Expression Of XRCC1 Research Articles

Effects of JWA, XRCC1 and BRCA1 mRNA expression on molecular staging for personalized therapy in patients with advanced esophageal squamous cell carcinoma.

BackgroundDNA damage repair genes JWA, XRCC1 and BRCA1 were associated with clinical outcomes and could convert the response to the cisplatin-based therapy in some carcinomas. The synergistic effects of JWA, XRCC1 and BRCA1 mRNA expression on personalized therapy remain unknown in advanced esophageal squamous cell carcinoma (ESCC).MethodsWe employed quantitative real-time polymerase chain reaction (qPCR) to determine the expression of JWA, XRCC1 and BRCA1 mRNA in paraffin-embedded specimen from 172 patients with advanced ESCC who underwent the first-line cisplatin-or docetaxel-based treatments.ResultsHigh JWA or XRCC1mRNA expression was correlated with longer median overall survival (mOS) in all the patients (both P < 0.001) or in subgroups with different regimens (all P < 0.05), but not correlated with response rate (RR, all P > 0.05). Multivariate analysis revealed that high JWA (HR 0.22; 95% CI 0.13-0.37; P < 0.001) or XRCC1 (HR 0.36; 95% CI 0.21-0.63; P < 0.001) mRNA expression emerged as the independent prognostic factors for ESCC patients in this cohort. But no significant difference in prognostic efficacy was found between JWA plus XRCC1 and JWA alone through ROC analysis. Further subgroup analysis showed cisplatin-based treatments could improve mOS of patients with low JWA expression (P < 0.05), especially in those with low BRCA1 expression simultaneously (P < 0.001); while in patients with high JWA expression, high BRCA1 mRNA expression was correlated with increased mOS in docetaxel-based treatments (P = 0.044).ConclusionJWA, XRCC1and BRCA1 mRNA expression could be used as predictive markers in molecular staging for personalized therapy in patients with advanced ESCC who received first-line cisplatin- or docetaxel-based treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1364-0) contains supplementary material, which is available to authorized users.

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. SiHa cells were cultured and treated with 10% Noni, 10 μg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice

Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1+/− mice than in wild-type mice. Additionally, a greater percentage of Xrcc1+/− mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery.

Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells

Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells.

TXNL1 induces apoptosis in cisplatin resistant human gastric cancer cell lines.

Cisplatin is one of the most commonly used drugs in the treatment of gastric cancer. However, drug resistance is a major obstacle for effective treatment and originates in multiple mechanisms such as enhanced DNA repair and anti-apoptosis. Our previous results demonstrated that XRCC1 was a key regulator of cisplatin induced DNA damage and apoptosis. TXNL1, a member of the thioredoxin family, negatively regulated the expression of XRCC1 via the ubiquitin-proteasome pathway. Here, we investigated the role of TXNL1 in the apoptosis induced by cisplatin. Our data showed that the expression of TXNL1 in the cisplatin resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP cells was significantly lower compared with the cisplatin sensitive cell lines BGC823 and SGC7901. Inhibition of the expression of TXNL1 in BGC823 and SGC7901 cells led to increased resistance to cisplatin induced apoptosis and cell death detected by Tunel and clonogenic assay, respectively. In contrast, over expression of TXNL1 in BGC823/DDP and SGC7901/DDP cells lead to higher cisplatin induced apoptosis and cell death. Moreover, our results demonstrated that the mechanism of TXNL1 regulating cisplatin-induced apoptosis was closely associated with Bcl-2 mediated mitochondria apoptosis pathway. In conclusion, these findings suggest that TXNL1 was a feasible modulator and potential chemotherapeutic target for the cisplatin resistant phenotype of human gastric cancer cells.

