NER Genes Research Articles

Impact of Polymorphism in Base Excision Repair and Nucleotide Excision Repair Genes and Risk of Cervical Cancer: A Case-Control Study.

Background:Last few years, several studies all over the world revealed the association of DNA repair genes with risk of developing different type of cancers, but were ambiguous to support the evidences in case of cervical cancer risk. These differences in earlier studies directed us to study the association of polymorphisms of BER genes (XRCC1, hOGG1, XPC) and NER genes (XPC, XPD) with cervical cancer susceptibility in the women of rural population of Maharashtra. Materials and Methods:The genetic polymorphism in BER and NER pathway genes was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using DNA isolated from intravenous blood samples of patients and normal controls. The study included 400 clinically confirmed cervical cancer patients and 400 healthy women from a tertiary care hospital (Krishna Hospital and Medical Research Centre) of south-western Maharashtra. The association of polymorphisms was confirmed by Odds ratio (OR) with 95% confidence interval. Results:The single nucleotide polymorphism (SNP) of BER genes including XRCC1, hOGG1 and APE1 were analyzed and the results were noted that 27466AA (OR=4.88; 95% CI: 3.61- 6.60; p<0.0001) and 28152AA (OR=2.89; 95% CI: 1.57- 5.31; p=0.0005) genotypes of XRCC1 (rs25489, rs25487) were significantly associated with cervical cancer risk. The 1245GG genotype of hOGG1 (rs1052133) (OR=45.30; 95% CI: 3.76- 7.46; p=0.001) also showed significant correlation, whereas 2197GG genotype of APE1 (rs1130409) gene showed negative association with cervical carcinogenesis (OR=0.59; 95% CI: 0.35- 0.97; p=0.005). Similarly when we studied SNPs of NER genes including XPC and XPD genes, 21151TT genotype of XPC (rs 2228000) was positively associated with cervical cancer development and 23591AA genotype of XPD (rs1799793) showed negative association (OR=0.34; 95% CI: 0.17- 0.64; p=0.001). Conclusion:The findings from this study supported that rs25489, rs25487SNPs of XRCC1, rs1052133 of hOGG1 and rs2228000 of XPC may increase cervical cancer risk, whereas rs1130409 SNP of APE1 and rs1799793 SNP of XPD gene lower the risk of cervical cancer in the studied population.

The multi-faceted high order polymorphic synergistic interactions among nucleotide excision repair genes increase the risk of lung cancer in North Indians

It is evident that gene-gene interactions are pervasive in the determination of the susceptibility of human diseases. Polymorphisms in nucleotide excision repair pathway (NER) genes can cause variations in the repair capacity and therefore, might lead to increase in susceptibility towards lung cancer through complex gene-gene and gene-smoking interactions.Logistic regression analysis, along with high order genetic interaction were analyzed using data mining tools such as multifactor dimensionality reduction (MDR) and classification and regression tree analysis (CART).Overall, a protective effect was reported when a combinatorial effect of SNPs were studied by applying logistic regression analysis. Multifactor dimensionality reduction (MDR) analysis, revealed that the four factor model i.e. XPC K939Q, XPA 5′UTR, XPG F670W and XPG D1104H had the best ability to predict lung cancer risk (CVC = 100, p < 0.0001). While a two factor model, including smoking and XPG F670W suggested smoking was associated with the risk of developing lung cancer (CVC = 100, p < 0.0001). Individually XPG F670W was identified as the primary risk factor. In classification and regression tree analysis (CART), we observed a 6-fold risk for SCLC patients carrying XPA 5′UTR (M), XPD K751Q (W) (OR: 6.20; 95%CI: 2.40–16.01, p = 0.0001).Polymorphic NER genes might jointly modulate lung cancer risk through gene-gene and gene-smoking interaction.

