alcohol, while XPD Lys751Gln variants indicated ∼2-fold increased risk of SCCHN in all the co-variate groups.Comparison of gene–gene interaction among XRCC1 Arg280His and XPD Lys751Gln suggested enhanced risk of SCCHN by ∼2.3-fold in group one and ∼6.1-fold in group two. In dichotomized groups of this combination, the risk was ∼2.4 times. Haplotype analysis revealed the frequency of C–G–G–G and C–A–G–G to be significantly associated with an increased risk of SCCHN. On the contrary, T–G–A–A significantly diminished the risk. CART analysis results showed that the terminal node that contains homozygous mutants of XPD Lys751Gln and XRCC1 Arg194Trp, wild type of XRCC1 Arg399Gln and homozygous mutant of XRCC1 Arg280His, represent the highest risk group.Our results demonstrate high degree of gene–gene interaction involving DNA repair genes of NER and BER pathways, namely XRCC1 and XPD. This study amply demonstrates positive association of XPD Arg751Gln polymorphism with an increased risk of SCCHN. Further, XRCC1 Arg280His variant though dormant individually, may also contribute to the development of cancer in combination with XPD Arg751Gln.

ERCC1 protein, mRNA expression, and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma (HNSCC) patients treated with surgery and adjuvant cisplatin-based chemoradiation (CRT).

5540 Background: Adjuvant cisplatin-based CRT improves overall survival (OS) in some HNSCC patients (pts) presenting with risk features, but toxicity is not negligible. ERCC1 is involved in NER pathway and is associated with resistance to chemo- and RT. We studied ERCC1 expression and the SNP T19007C in these pts. Methods: It is a retrospective study in HNSCC pts submitted to surgery and presenting with pathologic risk features. They were treated with 60-70 Gy and concurrent cisplatin 100 mg/m2d1, 22, 43. ERCC1 protein, mRNA expression and the SNP were studied in archived tissue blocks. ERCC1 expression was evaluated by immunohistochemistry, using a semi-quantitative H- score, and its mRNA expression was determined by quantitative RT-PCR. T19007C genotypes were detected using PCR-RFLP carried out in genomic DNA extracted from normal lymph nodes. Results: 69 pts (median age 56 y, 81% male) were studied. Primary sites: oral cavity 41%, larynx 32%, hypopharynx 16%, oropharynx 12%; stage IV 86%, pT3-pT4 78% and pN2-pN3 58%. 43 pts presented with ≥2 +ve lymph nodes, 27 with extracapsular spread and 18 with +ve margins. High-risk features were diagnosed in 40 pts (58%). During the median follow-up of 47 mo., we observed 30 deaths (22 disease-related). The 5-y OS was 40% and the 5-y disease-free survival was 31%. High H-score (>1.5) was seen in 32 pts (54%), who presented better 5-y OS (50% vs. 18%, HR 0.43, 95%CI 0.20-0.90, p=0.026). 15 out of 45 pts (33%), whose tumors had high ERCC1 mRNA expression (>3.1), presented better 5-y OS (86% vs. 30%, HR 0.26, 95%CI 0.14-1.01, p=0.052). Genotype distribution in 49 pts was 39% C/T, 37% C/C, and 24% T/T. No difference was detected among genotypes in terms of 5-y OS (45%, 46%, 46%; p=0.808). High H-score and ERCC1 mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Conclusions: High ERCC1 expression was associated with better prognosis in HNSCC pts submitted to adjuvant cisplatin-based CRT. It may be useful to better stratify these pts, however a prospective validation is needed. No significant financial relationships to disclose.

Relationship between ERCC1 C8092A and Mad1 G558A polymorphism with disease progression, and survival in patients with advanced epithelial ovarian cancer treated with induction chemotherapy with carboplatine paclitaxel (PC).

5101 Background: Platinum and taxane are active drugs in the treatment of ovarian cancer.The polymorphism C8092A of ERCC1 is involved in NER pathway of platinum-induced damage DNA.Mad1 G558A with m...

