Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H2O2 was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds

European ancestry and polymorphisms in DNA repair genes modify the risk of melanoma: A case-control study in a high UV index region in Brazil

Background: UV radiation is the major environmental factor related to development of cutaneous melanoma. Besides sun exposure and the influence of latitude, some host characteristics such as skin phototype and hair and eye color are also risk factors for melanoma. Polymorphisms in DNA repair genes could be good candidates for susceptibility genes, mainly in geographical regions exposed to high solar radiation. Objective: Evaluate the role of host characteristic.; and DNA repair polymorphism in melanoma risk in Brazil. Methods: We carried out a hospital-based case-control study in Brazil to evaluate the contribution of host factors and polymorphisms in DNA repair to melanoma risk. A total of 412 patients (202 with melanoma and 210 controls) were analyzed regarding host characteristics for melanoma risk as well as for 11 polymorphisms in DNA repair genes. Results: We found an association of host characteristics with melanoma development, such as eye and hair color, fair skin, history of pigmented lesions removed, sunburns in childhood and adolescence, and also European ancestry. Regarding DNA repair gene polymorphisms, we found protection for the XPG 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in the XPC gene (PAT+; IV-6A and 939Gln), which represent a haplotype for XPC. Melanoma risk was higher in individuals carrying the complete XPC haplotype than each individual polymorphism (OR 3.64; 95% CI 1.77-7.48). Conclusions: Our data indicate that the host factors European ancestry and XPC polymorphisms contributed to melanoma risk in a region exposed to high sun radiation. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systematic lupus erythematosus (SLE) patients has been reported ill some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy control were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls. SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features. Lupus (2008) 17, 988-995.

Polymorphisms of xenobiotic metabolizing enzymes and DNA repair genes and outcome in childhood acute lymphoblastic leukemia

The interindividual variation in the activity of xenobiotic metabolizing enzymes and DNA repair genes could modify an individual`s risk of recurrent malignancy and response to therapy. We investigated whether ALL outcome was related to polymorphisms in genes CYP2D6. MPO, EPHX1, NQO1, TS, XPD and XRCC1 in 95 consecutive ALL children by PCR or PCR-FRLP techniques. Polymorphisms in genes NQO1 and TS were associated with a significantly slow response to induction chemotherapy and NQO1 was also associated with a lower five-year event-free survival. This study suggests that polymorphisms of NQO1 and TS could be important for patient response to induction therapy and for treatment outcome. (C) 2009 Elsevier Ltd. All rights reserved.

Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Effects of UV radiation exposure on DNA and DNA repair enzymes

Dissertação para obtenção do Grau de Mestre em Biotecnologia

A glycine-arginine domain in control of the human MRE11 DNA repair protein.

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein.

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies.

BACKGROUND Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. OBJECTIVE To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. DESIGN SETTING AND PARTICIPANTS A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. RESULTS AND LIMITATIONS We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. CONCLUSION Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability.

DNA must constantly be repaired to maintain genome stability. Although it is clear that DNA repair reactions depend on cell type and developmental stage, we know surprisingly little about the mechanisms that underlie this tissue specificity. This is due, in part, to the lack of adequate study systems. This review discusses recent progress toward understanding the mechanism leading to varying rates of instability at expanded trinucleotide repeats (TNRs) in different tissues. Although they are not DNA lesions, TNRs are hotspots for genome instability because normal DNA repair activities cause changes in repeat length. The rates of expansions and contractions are readily detectable and depend on cell identity, making TNR instability a particularly convenient model system. A better understanding of this type of genome instability will provide a foundation for studying tissue-specific DNA repair more generally, which has implications in cancer and other diseases caused by mutations in the caretakers of the genome.

Epigenetic deregulation of DNA repair and its potential for therapy.

Epigenetic silencing of essential components of DNA repair pathways is a common event in many tumor types, and comprise O6-methylguanine-DNA methyltransferase (MGMT), human mut L homolog 1 (hMLH1), Werner syndrome gene (WRN), breast cancer susceptibility gene 1 (BRCA1), and genes of the Fanconi anemia pathway. Most interestingly, some of these alterations become the Achilles heel of the affected tumors upon treatment with certain classes of anticancer agents. That is, patients whose tumors carry such defects can be stratified for respective therapy rendering some classic DNA damaging agents, such as alkylators or DNA crosslinking agents, into "targeted therapies." Here we review some of the affected repair pathways that, when inactivated, sensitize the tumors to specific drugs and are thus exploitable for individualized therapy.

Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair.

ABSTRACT: BACKGROUND: Many studies have been published outlining the global effects of 17 beta-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap. RESULTS: We performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination. CONCLUSIONS: Our results show that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells.

INTRODUCTION: Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. METHODS: ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. RESULTS: ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. CONCLUSION: Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Colorectal cancer metastasis: the DNA repair inhibitor Dbait increases sensitivity to hyperthermia and improves efficacy of radiofrequency ablation.

PURPOSE: To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS: The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS: When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION: Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.

Association of DNA repair inhibition and radiofrequency ablation in the treatment of xenografted colorectal cancer

Purpose: Most of the patients with advanced colorectal cancer will develop liver metastasis, even after primary tumor resection. Although surgical resection remains the gold standard treatment of hepatic metastases, only few patients are eligible to curative resection. Radiofrequency ablation (RFA) is the most common curative alternative. Dbait are new molecules that inhibit DNA double-strand breaks repair. In vitro, Dbait has shown to increase cell death after hyperthermia. Here, we have assessed the combination of Dbait and RFA in the treatment of human colorectal cancer model xenografted in nude mice.Materials: 98 mice were flank-grafted with HT29 (human colon adenocarcinoma). When tumor reached 500 mm3, mice were sham treated (n=19), treated by Dbait via local injections (n=20), treated by RFA using an incomplete ablation scheme (n=20) or treated by combination of Dbait and RFA (n=39 separated in two Dbait regimens). After RFA, 39 mice were sacrificed for blinded pathological study, and 59 others were followed for survival analysis.Results: Mice treated by RFA-Dbait had significantly longer survival as compared to RFA alone (median survival: 56 vs 39 days, p<0.05) while RFA improved survival as compared to controls (median survival: 39 vs 28 days, p<0.05). Pathological studies of tumor slice have demonstrated significant decrease of tumor area and cancer cell viability in the RFA-Dbait group.Conclusions: While the implication of DNA repair activity in heat sensitivity remains unclear, our results show that the addition of Dbait to RFA enhances the antitumor response in this model and provide an experimental basis for the use of Dbait as an additional therapy to RFA.