Nucleotide excision repair is\xa0a predictor of early relapse in pediatric acute lymphoblastic leukemia

BackgroundNucleotide Excision Repair (NER) is a major pathway of mammalian DNA repair that is associated with drug resistance and has not been well characterized in acute lymphoblastic leukemia (ALL). The objective of this study was to explore the role of NER in relapsed ALL patients. We hypothesized that increased expression of NER genes was associated with drug resistance and relapse in ALL.MethodsWe performed secondary data analysis on two sets of pediatric ALL patients that all ultimately relapsed, and who had matched diagnosis-relapse gene expression microarray data (GSE28460 and GSE18497). GSE28460 included 49 precursor-B-ALL patients, and GSE18497 included 27 precursor-B-ALL and 14 T-ALL patients. Microarray data were processed using the Plier 16 algorithm and the 20 canonical NER genes were extracted. Comparisons were made between time of diagnosis and relapse, and between early and late relapsing subgroups. The Chi-square test was used to evaluate whether NER gene expression was altered at the level of the entire pathway and individual gene expression was compared using t-tests.ResultsWe found that gene expression of the NER pathway was significantly increased upon relapse in patients that took 3 years or greater to relapse (late relapsers, P = .007), whereas no such change was evident in patients that relapsed in less than 3 years (early relapsers, P = .180). Moreover, at diagnosis, the NER gene expression of the early relapsing subpopulation was already significantly elevated over that of the late relapsing group (P < .001). This pattern was validated by an ‘NER score’ established by averaging the relative expression of the 20 canonical NER genes. The NER score at diagnosis was found to be significantly associated with disease-free survival in precursor-B-ALL (P < .001).ConclusionPatients are over two times more likely to undergo early relapse if they have a high NER score at diagnosis, hazard ratio 2.008, 95% CI (1.256–3.211). The NER score may provide a underlying mechanism for “time to remission”, a known prognostic factor in ALL, and a rationale for differential treatment.

Photoprotective effects of phloretin sulfonate against UVB-induced damage in skin cell model and human volunteers

The phenolic compound phloretin is a prominent member of the chemical class of dihydrochalcones. Phloretin is specifically found in apple and apple juice and known for its biological properties. In particular we were attracted by potential dermo-cosmetic applications. However, practical limitations of phloretin do exist due to its poor water-solubility. To increase its water-solubility, phloretin was sulfonated with sulfuric acid (98 wt.%) and mixed with saturated salt water to produce phloretin sodium sulfonate. Here we report the photoprotective effect of phloretin sulfonate, a new semi-synthetic derivative of phloretin. Results showed that phloretin sulfonate can attenulate cyclobutane pyrimidine dimers (CPDs) formation, glutathione (GSH) depletion and apoptosis induced by UVB. The photoprotective effect of phloretin sulfonate is tightly correlated to the enhancement of NER genes expression. Furthermore, phloretin sulfonate had inhibitory effects on the UVB-induced release of the inflammatory mediators such as IL-6 and prostaglandin-E2. We also confirmed the safety and clinical efficacy of phloretin sulfonate on human skin. Overall, results demonstrated significant benefits of phloretin sulfonate on the protection of keratinocyte against UVB-induced injuries and suggested its potential use in skin photoprotection.

Attenuated NER expressions of XPF and XPC associated with smoking are involved in the recurrence of bladder cancer.

The varied NER genes and smoking are two important risk factors of bladder cancer, but the mechanism of the NER protein and smoking in cancer progression, however, remains unclear. In this report, we compared the expressions of NER genes in 79 bladder cancer tissues with or without any recurrence by real-time PCR and then analyzed the varied NER genes by immunochemistry in 219 bladder cancer tissue samples. Based on the clinical data, we analyzed the clinical value of varied NER genes and smoking in 219 bladder cancers by the Kaplan-Meier method and Cox proportional hazards regression. We found the expressions of the NER gene XPF and XPC were significantly lower in bladder cancer tissues with a recurrence compared with those without a recurrence at mRNA level. Also, the patients with the XPF and XPC defect had a statistically significant lower median recurrence-free survival time than those without the XPF and XPC defect, and smoking can make this difference more remarkable. Our results suggest that XPF and XPC expression may be a potential predictive factor for bladder cancer, and smoking can not only influence the recurrence of bladder cancer as a single factor but also aggravate the results of the XPF defect and XPC defect.