Abstract 3593: Genetic variants in NER pathway genes and ovarian cancer risk and outcomes

Abstract Nucleotide excision repair (NER) pathway is critical for protecting against damage from carcinogens. In this case-control study, we evaluated the associations between genetic variations in NER pathway and ovarian cancer risk in 339 Caucasian ovarian cancer cases and 349 Caucasian healthy controls. A comprehensive panel of 217 SNPs in 28 NER pathway genes was analyzed to assess their association with ovarian cancer risk. For the cases who received surgery and platinum-based chemotherapy, we also analyzed the association of these SNPs with overall survival and response to chemotherapy. Individually, 12 SNPs were significantly associated with the ovarian cancer risk. The most significant association was observed for SNP rs4150667 from GTF2H1 (OR = 0.64, 95% CI 0.47- 0.86). A strong gene-dosage effect was observed when these top 12 SNPs were combined for unfavorable genotype analysis. Compared to subjects carrying ≤ 2 unfavorable genotypes, individuals carrying 3 (OR = 2.11, 95% CI = 1.32−3.39), 4 (OR = 2.32, 95% CI = 1.47 − 3.66) or ≥ 5 (OR = 4.31, 95% CI = 2.75 − 6.78) unfavorable genotypes exhibited significantly increased risk for ovarian cancer (P for trend < 0.0001). Classification and regression tree (CART) analysis further revealed potential high order gene-gene interaction among the top 12 SNPs and identified 7 terminal nodes with the percentage of ovarian cancer cases in each terminal node range from 27% to 64%. In the clinical outcome study, there were 10 SNPs that exhibited significant association with overall survival. The unfavorable genotype analysis showed the median survival times for individuals carrying ≤3, 4∼7, and ≥ 8 unfavorable genotypes were 83.3, 40.0, and 24.7 months, respectively, indicating a strong gene-dosage effect (P for trend < 0.0001). We also observed significant gene-dosage effect in chemotherapy response. In summary, this pathway-based approach demonstrated that increasing numbers of unfavorable genotypes in the NER predispose susceptible individuals to increased risk of ovarian cancer and affect clinical outcomes in ovarian cancer patients. These findings warrant further replications in independent populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3593.

Association between Mad1 G558A and ERCC1 C8092A polymorphisms and response to induction chemotherapy in advanced ovarian carcinoma

5568 Background: Platinum and taxane remain active drugs in the treatment of ovarian cancer.The polymorphism C8092A of ERCC1 is involved in NER pathway of platinum induced damage DNA and Mad1 G558A with modulation of the chromosomal segregation. As primary aim we evaluated the influence of these polymorphisms in the chemotherapy response. Methods: ERCC1 C8092A and Mad1 G558A polymorphism allelic and genotypic frequency was determined on DNA isolated from blood tumor samples by PCR and specific digestion in 56 patients with cancer of advanced ovary who received induction chemotherapy with 3 courses of paclitaxel 175 mg/m2 and AUC6 carboplatin every 3 weeks. Informed consent was obtained for all subjects and the study was approved by the hospital ethic committee. Results: Between January 2008 to June 2008, 56 women were evaluated, with age media was 53 years, majority characteristics were stages III (66.9%) 37 and IV (33.1%) 19 patients, histology serous papillary (94.6%) high-grade differenced (73.2%). Mad1 genotype were 7 G/G (12.5%), 30 were G/A (53.6%), 19 were A/A( 33.9%), and ERCC1 genotype were 20 C/C (35.7%), 30 were C/A (53.6%) and 6 were A/A (10.7%). All biochemical response by Rustin criteria was noted in 89.28%. Allele A for Mad1 is more frequently observed in patients with ovarian carcinoma versus in mexican healthy women (61% vs. 49%) and suggests the possibility that the polymorphism of Mad1 is a marker of risk for ovary cancer (RR = 1, 15, p < 0, 05). According our aim we observed a higher response in allele G (91.89%) than allele A (87.75%) of Mad1 (p < 0.10).Interestingly allele A of ERRC1 was not associated greater response (88.88% vs. 92%, p > 0.10). Additionally we evaluated the predictive value of Ca 125 return to normal range after 3 chemotherapy courses, showed in 22 patients (39.28%) in univariate analysis we noted that allele G of Mad1 has been linked to improved response (p = 0.041). Conclusions: This is the first study in ovary cancer that evaluates predictive value of two SNP, one of them involved in the spindle checkpoint and the other in DNA repair mechanism (NER), suggesting to Mad1 G558A likely risk polymorphism for ovary cancer and response to chemotherapy based on taxanes, which may be a predictive biomarker. No significant financial relationships to disclose.

