Urinary 8-oxo-2′-deoxyguanosine:Redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2′-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G → T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically. © 2002 Elsevier Science Inc.

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repair

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repair

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control

Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. 

Maximizing efficacy of the hypomethylating drug 5-aza-2'-deoxycytidine in human leukemia

5-aza-2'-deoxycytidine (DAC) is a cytidine analogue that strongly inhibits DNA methylation, and was recently approved for the treatment of myelodysplastic syndromes (MDS). To maximize clinical results with DAC, we investigated its use as an anti-cancer drug. We also investigated mechanisms of resistance to DAC in vitro in cancer cell lines and in vivo in MDS patients after relapse. We found DAC sensitized cells to the effect of 1-β-D-Arabinofuranosylcytosine (Ara-C). The combination of DAC and Ara-C or Ara-C following DAC showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of global methylation. RIL gene activation and H3 lys-9 acetylation of short interspersed elements (Alu). One possible explanation is that hypomethylated cells are sensitized to cell killing by Ara-C. Turning to resistance, we found that the IC50 of DAC differed 1000 fold among and was correlated with the dose of DAC that induced peak hypomethylation of long interspersed nuclear elements (LINE) (r=0.94, P<0.001), but not with LINE methylation at baseline (r=0.05, P=0.97). Sensitivity to DAC did not significantly correlate with sensitivity to another hypomethylating agent 5-azacytidine (AZA) (r=0.44, P=0.11). The cell lines most resistant to DAC had low dCK, hENT1, and hENT2 transporters and high cytosine deaminase (CDA). In an HL60 leukemia cell line, resistance to DAC could be rapidly induced by drug exposure, and was related to a switch from monoallelic to biallelic mutation of dCK or a loss of wild type DCK allele. Furthermore, we showed that DAC induced DNA breaks evidenced by histone H2AX phosphorylation and increased homologous recombination rates 7-10 folds. Finally, we found there were no dCK mutations in MDS patients after relapse. Cytogenetics showed that three of the patients acquired new abnormalities at relapse. These data suggest that in vitro spontaneous and acquired resistance to DAC can be explained by insufficient incorporation of drug into DNA. In vivo resistance to DAC is likely due to methylation-independent pathways such as chromosome changes. The lack of cross resistance between DAC and AZA is of potential clinical relevance, as is the combination of DAC and Ara-C. ^

The major chromatin protein histone H1 binds preferentially to cis-platinum-damaged DNA

Both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum(II) form covalent adducts with DNA. However, only the cis isomer is a potent anticancer agent. It has been postulated that the selective action of cis-DDP occurs through specific binding of nuclear proteins to cis-DDP-damaged DNA sites and that binding blocks DNA repair. We find that a very abundant nuclear protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1, which had been previously considered a major candidate for such binding in vivo.

Assessment of in vivo genotoxicity of the rodent carcinogen furan: evaluation of DNA damage and induction of micronuclei in mouse splenocytes

In recent years, several surveys have highlighted the presence of the rodent carcinogen furan in a variety of food items. Even though the evidence of carcinogenicity of furan is unequivocal, the underlying mechanism has not been fully elucidated. In particular, the role of genotoxicity in furan carcinogenicity is still not clear, even though this information is considered pivotal for the assessment of the risk posed by the presence of low doses of furan in food. In this work, the genotoxic potential of furan in vivo has been investigated in mice, under exposure conditions similar to those associated with cancer onset in the National Toxicology Program long-term bioassay. To this aim, male B6C3F1 mice were treated by gavage for 4 weeks with 2, 4, 8 and 15 mg furan/kg b.w./day. Spleen was selected as the target organ for genotoxicity assessment, in view of the capability of quiescent splenocytes to accumulate DNA damage induced by repeat dose exposure. The induction of primary DNA damage in splenocytes was evaluated by alkaline single-cell gel electrophoresis (comet assay) and by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX). The presence of cross-links was probed in a modified comet assay, in which cells were irradiated in vitro with gamma-rays before electrophoresis. Chromosome damage was quantitated through the detection of micronuclei in mitogen-stimulated splenocytes using the cytokinesis-block method. Micronucleus induction was also assessed with a modified protocol, using the repair inhibitor 1-beta-arabinofuranosyl-cytosine to convert single-strand breaks in micronuclei. The results obtained show a significant (P < 0.01) increase of gamma-H2AX foci in mitogen-stimulated splenocytes of mice treated with 8 and 15 mg furan/kg b.w. and a statistically significant (P < 0.001) increases of micronuclei in binucleated splenocytes cultured in vitro. Conversely, no effect of in vivo exposure to furan was observed when freshly isolated quiescent splenocytes were analysed by immunofluorescence and in comet assays, both with standard and radiation-modified protocols. These results indicate that the in vivo exposure to furan gives rise to pre-mutagenic DNA damage in resting splenocytes, which remains undetectable until it is converted in frank lesions during the S-phase upon mitogen stimulation. The resulting DNA strand breaks are visualized by the increase in gamma-H2AX foci and may originate micronuclei at the subsequent mitosis.

The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin

Resistance to chemotherapeutic drugs can be an obstacle to a successful treatment of cancer patients in part associated with individual response and differences in the DNA repair system. The Comet assay is an informative test to investigate DNA damage and repair in cells in response to a variety of DNA-damaging agents, including chemotherapeutic drugs. The aim of this study was to assess leukocytes damage after in-vitro cisplatin treatment and DNA repair action using the Comet assay in 20 patients with melanoma and 20 cancer-free individuals. Leukocytes` DNA damage before and after cisplatin treatment, in three different concentrations, was analyzed. The DNA repair capability was investigated after 1-5 h of in-vitro cells growing without cisplatin. The Comet score of the patients` basal DNA damage was higher than that observed in controls, but the difference was not statistically significant (P=0.85). Although both groups had similar Comet scores to all cisplatin concentrations tested and the DNA repair times, the basal DNA damage (P < 0.001) and cisplatin damages (P < 0.005) were statistically lower than the different repair times investigated. Considering the progressive increase in the Comet score due to repair time, the negative results here observed could be associated with the reduced cell culture incubation that should be better evaluated. Considering the mutagenic action of cisplatin on tumor cells and the importance of individual DNA repair mechanisms in the chemotherapeutic melanoma treatment, the peripheral leukocytes could be particularly useful as a tool for DNA repair response identified by the Comet assay. Melanoma Res 21:99-105 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.