Variants in nucleotide excision repair core genes and susceptibility to recurrence of squamous cell carcinoma of the oropharynx

Genetically determined capacity for NER may modulate both cancer risk and prognosis. Thus, we evaluated associations of seven selected variants in the NER core genes with recurrence risk in 658 squamous cell carcinoma of the oropharynx (SCCOP) patients treated principally by radiation. The seven polymorphisms in the core NER genes (XPC-rs2228000, XPC-rs2228001, XPD-rs1799793, XPD-rs13181, XPG-rs17655, ERCC1-rs3212986 and XPA-rs1800975) were genotyped using PCR-RFLP method and log-rank test and multivariable Cox models were used to evaluate the associations in both dominant and recessive genetic models. In a dominant model, we found that polymorphisms of XPC-rs2228000, XPD-rs1799793 and XPG-rs17655 were significantly associated with disease-free survival (log-rank, p = 0.014; p = 0.00008; p = 0.0007, respectively), and these polymorphisms were significantly associated with recurrence risk of SCCOP (hazard ratio (HR) = 1.6, 95% confidence interval (CI) 1.1-2.3 for XPC-rs2228000; HR = 0.4, 95% 0.3-0.6 for XPD-rs1799793 and HR = 0.5, 95% CI 0.4-0.8 for XPG-rs17655) after multivariable adjustment. Moreover, the borderline significant or significant associations were also found for these three polymorphisms in HPV16/18-positive SCCOP patients (HR = 1.6, 95% CI 1.0-4.1 for XPC-rs2228000; HR = 0.2, 95% CI 0.1-0.5 for XPD-rs1799793 and HR = 0.1, 95% CI 0.0-0.9 for XPG-rs17655). However, similarly significant associations were not found for these polymorphisms in a recessive model. These findings suggest that polymorphisms of XPC-rs2228000, XPD-rs1799793 and XPG-rs17655 in the NER core genes may contribute to recurrence risk of SCCOP, particularly HPV-positive SCCOP, in a dominant but not in a recessive model. However, validation of these results is warranted.

Functional, Genetic, and Epigenetic Aspects of Base and Nucleotide Excision Repair in Colorectal Carcinomas

DNA repair capacity (DRC) is a determinant not only of cancer development but also of individual response to therapy. Previously, altered base and nucleotide excision repair (BER and NER) have been described in lymphocytes of patients with sporadic colorectal cancer. We, for the first time, evaluate both excision repair capacities in human colon biopsies to study their participation in colorectal tumorigenesis. Seventy pairs of tumor and adjacent healthy tissues were analyzed for BER- and NER-specific DRC by a comet repair assay. Tissue pairs were further compared for expression levels of a panel of 25 BER and NER genes complemented by their promoter methylation status. We observed a moderate increase of NER-DRC (P = 0.019), but not of BER-DRC in tumors. There was a strong correlation between both tissues for all investigated parameters (P < 0.001). However, 4 NER (CSB, CCNH, XPA, XPD) and 4 BER (NEIL1, APEX1, OGG1, PARP1) genes showed a 1.08- to 1.28-fold change difference in expression in tumors (P < 0.05). Individual gene expression levels did not correlate with overall DRC, and we did not detect any aberrant methylation of the investigated genes. Our complex analysis showed that tumor cells are not deficient in BER and NER, but rather follow patterns characteristic for each individual and are comparable with adjacent tissue. Alteration of excision repair pathways is not a pronounced event in colorectal carcinogenesis. This study shows the feasibility of DRC evaluation in human solid tissues, representing a complex marker of multigene DNA repair processes.

Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage

Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients.

Genetic Polymorphisms in Detoxification and DNA Repair Genes and Susceptibility to Glycidamide-Induced DNA Damage

Acrylamide (AA) is a probable human carcinogen formed in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), the AA metabolite formed by epoxidation, is considered the ultimate genotoxic agent. In this study, the in vitro genotoxic potential of AA and GA in human whole blood leukocytes was compared using the alkaline comet assay. Although AA did not induce significant DNA damage in the concentrations tested (up to 1000 μM), GA markedly increased the percentage of tail DNA at concentrations ≥250 μM. Further, this study addressed the role of genetic polymorphisms in key genes involved in metabolism and DNA repair pathways (BER, NER, HRR, and NHEJ) on GA-induced genotoxicity assessed by the alkaline comet assay. The results obtained suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val and Lys939Gln) genes, either alone or in combination.