Non–Small Cell Lung Cancer Exhibits Transcript Overexpression of Genes Associated with Homologous Recombination and DNA Replication Pathways

Genes involved in DNA repair and replication have been recently investigated as predictive markers of response to chemotherapy in non-small cell lung cancer (NSCLC). However, few data on the expression of these genes in tumor compared with corresponding normal lung are available. The aim of this study was to evaluate differential mRNA levels of 22 DNA repair genes of five different DNA repair pathways: direct, base excision, nucleotide excision (NER), double-strand break (DSBR), and postreplicative repair. In addition, six genes involved in DNA replication (REP) and three telomere maintenance genes were investigated. Total RNAs extracted from fresh-frozen tumors and corresponding normal tissues of 50 consecutive chemo-naïve resected NSCLC patients were analyzed. Transcript levels were quantified by real-time PCR. A significant overexpression was detected in 20 of 30 (67%) genes, mostly belonging to DSBR pathways, whereas others (XPA, XPC, and UBE2N; 10%) were significantly underexpressed. For 7 of 30 (23%) genes, mostly belonging to NER pathway, no significant difference between paired tumor and normal samples was observed. Transcript overexpression of DSBR and REP genes was significantly higher in poorly differentiated carcinomas and DSBR levels were higher in men compared with women. The transcriptional overexpression of four genes (XRCC5, TOP3B, TYMS, and UNG) showed significant correlation with a shorter patients' outcome at the univariate, whereas only stage of disease appeared as an independent factor affecting prognosis, as assessed by multivariate analysis. In conclusion, genes belonging to DNA repair/replication pathways are overexpressed in NSCLC and are associated with a more aggressive phenotype.

Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene

Benzo[a]pyrene is a ubiquitously distributed environmental pollutant known to cause DNA damage, whereas PARP-1 is a nuclear enzyme that is activated by damaged DNA and plays an important role in base excision repair and genomic stability. Here, 16HBE and its PAPR1-deficient cells were exposed to BaP, and the DNA damage level and repair ability of both cell lines were measured by alkaline comet assay. The results showed that cell viability of both cell lines decreased in a dose-dependent manner when exposed to BaP, but there was no significant difference between two cell lines. Comet assay showed that BaP caused DNA damage in both cell lines at an obvious dose- and time-dependent manner. Compare with 16HBE, the PARP1-deficient cells were more sensitive to the damage caused by BaP. The results of DNA repair experiment showed that both cell lines can recover from the damage in a time-dependent pattern. The relative repair percentage of PARP1-deficient cells were generally lower than that of 16HBE at all exposed concentrations at the early stage of repair, but tended to be closer between two cell lines at the later period. According to results, we came to the conclusion that PARP1-deficient cells were more sensitive to BaP in contrast to normal 16HBE; DNA repair capacity in PARP1-deficient cells decreased significantly at the early stage of repair, but increased to the equivalent level of normal 16HBE in the later period. PARP-1 plays an important role in early repair of DNA damage caused by BaP in 16HBE notwithstanding the main repair work is taken by NER pathway.

Distortion-Dependent Unhooking of Interstrand Cross-Links in Mammalian Cell Extracts

Interstrand cross-links (ICLs) are formed by many chemotherapeutic agents and may also arise endogenously. The mechanisms used to repair these lesions remain unclear in mammalian cells. Repair in Escherichia coli and Saccharomyces cerevisiae requires an initial unhooking step to release the tethered DNA strands. We used a panel of linear substrates containing different site-specific ICLs to characterize how structure affects ICL processing in mammalian cell extracts. We demonstrate that ICL-induced distortions affect NER-dependent and -independent processing events. The NER-dependent pathway produces dual incisions 5' to the site of the ICL as described previously [Bessho, T., et al. (1997) Mol. Cell. Biol. 17 (12), 6822-6830] but does not release the cross-link. Surprisingly, we also found that the interstrand cross-linked duplexes were unhooked in mammalian cell extracts in a manner independent of the NER pathway. Unhooking occurred identically in extracts prepared from human and rodent cells and is dependent on ATP hydrolysis and metal ions. The structure of the unhooked product was characterized and was found to contain the remnant of the cross-link. Both the NER-mediated dual 5' incisions and unhooking reactions were greatly stimulated by ICL-induced distortions, including increased local flexibility and disruption of base pairs surrounding the site of the ICL. These results suggest that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL could contribute significantly to initial cross-link recognition and processing.

Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

1,N6-Ethenoadenine (epsilonA) is generated endogenously by lipid peroxidation and exogenously by tumorigenic industrial agents, vinyl chloride, and vinyl carbamate. epsilonA detected in human tissues causes mutation and is implicated in liver, colon and lung cancers. N-methyl purine DNA-glycosylase (MPG) is the only enzyme known so far to repair epsilonA. However, the mechanism of in vivo repair of epsilonA and the role of MPG remain enigmatic. Moreover, previous in vivo repair studies for DNA lesions, including epsilonA, focused only on the step of the removal of the base lesion without further insight into the completion of the repair process. This may be in part due to the unavailability of an appropriate in vivo quantitative method to evaluate complete BER process at the basal level. Our newly developed in vivo method is highly sensitive and involves phagemid M13mp18, containing epsilonA at a defined position. The complete repair events have been estimated by plaque assay in E. coli with the phagemids recovered from the human cells after cellular processing. We found that the detectable complete (removal and replacement of epsilonA with adenine) repair was observed only 18% in 16 h, but with the repair nearing completion within 24 h in colon cancer, HCT-116, cells. Moreover, MPG is the predominant enzyme for the BER process to remove epsilonA in mammalian cells. Although, the epsilonA is fairly a bulky adduct compared to other small BER substrate lesions, NER pathway is not involved in repair of this adduct. Furthermore, the epsilonA repair in vivo and in vitro is predominant in the G0/G1 phase of the cell cycle.