Microrna Expression and Drug-Induced Changes in Gene Expression Correlate with Ara-C Chemosensitivity in AML Cell Lines

Abstract:Acute myeloid leukemia (AML) is a heterogeneous disorder, which is characterized by chromosomal abnormalities and genetic alterations. Cytarabine (Ara-C) is the most commonly used nucleoside analog for treatment of AML. However, the use of Ara-C is associated with two important clinical complications namely, inter-patient variability in response and development of intrinsic resistance. The inter-patient variability in response can be partly associated with polymorphisms in proteins that are required for intracellular uptake and activation of Ara-C to its phosphorylated form. Apart from genetic polymorphisms, expression of proteins involved in the uptake, activation, and inactivation of Ara-C have been shown to correlate with the overall patient survival. Over the past decade, various studies have identified microRNAs as important post-transcriptional regulators of gene expression. However, there are no studies till date that have identified key miRNAs involved in regulation of Ara-C pathway genes. Identification of these miRNAs will help in targeting these miRNAs to further understand inter-patient differences in gene expression. Additionally, drugs can also influence gene expression. However, there is critical gap in literature regarding role of Ara-C in inducing changes in gene expression. Understanding the dynamics of gene expression due to miRNAs and drug-induced changes would help open new opportunities for development of improved treatment strategies.Thus, the objective of this study is to understand the role of microRNAs in altering cytarabine cytotoxicity by influencing expression of pharmacokinetics (PK) and pharmacodynamics (PD) genes (n=18) in AML cell lines representing different risk groups. We evaluated genome-wide miRNA expression in 7 AML cell lines from different risk groups (favorable risk group: Kasumi-1, ME-1; intermediate risk group: AML-193, KG-1; adverse risk group: HL-60, MV-4-11, MOLM-16). We also evaluated the impact of cytarabine-induced gene expression changes in these AML cell lines. The gene expression changes were correlated with the in vitro chemosensitivity.Our preliminary results indicate that there was a significant correlation between the baseline miRNA expression for 16 miRNAs and Ara-C IC50 values (selected shown in Figure 1). We also observed that 57 microRNAs were associated with gene expression levels of the selected 18 Ara-C pharmacogenes (selected shown in Figure 1). Four miRNAs (miR-425-5p, miR-517a-3p, miR-519b-5p+hsa-miR-519c-5p, miR-522-3p) were found to be significantly associated with both gene expression and Ara-C IC50 values. We found that there were significant changes in gene expression of Ara-C pathway genes following treatment with 1uM or 10uM Ara-C. Briefly, there were significant changes in DCK, SLC29A1, CTPS1, CMPK1, NME1 and XRCC1 expression when treated with 10uM Ara-C and RRM2, NME1 and XRCC1 expression when treated with 1uM Ara-C.In conclusion, drug-induced changes in gene expression and miRNAs expression were found to correlate with chemosensitivity of AML cell lines. The preliminary results from our study help provide an insight into potential/additional molecular mechanisms associated with resistance observed in AML patients. Such knowledge is clinically significant, as identification of factors that contribute to the variable drug response would help in understanding and thus improving the variability in efficacy associated with cytarabine therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNA polymerase β-dependent cell survival independent of XRCC1 expression.

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.

XRCC2 promotes colorectal cancer cell growth, regulates cell cycle progression, and apoptosis.

X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2) and poly(ADP-ribose) polymerase 1 (PARP1) both play important roles in homologous recombination DNA repair. According to the theory of synthetic lethality, XRCC2-deficient cells are more sensitive to PARP1 inhibitors compared to XRCC2-expressing cells. We investigated XRCC2 expression and function in colorectal cancer (CRC), and the characteristics of sensitivity to PARP1 inhibitor in CRC cells with different XRCC2 levels. We enrolled 153 patients with CRC who had undergone surgery in this study. XRCC2 expression was assessed using immunohistochemistry. Stable CRC SW480 cell lines with low or high XRCC2 expression were constructed. Following treatment with the PARP1 inhibitor olaparib, the viability of cells with different XRCC2 levels was determined; cell cycle distribution and apoptosis were analyzed using flow cytometry. B-cell lymphoma-2 (Bcl-2) protein expression was measured by Western blotting. The positive rates of XRCC2 in primary CRC tissue were significantly higher than that in the matched adjacent noncancerous tissue, and XRCC2 expression status in primary CRC was related to tumor site, Dukes' stage, and tumor-nodes-metastasis (TNM) stage. XRCC2 overexpression inhibited CRC cell apoptosis and promoted proliferation by enriching cells in the G0/G1 phase. Moreover, olaparib suppressed proliferation, and olaparib sensitivity in CRC cells with high XRCC2 expression was greater. High XRCC2 expression promotes CRC cell proliferation and enriches cells in the G0/G1 phase but inhibits apoptosis. High XRCC2 expression cells are more sensitive to olaparib, which inhibits their viability.