Zebrafish (Danio rerio) life-cycle exposure to chronic low doses of ethinylestradiol modulates p53 gene transcription within the gonads, but not NER pathways

Parental full life-cycle exposure to ethinylestradiol (EE₂) significantly affects embryo development and survival. One of the possible mechanisms of action of EE₂ may involve the impairment of an organism's ability to repair DNA damage. DNA repair mechanisms have sophistically evolved to overcome DNA damaging hazards that threaten the integrity of the genome. In the present study, changes in the transcription levels of key genes involved in two of the most thoroughly studied DNA repair systems in mammals were evaluated in adult zebrafish (Danio rerio) gonad upon full life-cycle exposure to chronic environmentally low levels of EE₂ (i.e., 0.5, 1 and 2 ng/L EE₂). Real time PCR was used to analyse the expression levels of nucleotide excision repair genes (NER) as well as the tumor suppressor p53 and downstream selected effectors, i.e., p21 (cyclin-dependent kinase inhibitor), GADD45α (growth arrest and DNA damage induced 45, alpha), bax (bcl2-associated X protein) and p53 key regulator MDM2 (murine double minute 2 protein). NER genes transcription levels in gonads did not differ significantly among treatments. In contrast, the number of transcripts of p53 gene was significantly increased in male gonads at all EE₂ exposure concentrations and in females at 1 ng/L EE₂. Despite the increase in p53 transcripts, transcription levels of p21, GADD45α and bax genes were not affected upon EE₂ treatment, whereas MDM2 gene expression significantly increased in females at the intermediate EE₂ dose (1 ng/L). Overall, the present study indicate that chronic low levels of EE₂ significantly modulates the transcription of p53, a key gene involved in DNA repair, particularly in male zebrafish gonads, which supports the hypothesis of an impact of EE2 in male gonad DNA repair pathways.

Abstract 2639: A community-based study of nucleotide excision repair polymorphisms in relation to risk of non-melanoma skin cancer

Abstract Nucleotide excision repair (NER) is responsible for protecting DNA in skin cells against ultraviolet radiation-induced damage. Using a candidate pathway approach, a matched case-control study nested within a prospective, community-based cohort was carried out to test the hypothesis that single nucleotide polymorphisms (SNPs) in NER genes are associated with susceptibility to non-melanoma skin cancer (NMSC). Histologically-confirmed cases of NMSC (n=900) were matched to controls (n=900) on age, gender, and skin type. Associations were measured between NMSC and 221 SNPs in 26 NER genes. Using the additive model, two tightly linked functional SNPs in ERCC6 were significantly associated with increased risk of NMSC: rs2228527 (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.20 - 2.05), and rs2228529 (OR 1.57, 95% CI 1.20 - 2.05). These associations were confined to basal cell carcinoma of the skin (BCC) (rs2228529, OR 1.78, 95% CI 1.30 - 2.44; rs2228527 OR 1.78, 95% CI 1.31 - 2.43). These novel, hypothesis-generating findings suggest functional variants in ERCC6 may be associated with an increased risk of NMSC that may be specific to BCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2639. doi:1538-7445.AM2012-2639

Expression of genes involved in DNA repair and cell cycle checkpoint pathways in Triple Negative compared to Luminal A Breast Cancer: a molecular characterization

Results Among the NER genes, ERCC1 (p < 0.0001) and XPA (p = 0.03) genes were significantly less expressed in TNBC than in LABC; among the FA genes, BRCA1 (p < 0.0001), FANCD2 (p < 0.0001), FANCF (p < 0.0001) and PALB2 (p = 0.0006) genes were significantly less expressed in TNBC than in LABC. Also CHK1 was less expressed in TNBC (p < 0.0001). In relation to the clinical-pathological characteristics, lower level of XPG and FANCA were associated with larger tumor size (≥ pT2) at definitive surgery in TNBC. The high expression of FANCA was related to an increase of either the Overall Survival (p = 0.0045) or the Event-Free Survival (p = 0.0141) on univariate analysis in TNBC. Conclusions DNA repair and cell cycle checkpoint related genes with particular regards to ERCC1/XPA in NER family, BRCA/FANC family and CHK1 may be useful as prognostic markers in TNBC and likely to be important in

Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls

Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P < 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups (P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC, XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P < 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

Abstract 886: Germline variation in nucleotide excision repair genes and lung cancer risk in smokers

Abstract Nucleotide excision repair (NER) is the primary mechanism for detecting and repairing bulky DNA lesions, which in the respiratory tract can be induced by tobacco smoke and other environmental exposures. By altering DNA repair capacity, single nucleotide polymorphisms (SNPs) in NER protein-encoding genes may affect lung cancer risk, especially in smokers. Although meta-analyses have suggested some associations between NER SNPs and lung cancer (e.g., ERCC2 rs13181 and XPA rs1800975), they are largely built upon studies that have examined a limited number of SNPs in a few genes in fairly small samples. To comprehensively assess the influence of NER gene variation on lung cancer risk, we genotyped 78 SNPs tagging the regions spanning ±2500 base pairs of the ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, LIG1, POLE, XPA and XPC genes in lung cancer cases and controls selected from the Beta-Carotene and Retinol Efficacy Trial, a randomized chemoprevention trial in smokers. Controls were matched to cases on age, sex, race, enrollment year, and baseline measures of smoking status and occupational asbestos exposure. Gene coverage by the tag SNPs selected using HapMap CEU data (dbSNP build 129, ≫5% minor allele frequency, pairwise r2 &amp;gt;0.8) ranged from 91.7% to 100%. To assess SNP-level associations, we computed odds ratios (OR) and 95% confidence intervals (CI) adjusted for matching variables using logistic regression. To assess gene-level associations, we computed global p-values for likelihood ratio tests comparing each model with all SNPs in a given gene to that with no SNPs. Analyses were limited to Caucasians (746 cases, 1477 controls). At the gene level, XPA (pglobal=0.008) and LIG1 (pglobal=0.039) were associated with lung cancer risk. Associations were detected for two of the nine XPA SNPs examined, rs3176683 (OR per G allele: 0.77, 95% CI: 0.58-1.00, p=0.05) and rs3176658 (OR per A allele: 0.83, 95% CI: 0.69-1.00, p=0.05), and three of the eleven LIG1 SNPs examined, rs156640 (OR per G allele: 1.2, 95% CI: 1.1-1.4, p=0.002), rs156641 (OR per A allele: 1.2, 95% CI: 1.1-1.4, p=0.003), and rs8100261 (OR per A allele: 0.87, 95% CI: 0.77-0.99, p=0.03). The two XPA SNPs (r2=0.01) exhibited stronger associations when included together in a single regression model. In diplotype analyses, lung cancer risk was lower for those carrying the homozygous major genotypes of rs3176683 and rs3176658, relative to both heterozygous genotypes (OR: 0.38, 95% CI: 0.17-0.87). Lung cancer risk was higher for those carrying the homozygous minor genotypes of rs156640 and rs156641 and homozygous major genotype of rs8100261, relative to the homozygous major genotypes of rs156640 and 156641 and homozygous minor genotype of rs8100261 (OR: 1.7, 95% CI: 1.2-2.3). Although reported ERCC2 rs13181 and XPA rs1800975 associations could not be confirmed, our data nonetheless support the hypothesis that germline variation in NER genes influences lung cancer risk. 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 886. doi:10.1158/1538-7445.AM2011-886

Abstract LB-281: Inhibition of Hedgehog results in altered regulation of c-jun and of ERCC1 in cisplatin-resistant A2780-CP70 human ovarian cancer cells