Comparative analysis of DNA repair pathways in mammals.

In our study we used three groups of animals: rodents, wild mice and white footed mice; bats, little brown bat and Brazilian free‐tailed bat; primate, marmoset and humans. These groups have different longevity quotient: rodents (0.8 and 1.6); bats (8.1 and 2.7?); primates (4.2 and 2.3) respectively. We hypothesize that DNA repair ability and DNA stability might correlate with species maximum longevity. In order to study different pathways of DNA repair we treated cells with gamma‐radiation (targeting base excision repair, BER) or UV‐C light (targeting nucleotide excision repair, NER). We monitored cell survival as well as DNA‐damage level with comet‐assay. Our results indicate that bat's cells are similar to primate cells following gamma radiation – they survive better and have less damage then rodent's cells. This suggests that bats and primates have superior BER compared with rodents. However, following UV‐C treatment, bat's cells are similar to rodent's cells – they are very sensitive to UV‐type of damage; at same time both primate species show comparable resistance to UV, superior to rodent or bat. These results are indicating that NER pathway is not efficient in repairing DNA in rodents and bats. Study with caspase activation and ROS production are supporting our conclusion ‐ exceptional bat's longevity is not dependant from NER pathway.

DNA Repair Polymorphisms Modify Bladder Cancer Risk: A Multi-factor Analytic Strategy

Objectives: A number of common non-synonymous single nucleotide polymorphisms (SNPs) in DNA repair genes have been reported to modify bladder cancer risk. These include: APE1-Asn148Gln, XRCC1-Arg399Gln and XRCC1-Arg194Trp in the BER pathway, XPD-Gln751Lys in the NER pathway and XRCC3-Thr241Met in the DSB repair pathway. Methods: To examine the independent and interacting effects of these SNPs in a large study group, we analyzed these genotypes in 1,029 cases and 1,281 controls enrolled in two case-control studies of incident bladder cancer, one conducted in New Hampshire, USA and the other in Turin, Italy. Results: The odds ratio among current smokers with the variant XRCC3-241 (TT) genotype was 1.7 (95% CI 1.0–2.7) compared to wild-type. We evaluated gene-environment and gene-gene interactions using four analytic approaches: logistic regression, Multifactor Dimensionality Reduction (MDR), hierarchical interaction graphs, classification and regression trees (CART), and logic regression analyses. All five methods supported a gene-gene interaction between XRCC1-399/XRCC3-241 (p = 0.001) (adjusted OR for XRCC1-399 GG, XRCC3-241 TT vs. wild-type 2.0 (95% CI 1.4–3.0)). Three methods predicted an interaction between XRCC1-399/XPD-751 (p = 0.008) (adjusted OR for XRCC1-399 GA or AA, XRCC3-241 AA vs. wild-type 1.4 (95% CI 1.1–2.0)). Conclusions: These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and highlight the value of using multiple complementary analytic approaches to identify multi-factor interactions.

Polymorphism in nucleotide excision repair gene XPC correlates with bleomycin‐induced chromosomal aberrations

Chromosomal aberrations (CAs) are important genetic alterations in the development and progression of the majority of human cancers. The frequency with which such alterations occur depends to a large extent on polymorphisms of DNA-repair genes and in genes coding for xenobiotic metabolizing enzymes, which are involved in the processes of activation and inactivation of xenobiotics. The frequency of bleomycin (BLM)-induced CAs is an indirect measure of the effectiveness of DNA repair mechanisms, and a predictor of environment-related risk of cancer. Our study was conducted on the human peripheral blood lymphocytes of 82 healthy volunteers. The aim of the study was to elucidate whether the frequency of BLM-induced CAs is correlated with polymorphisms of selected genes involved in different mechanisms of DNA repair such as: XRCC1 [base excision repair]; XPA, XPC, XPG, XPD, XPF, ERCC1 [nucleotide excision repair], NBS1, RAD51, XRCC2, XRCC3, RAD51, and BRCA1 [homologous recombination], as well as in genes encoding xenobiotic metabolizing enzymes, such as CYP1A, CYP2E1, NAT2, GSTT1, and EPHX (mEH). Our study indicated that, of the polymorphisms studied, only XPC (exon 15 and intron 11) is associated with BLM-induced CAs, suggesting a role of the NER pathway in the repair of BLM-induced chromosomal aberrations.