JWA reverses cisplatin resistance via the CK2-XRCC1 pathway in human gastric cancer cells.

Gastric cancer is the third most common malignancy in China, with a median 5-year survival of only 20%. Cisplatin has been used in first-line cancer treatment for several types of cancer including gastric cancer. However, patients are often primary resistant or develop acquired resistance resulting in relapse of the cancer and reduced survival. Recently, we demonstrated that the reduced expression of base excision repair protein XRCC1 and its upstream regulator JWA in gastric cancerous tissues correlated with a significant survival benefit of adjuvant first-line platinum-based chemotherapy as well as XRCC1 playing an important role in the DNA repair of cisplatin-resistant gastric cancer cells. In the present study, we demonstrated the role of JWA in cisplatin-induced DNA lesions and aquired cisplatin resistance in five cell-culture models: gastric epithelial cells GES-1, cisplatin-sensitive gastric cancer cell lines BGC823 and SGC7901, and the cisplatin-resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP. Our results indicated that JWA is required for DNA repair following cisplatin-induced double-strand breaks (DSBs) via XRCC1 in normal gastric epithelial cells. However, in gastric cancer cells, JWA enhanced cisplatin-induced cell death through regulation of DNA damage-induced apoptosis. The protein expression of JWA was significantly decreased in cisplatin-resistant cells and contributed to cisplatin resistance. Interestingly, as JWA upregulated XRCC1 expression in normal cells, JWA downregulated XRCC1 expression through promoting the degradation of XRCC1 in cisplatin-resistant gastric cancer cells. Furthermore, the negative regulation of JWA to XRCC1 was blocked due to the mutation of 518S/519T/523T residues of XRCC1, and indicating that the CK2 activated 518S/519T/523T phosphorylation is a key point in the regulation of JWA to XRCC1. In conclusion, we report for the first time that JWA regulated cisplatin-induced DNA damage and apoptosis through the CK2—P-XRCC1—XRCC1 pathway, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.

Putative functional variants of XRCC1 identified by RegulomeDB were not associated with lung cancer risk in a Korean population

The Encyclopedia of DNA elements (ENCODE) project revealed that nearby or distantly located non-coding DNA regulates the expression of coding genes. RegulomeDB (http://regulome.stanford.edu) is a new database that can be used to predict whether a variant affects transcription factor binding and gene expression. We investigated the association between lung cancer risk and potentially functional polymorphisms of XRCC1 that were selected using RegulomeDB in a Korean population. A total of 185 polymorphisms of XRCC1 were evaluated using RegulomeDB. Strong evidence suggested that 10 polymorphisms, from among the 185, affected XRCC1 expression with scores of 1a-1f that were based on the RegulomeDB scoring system. The rs2854510 polymorphism was rare in Asians (minor allele frequency < 0.05). Eight polymorphisms were in strong linkage disequilibrium (LD). The rs2854509 polymorphism, which was one of the 8 polymorphisms in LD, and rs7248167, which was not in the LD block, were genotyped in 610 lung cancer patients and 607 age- and sex-matched controls. Additionally, four polymorphisms of XRCC1 (rs25487, rs25489, rs1799782, and rs3213245), which were investigated with regard to their association with lung cancer risk in previous studies, were also genotyped. Two polymorphisms (rs2854509 and rs7248167) that were predicted to affect XRCC1 expression based on their RegulomeDB scores were not associated with lung cancer risk (P = 0.31 and 0.93, respectively). When stratified according to age, gender, smoking status, and tumor histology, the two polymorphisms of XRCC1 were not associated with lung cancer risk. Among the four polymorphisms that were previously studied, only rs25489 of XRCC1 was significantly associated with lung cancer risk (dominant model, adjusted odds ratio = 0.61, 95% confidence interval = 0.46-0.83, P = 0.002). Although RegulomeDB is an attractive tool for predicting the regulatory potential of variants, the two polymorphisms that were selected using RegulomeDB were not associated with lung cancer risk.