Abstract Cancer stem cells are presumed to be highly resistant to chemotherapy. Activation of the Hedgehog pathway has been reported as necessary, for the cancer stem cell state. A key gene in the Hedgehog pathway is Gli1. Resistance to platinum compounds is due in part, to up-regulated nucleotide excision repair, NER. ERCC1 is a key marker for NER activity. Transcriptional control of ERCC1 occurs in part, through c-Jun. Upon treatment with cisplatin, cisplatin-resistant A2780-CP70 cells normally up-regulate c-jun and c-fos, which then up-regulate ERCC1 and other NER genes. More specifically, the c-jun Ser63/73 cascade is up-regulated, with up-regulation of activator protein 1 (Reed, JBC 1998). We studied the relationship between Gli1 expression, ERCC1 expression, and cisplatin resistance in the human ovarian cancer cell lines A2780 and A2780-CP70. A2780-CP70 cells are 10-15 fold more resistant to cisplatin than A2780 cells. We used two different probes to modulate the Hedgehog pathway: a) cyclopamine as a pharmacologic inhibitor of the Smoothened; and, b) an anti-Gli1 shRNA to specifically inhibit Gli1. At baseline, cisplatin-resistant cells, expressed &amp;gt; 10-fold more Gli1 protein than cisplatin-sensitive cells. In cisplatin-resistant cells, Gli1 protein was down-regulated in response to cyclopamine, and to anti-Gli1 shRNA. When A2780-CP70 cisplatin-resistant cells are pretreated with cyclopamine, or anti-Gli1 shRNA, the following events occur. The c-jun Thr91/93 cascade is upregulated, which is associated with pro-apoptotic processes. The c-jun Ser63/73 cascade is suppressed. This cascade is associated with pro-growth processes. If cisplatin is given after cyclopamine or anti-Gli1 pretreatment; Gli1 protein is down-regulated; the c-jun Thr91/93 cascade is upregulated; the c-jun Ser63/73 cascade is suppressed; and cells do not up-regulate ERCC1, XPD, or XRCC1. Thus, the normal cisplatin-induced up-regulation of these three genes involved in NER (ERCC1, XPD) and in base excision repair (XRCC1), is suppressed by suppression of the Hedgehog pathway. We conclude that Gli1 and/or other genes involved in the Hedgehog pathway (which is critical for cancer stem cells), may play key roles in the up-regulation of DNA repair genes, specifically in response to DNA damaging agents such as cisplatin. 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 LB-281.

Abstract CN4-3: Factors that influence the regulation of ERCC1 in drug-resistant cells

Abstract ERCC1 is an excellent marker for nucleotide excision (NER) repair activity. ERCC1 expression in tumor tissues has been closely correlated with clinical resistance to platinum coordination complexes in lung cancer, ovarian cancer, gastric cancer, and other malignancies. We have previously demonstrated that up-regulation of ERCC1 is coordinated with up-regulation of other genes in the NER excision repair protein complex, such as XPA, XPD, XPB, etc (Reed and colleagues; Mut Res 1993, J Clin Invest 1994, Cancer Res 1995, Biochem Pharm 2000). The positive transcriptional regulator for ERCC1 is activator protein 1, or AP1, a heterodimer of c-jun and c-fos (Reed, JBC 1998). The negative transcriptional regulator for ERCC1 is the myeloid zinc finger gene MZF1 (Reed, Biochem Pharm 2006). Cancer stem cells are presumed to be highly resistant to chemotherapy. Activation of the Hedgehog pathway has been reported as necessary, for the cancer stem cell state. A key gene in the Hedgehog pathway is Gli1. Resistance to platinum compounds is due in part, to up-regulated nucleotide excision repair, NER. Upon treatment with cisplatin, cisplatin-resistant A2780-CP70 cells normally up-regulate c-jun and c-fos, which then up-regulate ERCC1 and other NER genes. More specifically, the c-jun Ser63/73 cascade is up-regulated, with up-regulation of activator protein 1 (Reed, JBC 1998). We therefore studied the relationships between Gli1 expression, c-jun expression, ERCC1 expression, and cisplatin resistance in the human ovarian cancer cell lines A2780 and A2780-CP70. A2780-CP70 cells are 10–15 fold more resistant to cisplatin than A2780 cells. We used two different probes to modulate the Hedgehog pathway: a) cyclopamine as a pharmacologic inhibitor of the Smoothened; and, b) an anti-Gli1 shRNA to specifically inhibit Gli1. At baseline, cisplatin-resistant cells, expressed &amp;gt;10-fold more Gli1 protein than cisplatin-sensitive cells. In cisplatin-resistant cells, Gli1 protein was down-regulated in response to cyclopamine, and to anti-Gli1 shRNA. When A2780-CP70 cisplatin-resistant cells are pretreated with cyclopamine, or anti-Gli1 shRNA, the following events occur. The c-jun Thr91/93 cascade is upregulated, which is associated with pro-apoptotic processes. The c-jun Ser63/73 cascade is suppressed. This cascade is associated with pro-growth processes. If cisplatin is given after cyclopamine or anti-Gli1 pretreatment; Gli1 protein is down-regulated; the c-jun Thr91/93 cascade is upregulated; the c-jun Ser63/73 cascade is suppressed; and cells do not up-regulate ERCC1, XPD, or XRCC1. Thus, the normal cisplatin-induced up-regulation of these three genes involved in NER (ERCC1, XPD) and in base excision repair (XRCC1), is suppressed by suppression of the Hedgehog pathway. We conclude that Gli1 and/or other genes involved in the Hedgehog pathway (which is critical for cancer stem cells), may play key roles in the up-regulation of DNA repair genes, specifically in response to DNA damaging agents such as cisplatin. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN4-3

Genetic Characterization of the Nucleotide Excision Repair System of Neisseria gonorrhoeae

Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen.

NAT2 and NER genetic variants and sporadic prostate cancer susceptibility in African Americans

Prostate cancer is a common malignancy that disproportionately affects African-American men. Environmental factors and variation in genes responsible for chemical and dietary carcinogen metabolism and DNA damage repair may modulate risk. Fourteen single nucleotide polymorphisms in NAT2 and four NER genes (ERCC1, XPF/ERCC4, XPG/ERCC5 and CSB/ERCC6) were genotyped in a case-control study of 254 African-American prostate cancer cases and 301 healthy controls from Washington, DC. Smoking status, BMI, age and genetic ancestry were included as covariates in the association analyses. We found that individuals homozygous for the XPG/ERCC5 -72C/T promoter polymorphism had a significant reduction in risk, for prostate cancer (OR=0.12; 95% CI=0.03-0.48). A haplotype trend regression test also revealed a protective effect for the haplotype bearing the T allele (P=0.003). In silica analyses suggest a functional implication for the promoter variant since it deletes a GCF transcriptional factor-binding site responsible for the downregulation of transcription. The protective effect of the promoter SNP on risk for prostate cancer was independent of smoking. In contrast, none of the SNPs typed for NAT2, ERCC1, ERCC4 and ERCC6 showed significant association with risk. Additional tests for genotype interactions were not significant. We note that there may be other factors, such as dietary exposures, which may modulate prostate cancer risk in combination with genetic variation within the NAT2 and NER genes. Our results, in combination with previous observations of LOH for ERCC5 in prostate tumors, provide further evidence for a role of XPG/ERCC5 in the etiology of prostate cancer.

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers

Transcription Domain-Associated Repair in Human Cells

Nucleotide excision repair (NER), which is arguably the most versatile DNA repair system, is strongly attenuated in human cells of the monocytic lineage when they differentiate into macrophages. Within active genes, however, both DNA strands continue to be proficiently repaired. The proficient repair of the nontranscribed strand cannot be explained by the dedicated subpathway of transcription-coupled repair (TCR), which is targeted to the transcribed strand in expressed genes. We now report that the previously termed differentiation-associated repair (DAR) depends upon transcription, but not simply upon RNA polymerase II (RNAPII) encountering a lesion: proficient repair of both DNA strands can occur in a part of a gene that the polymerase never reaches, and even if the translocation of RNAPII is blocked with transcription inhibitors. This suggests that DAR may be a subset of global NER, restricted to the subnuclear compartments or chromatin domains within which transcription occurs. Downregulation of selected NER genes with small interfering RNA has confirmed that DAR relies upon the same genes as global genome repair, rather than upon TCR-specific genes. Our findings support the general view that the genomic domains within which transcription is active are more accessible than the bulk of the genome to the recognition and repair of lesions through the global pathway and that TCR is superimposed upon that pathway of NER.