Prognostic significance of nuclear excision (NER) and base excision (BER) DNA repair gene polymorphisms in esophageal cancer

2511 Background: DNA repair pathways are involved in cisplatin-induced damage (NER pathway) & radiation damage (BER pathway). Some single nucleotide polymorphisms (SNPs) of DNA repair genes are associated with DNA repair capacity, cancer risk & outcomes. We investigated the prognostic significance of 7 NER/BER SNPs on disease free (DFS) & overall survival (OS) in esophageal cancer. Methods: 150 patients with esophageal cancer treated with cisplatin-based chemoradiation & surgery were genotyped for BER (XRCC1 Arg399Gln; APE1 Asp148Glu; hOGG1 Ser326Cys) & NER (ERCC1 8092C/A; ERCC1 codon 118 C/T; XPD Asp312Asn; XPD Lys751Gln) SNPs. Analysis involved Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. Results: Median age: 63 years (range 28–80); 91% male; 100% ECOG performance status (PS) 0–1; adenocarcinoma 79%; stages IIA 22%, IIB 30%, III 33%, and IVA 15%. No SNPs were associated with stage or PS. Multiple NER SNP was independently prognostic for OS and DFS (see Table ). When compared to individuals who were wildtype in all four studied NER SNPs, individuals with variants in all four NER SNPs were associated with substantial improvement in OS (Adjusted Hazard Ratio (AHR) = 0.40, 95% confidence interval (CI) = 0.2–0.7) and DFS (AHR = 0.44, 95%CI = 0.2–0.8). Furthermore, increasing numbers of variant genotypes were associated with a progressive increase in OS & DFS when all seven NER/BER pathway SNPs were analysed together ( Table ). There was a 3.8- fold increase in OS (75 vs. 20 months) and five-fold increase in DFS (51 vs. 10 months) when comparing individuals with 6–7 SNPs with variant alleles to individuals with 0–1 SNPs with variant alleles. Conclusions: The ERCC1 8092 C/A, XPD Asp312Asn & XPD Lys751Gln SNPs in the NER pathway are associated individually with prognosis in esophageal cancer patients treated with cisplatin-based trimodality regimens. In addition, as the number of NER and BER SNPs carrying variant alleles increased, OS and DFS improved dramatically. [Table: see text] No significant financial relationships to disclose.

Genetic Variation in the Nucleotide Excision Repair Pathway and Bladder Cancer Risk

Nucleotide excision repair (NER) is critical for protecting against damage from carcinogens in tobacco smoke. We evaluated the influence of common genetic variation in the NER pathway on bladder cancer risk by analyzing 22 single nucleotide polymorphisms (SNP) in seven NER genes (XPC, RAD23B, ERCC1, ERCC2, ERCC4, ERCC5, and ERCC6). Our study population included 1,150 patients with transitional cell carcinoma of the urinary bladder and 1,149 control subjects from Spain. Odds ratios (OR) and 95% confidence intervals (95% CI) were adjusted for age, gender, region, and smoking status. Subjects with the variant genotypes for SNPs in four of the seven genes evaluated had small increases in bladder cancer risk compared to subjects with the homozygous wild-type genotypes: RAD23B IVS5-15A>G (OR, 1.3; 95% CI, 1.1-1.5; P = 0.01), ERCC2 R156R (OR, 1.3; 95% CI, 1.1-1.6; P = 0.006), ERCC1 IVS5+33A>C (OR, 1.2; 95% CI, 1.0-1.5; P = 0.06; P(trend) = 0.04), and ERCC5 M254V (OR, 1.4; 95% CI, 1.0-2.0; P = 0.04). A global test for pathway effects indicated that genetic variation in NER characterized by the 22 SNPs analyzed in this study significantly predicts bladder cancer risk (P = 0.04). Pairwise comparisons suggested that carrying variants in two genes could result in substantial increases in risk. Classification tree analyses suggested the presence of subgroups of individuals defined by smoking and NER genotypes that could have substantial increases in risk. In conclusion, these findings provide support for the influence of genetic variation in NER on bladder cancer risk. A detailed characterization of genetic variation in key NER genes is warranted and might ultimately help identify multiple susceptibility variants that could be responsible for substantial joint increases in risk.