XRCC2 gene polymorphisms and its protein are associated with colorectal cancer susceptibility in Chinese Han population.

XRCC2 is an essential part of the homologous recombination repair pathway. However, relatively little is known about the effect of XRCC2 gene C41657T and G4234C polymorphisms on the individual susceptibility to colorectal cancer (CRC). The purpose of this study was to investigate the association between XRCC2 gene C41657T and G4234C polymorphisms and CRC and to explore the relationship among the polymorphisms and clinicopathologic parameters and protein expression levels of XRCC2. A hospital-based case-control study was conducted with 246 CRC cases and 262 healthy controls. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. XRCC2 protein was analyzed by immunohistochemistry for the paraffin sections of 120 CRC cases. The study data showed that the C41657T genotypes were associated with the risk of CRC. The CT/TT genotypes and T allele were overrepresented among the CRC cases. Compared with CC, CT/TT enhanced the risk of CRC (odds ratio = 1.646, 95 % confidence interval = 1.127-2.404, P = 0.010). XRCC2 protein expression of CRC patients with CT/TT genotypes was significantly higher than that of the patients with CC genotype (χ (2) = 4.887, P = 0.027). XRCC2 gene G4234C polymorphisms have no relevance to the risk of CRC. Our findings suggest that XRCC2 C41657T polymorphism may adjust the XRCC2 expression and might influence susceptibility of CRC.

Abstract 902: XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay

Abstract The objective of this study was to use nanoimmunoassay (NIA) to quantify XRCC1 levels and correlate these with response to platinum-based chemotherapeutic agents in HNSCC cell lines. NIA is a developing technique that uses nanogram-scale samples to quantify specific proteins by immunoassay after separation by isoelectric focusing. Detection is more sensitive and quantification more accurate than with standard immunoblot techniques. XRCC1 is a base-excision repair protein that has been identified as a possible mediator of resistance to platinum-based chemotherapeutic agents, but its role and predictive utility have undergone limited investigation in head and neck squamous cell carcinoma (HNSCC). Detection of XRCC1 by NIA was first optimized. In lysates of HNSCC cell lines, XRCC1 was consistently identified at a pI of 5.45 ± 0.1. Peak intensity was improved by the addition of 7 M urea to the specimen prior to loading. In order to correct for sample and loading variability, and detection of a simultaneous internal loading control was also optimized. Under conditions that were optimal for detection of XRCC1, thioredoxin as consistently identified at a pI of 4.90 ± 0.1. Using 9 HNSCC cell lines, XRCC1 levels were evaluated using whole cell lysates in the untreated (basal) state as well as 24 h after treatment with cisplatin or vehicle. NIA was performed on 200 ng of whole cell lysate and XRCC1 and thioredoxin peak areas were quantified using the associated software. Proliferation assays were performed using alamarBlue to determine the IC50 for cisplatin in each cell line. Basal XRCC1 expression levels did not correlate with sensitivity to cisplatin. However, XRCC1 levels were noted to be significantly altered after treatment. When XRCC1 levels were normalized to the thioredoxin loading control, the ratio of treated to basal XRCC1 was correlated to the IC50 for cisplatin across all cell lines (R2=0.428). In cell lines identified as resistant to cisplatin, XRCC1 levels increased upon treatment, whereas in sensitive cell lines, levels remained unchanged. These NIA findings were confirmed using XRCC1 immunoblot with beta-actin as a loading control; XRCC1 levels obtained using this approach also correlated with the IC50 for cisplatin (R2=0.518). While timing and methodology need to be further elucidated, the change in XRCC1 protein level in response to a cisplatin challenge has the potential to be used as a biomolecular predictor of sensitivity that could direct treatment modality selection early in the course of therapy for HNSCC patients. The quantities of protein used allow for potential evaluation of fine-needle aspirates in patients undergoing treatment. Citation Format: Stephen S. Schoeff, Dane M. Barrett, James Teng, Ashraf Khalil, Matthew A. Hubbard, Anne K. Maxwell, Amir Allak, Rolando E. Mendez, Mark Axelrod, Mark J. Jameson. XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 902. doi:10.1158/1538-7445.AM2014-902

Effect of APE1 T2197G (Asp148Glu) polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in patients with colorectal cancer.

It has been hypothesized that genetic variation in base excision repair (BER) might modify colorectal adenoma risk. Thus, we evaluated the influence of APE1 T2197G (Asp148Glu) polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in normal and tumor samples from patients with colorectal cancer. The results indicate a downregulation of OGG1 and an upregulation of XRCC1 expression in tumor tissue. Regarding the anatomical location of APE1, OGG1 and PARP-1, a decrease in gene expression was observed among patients with cancer in the rectum. In patients with or without some degree of tumor invasion, a significant downregulation in OGG1 was observed in tumor tissue. Interestingly, when taking into account the tumor stage, patients with more advanced grades (III and IV) showed a significant repression for APE1, OGG1 and PARP-1. XRCC1 expression levels were significantly enhanced in tumor samples and were correlated with all clinical and histopathological data. Concerning the polymorphism T2197G, GG genotype carriers exhibited a significantly reduced expression of genes of the BER repair system (APE1, XRCC1 and PARP1). In summary, our data show that patients with colorectal cancer present expression changes in several BER genes, suggesting a role for APE1, XRCC1, PARP1 and OGG1 and APE1 polymorphism in colorectal carcinogenesis.

Association of reduced XRCC2 expression with lymph node metastasis in breast cancer tissues.

The main purpose of this study was to evaluate the association between reduction in XRCC2 gene and involvement of lymph node metastasis in breast cancer. In first part of the study, meta-analysis of 14 published XRCC2 studies was performed to define the role of XRCC2 gene as diagnostic marker and in second part of the study XRCC2 gene expression was observed using real time PCR in study cohort of 100 females (50 breast cancer patients and 50 controls). A statistically significant down regulation of XRCC2 (p < 0.04) and up-regulation of ki-67 (p < 0.05) was observed in breast cancer tissues compared to non-cancerous healthy tissues. In order to explore gene-gene and gene-clinicopathological parameters relationship Spearmen correlation was performed. We observed a significantly negative correlation between XRCC2 and Ki-67 expression (r = -0.376**, p < 0.01). In case of gene-clinicopathological parameters relationship, we observed a significant correlation between XRCC2 expression and lymph node status (r = -0.521***, p < 0.002) and metastatic status (r = -0.303*, p < 0.04) of breast cancer patients. Our data suggests that deregulation of XRCC2 in breast cancer has the potential to predict lymph node metastasis and may serve as a therapeutic target for breast cancer patients at risk of metastasis.

Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy

BRCA1, a key factor in homologous recombination (HR) repair may also regulate base excision repair (BER). Targeting BRCA1-BER deficient cells by blockade of ATM and DNA-PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression in two cohorts (n = 1602 sporadic and n = 50 germ-line BRCA1 mutated) and mRNA expression in two cohorts (n = 1952 and n = 249). Artificial neural network analysis for BRCA1-DNA repair interacting genes was conducted in 249 tumours. Pre-clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA-PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol β at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol β expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol β expressing BRCA1 negative tumours (ps < 0.05). Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol β. BRCA1-BER deficient cells were sensitive to ATM and DNA-PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol β expression status in BRCA1 negative tumours may have prognostic significance. BRCA1-BER deficient cells could be targeted by ATM or DNA-PKcs inhibitors for personalized therapy.

Role of the functional variant (-652T>G) in the XRCC4 promoter in prostate cancer.

Several genes encoding DNA repair molecules have been proposed as cancer-susceptibility genes. Many studies have suggested that SNPs in XRCC4 could be implicated in altering the risk of prostate cancer (PCa). We examined the role of the functional variant (-652T>G) in the XRCC4 promoter in PCa. The transcriptional activity of XRCC4 gene was measured by luciferase assay. We performed real-time PCR/immunohistochemical assay to verify the association between expression level of XRCC4 mRNA/protein and XRCC4 -652T>G polymorphism. In addition, electrophoretic mobility shift assay (EMSA) was used to confirm whether this polymorphism has an effect on binding ability of the transcription factor. We found that the G variant significantly increased the transcription activity of the XRCC4 gene and the binding ability of transcriptional factor GATA-1 to the XRCC4 promoter. Furthermore, the results suggested that the XRCC4 protein and mRNA were overexpressed in individuals who carried the -652G allele compared to carriers of the -652T allele. In addition, the expression of XRCC4 in PCa tissues was lower than in adjacent normal tissues. Our data suggest that the XRCC4 promoter -652G>T polymorphism is functional and may influence genetic susceptibility to prostate cancer. Case-control studies are required to validate our findings in the future.

ERCC1 expression and tumor regression predict survival in esophageal squamous cell carcinoma patients receiving combined trimodality therapy

PurposeCombined trimodality therapy with neoadjuvant chemoradiation followed by surgery has shown promising results for locally advanced operable esophageal cancer. DNA repair proteins may affect treatment efficacy through repairing DNA damage induced by chemotherapy and radiation therapy. We evaluated the associations of XRCC1, ERCC1 and MGMT expression with histopathologic response and survival in patients with locally advanced operable esophageal squamous cell carcinoma (ESCC) who received neoadjuvant chemoradiation. MethodsParaffin-embedded pre-treatment tissue samples, collected by endoscopic biopsy from patients treated with cisplatin-based neoadjuvant chemoradiation followed by surgery, were immunohistochemically stained for XRCC1, ERCC1 and MGMT expression. ResultsOf the 44 patients, major histopathologic response was noted in 26 (59.1%) patients. 68.8% of patients with ERCC1-negative tumors had major histopathologic response, compared to 53.6% of those who expressed positive ERCC1, though the difference was not statistically significant (P=0.361). The patients with ERCC1-negative tumor presented much better overall survival than those positive for ERCC1 expression (P=0.018). Patients with major histopathologic response had a 3-year survival rate of 96.2% versus those with minor response, with a 3-year survival rate of 41.5% (P=0.000). Multivariate analysis showed that ERCC1 expression and histopathologic response were independent predictive factors of overall survival in patients with locally advanced operable ESCC receiving neoadjuvant chemoradiation. ConclusionPatients with ERCC1-negative tumors show a benefit from neoadjuvant chemoradiation, ERCC1 expression and tumor regression are useful predictive markers in patients with locally advanced operable ESCC receiving neoadjuvant chemoradiation followed by surgery.

XRCC2 rs3218536 polymorphism decreases the sensitivity of colorectal cancer cells to poly(ADP-ribose) polymerase 1 inhibitor

Single nucleotide polymorphisms (SNPs) are associated with the development of certain types of cancer. The present study aimed to investigate the association between X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2) SNPs and colorectal cancer (CRC) cell sensitivity to the poly(ADP-ribose) polymerase (PARP) 1 inhibitor olaparib (AZD2281). SNaPshot® analysis of XRCC2 SNPs was performed in five CRC cell lines. The AZD2281-sensitivities of the CRC cells were also analyzed using MTT assays. The effect of AZD2281 on XRCC2 and PARP1 expression was investigated in the five cell lines using quantitative polymerase chain reaction and western blot analyses. Parallel investigations were performed using a cisplatin (DDP) model of DNA damage. The XRCC2 rs3218536 SNP was found to be associated with the LoVo microsatellite instability CRC cell line. The relative rate of growth inhibition was found to be lower in the LoVo cells following treatment with AZD2281 compared with the other four cell lines (P=0.002). Furthermore, the XRCC2 mRNA level in the LoVo cells was observed to be significantly higher than that in the other four cell lines (P<0.05). Similar results were found using the DDP model of DNA damage (P<0.05). The present study indicated that the XRCC2 rs3218536 polymorphism decreases the sensitivity of CRC cells to AZD2281.

Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24h with the following concentrations of tested chemicals: B[a]P: 1μM, 10μM, 25μM; EOMs: 1μg/ml, 10μg/ml, 25μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatment of human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs.