DNA Methylation Damage Research Articles

Proficient repair in chromatin remodeling defective ino80 mutants of Saccharomyces cerevisiae highlights replication defects as the main contributor to DNA damage sensitivity

Ino80 is an evolutionarily conserved member of the SWI2/ SNF2-family of ATPases in Saccharomyces cerevisiae. It resides in a multiprotein helicase/chromatin remodeling complex, and has been shown to play a key role in the stability of replication forks during replication stress. Though yeast with defects in ino80 show sensitivity to killing by a variety of DNA-damaging agents, a role for the INO80 protein complex in the repair of DNA has only been assessed for double-strand breaks, and the results are contradictory and inconclusive. We report that ino80Δ cells are hypersensitive to DNA base lesions induced by ultraviolet (UV) radiation and methyl methanesulfonate (MMS), but show little (or no) increased sensitivity to the DNA double-strand break (DSB)-inducing agents ionizing radiation and camptothecin. Importantly, ino80Δ cells display efficient removal of UV-induced cyclobutane pyrimidine dimers, and show a normal rate of removal of DNA methylation damage after MMS exposure. In addition, ino80Δ cells have an overall normal rate of repair of DSBs induced by ionizing radiation. Altogether, our data support a model of INO80 as an important suppressor of genome instability in yeast involved in DNA damage tolerance through a role in stability and recovery of broken replication forks, but not in the repair of lesions leading to such events. This conclusion is in contrast to strong evidence for the DNA repair-promoting role of the corresponding INO80 complexes in higher eukaryotes. Thus, our results provide insight into the specialized roles of the INO80 subunits and the differential needs of different species for chromatin remodeling complexes in genome maintenance.

Abstract 1154: Direct detection of DNA methylation and mutagenic damage through single-molecule, real-time (SMRTTM) DNA sequencing

Abstract Changes in genomic methylation patterns are often associated with cancer processes. The development of new, high-throughput techniques for mapping aberrant methylation patterns, along with various forms of DNA damage, will be important for advancing cancer research. Single-molecule real-time (SMRTTM) sequencing yields a rich set of information about nucleic acid structure that allows direct detection of various forms of modified nucleotides, including 5-methylcytosine, 5-hydroxymethylcytosine, N6-methyladenosine, and 8-oxoguanosine. We have studied DNA templates with a range of CpG methylation patterns, and we describe how this approach, in combination with circular consensus sequencing of individual DNA molecules, can be used for base-pair resolution identification of epigenetic modifications. Unlike current bisulfite-sequencing techniques, which are limited by short read lengths and by the reduction in genomic complexity, our method will enable mapping of methylation patterns within even highly repetitive genomic regions. Overall, these results highlight the potential applications of the SMRTTM technology to high-throughput sequencing of methylation and mutagenic DNA lesions. 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 1154.

In vitro basis for treatment with hypomethylating agents and histone deacetylase inhibitors: can epigenetic changes be used to monitor treatment?

Hematopoietic disorders such as myelodysplastic syndromes (MDS) show a high frequency of methylation of tumor suppressor genes. DNA methyltransferase (DNMT) inhibitors such as azacitidine and decitabine are used to target DNA methylation in MDS patients. Combining these drugs with histone deacetylase (HDAC) inhibitors in vitro resulted in synergistic tumor suppressor gene re-expression. Several phase I trials have examined methylation, gene expression and DNA damage as markers of clinical response to DNMT and HDAC inhibitors, with conflicting results. Trials are ongoing to investigate early methylation changes and DNA damage markers to understand the mechanisms of these drugs and as potential predictors of clinical response.

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM

By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited the duration of the S phase delay. The combination of MMS and 4-AN resulted in ATM and Chk2 phosphorylation and the time course of phosphorylation for both kinases correlated with the S phase delay. Chk2 phosphorylation was reduced in the absence of ATM activity. The Chk2 phosphorylation that remained in the absence of ATM appeared to be dependent on ATR and DNA-PK. The results demonstrate that, following initiation of base excision repair and inhibition of PARP activity, ATM activation is critical for preventing the cell from progressing through S phase, and for protection against MMS-induced cytotoxicity.

Effect of age, smoking and other lifestyle factors on urinary 7‐methylguanine and 8‐hydroxydeoxyguanosine

Urinary 8-hydroxydeoxyguanosine (8-OH-dG) and 7-methylguanine (m7Gua) were measured by a column-switching high performance liquid chromatography method as markers of oxidative and methylating DNA damage, respectively. We investigated the associations between urinary 8-OH-dG or m7Gua and various lifestyle and demographic factors, such as age and sex. The urinary 8-OH-dG excretion level was positively correlated with cigarette smoking, but inversely correlated with fruit consumption, physical activity and total energy consumed per day. A multiple regression analysis revealed that daily physical activity and healthy meal combinations decreased the urinary 8-OH-dG level, whereas alcohol consumption increased it. In terms of the urinary m7Gua measurement, cigarette smoking, age and consumption of meat, fish, egg, soybean, etc. were positively correlated with the urinary m7Gua level, whereas body weight, BMI, physical activity, and dietary index score, which indicates good nutritional balance, were negatively correlated with the amount of m7Gua. Based on a multiple regression analysis, cigarette smoking and age correlated with the m7Gua level, while high BMI and healthy meal combinations have significant reducing effects on m7Gua level. Therefore, the urinary m7Gua level is considered to be a useful marker of DNA methylation, not only from smoking, but also from aging and unhealthy dietary habits.

Dynamical effects of epigenetic silencing of 14-3-3σ expression

The development and progression of malignant tumours are often due to deregulated cell cycle control involving a plethora of different molecules. Among these, tumour suppressor proteins like p53 play a crucial role. p53 induces 14-3-3sigma, a multifunctional protein kinase inhibitor, centrally involved in cell cycle control and DNA damage repair after genotoxic stress. Recently, it has been shown that 14-3-3sigma is epigenetically silenced in a variety of tumours, which might contribute to tumour development and progression via impaired cell cycle control. In addition, p53, its inhibitor MDM2 and 14-3-3sigma form a signalling module in which 14-3-3sigma positively regulates the activity of p53 through feedback regulation. Here we present a mathematical model integrating the effects of 14-3-3sigma gene silencing, the dynamics of 14-3-3sigma induction and compartmentalisation by genotoxic stress and the role of interacting molecules p53 and MDM2. In vitro experiments with different melanoma cell lines were performed and our mathematical model was subjected to computer simulations to analyse different scenarios of activation depending on gene methylation status and DNA damage levels. Our analysis indicates that 14-3-3sigma expression is silenced by high gene methylation, but also that strong stimulation is necessary to induce 14-3-3sigma expression in cases of intermediate levels of gene methylation. More intriguingly, the model suggests that epigenetic silencing of 14-3-3sigma affects p53 dynamics in a synergistic way, such that the accumulative effect of partial downregulation of p53 expression and reduction of its nuclear fraction could affect drastically the activity of p53 as a transcription factor.

Distinct Phosphatases Mediate the Deactivation of the DNA Damage Checkpoint Kinase Rad53

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.

Role of Mismatch Repair and MGMT in Response to Anticancer Therapies

Tumor resistance to cytotoxic chemotherapy drugs and their toxicity to normal cells are major clinical obstacles to anticancer therapy effectiveness. Alterations in various DNA repair pathways play a key role in the development of both mechanisms of drug resistance and toxicity. Since deregulation of the DNA damage response and alterations in DNA repair pathways are relatively common in human cancer, the knowledge of these alterations in cancer cells would be an important predictive factor for the clinical response to chemotherapy and a useful guide in designing an appropriate therapeutic strategy. This review is focused on the mismatch repair (MMR) pathway and the O(6)-methylguanine-DNA-methyltransferase (MGMT) repair protein. In particular, we examine how inactivation of these DNA repair mechanisms might affect the response of tumor cells to chemotherapy, with a special emphasis on agents inducing methylation and oxidative DNA damage and interstrand DNA cross-links (ICLs). In addition, we provide novel experimental evidence indicating that MMR is required for efficient repair of ICLs via stabilization of RAD51 containing repair intermediates. Finally, we discuss possible emerging therapeutical strategies for treating MMR-defective tumors.

Methylation damage response in hematopoietic progenitor cells

The cellular response to methylation DNA damage was compared in multipotent CD34 + hematopoietic stem cells and mature CD34 − cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34 + cells compared to their terminally differentiated CD34 − counterparts. The overexpressed genes included members of the mismatch repair (MMR) ( MSH2, MSH6, MLH1, PMS2), base excision repair ( AAG, APEX), DNA damage reversal (O 6-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34 + and CD34 − cells obtained following expansion of CD34 + cells in a medium containing early acting cytokines. Early progenitor CD34 + and early precursor CD34 − cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34 + and CD34 − cells. Cytotoxicity required the presence of the MGMT inhibitor O 6-benzylguanine and the timing of MNU cell death (48 and 72 h) was similar in CD34 + and CD34 − cells. These data indicate that cycling CD34 + and CD34 − cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.

Human Monocytes, but not Dendritic Cells Derived from Them, Are Defective in Base Excision Repair and Hypersensitive to Methylating Agents

Monocytes and dendritic cells are key players in the immune response. Because dendritic cells drive the tumor host defense, it is important that monocytes and dendritic cells survive cytotoxic tumor therapy. Although most of the anticancer drugs target DNA, the DNA repair capacity of monocytes and dendritic cells has not yet been investigated. We studied the sensitivity of monocytes and monocyte-derived dendritic cells against various genotoxic agents and found monocytes to be more sensitive to overall cell kill and apoptosis upon exposure to methylating agents (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and the anticancer drug temozolomide). On the other hand, upon treatment with the cross-linking chemotherapeutics fotemustine, mafosfamide, and cisplatin, monocytes and dendritic cells responded in the same way. Monocytes were also more sensitive than lymphocytes. The data indicate a defect in the repair of DNA methylation damage in monocytes. Because the expression of the repair protein O(6)-methylguanine-DNA methyltransferase was higher in monocytes than in dendritic cells, and because its inhibition by O(6)-benzylguanine had no effect on the sensitivity of monocytes, we investigated the base excision repair (BER) pathway. In contrast to dendritic cells, monocytes are unable to perform BER following exposure to methylating agents. Expression studies revealed that monocytes lack XRCC1 and ligase IIIalpha, whereas dendritic cells, similar to human lymphocytes, express these repair proteins at a high level. The data revealed a DNA repair defect in a specific human cell population. The BER defect in monocytes may cause them to be selectively killed during tumor therapy with alkylating agents, provoking hematotoxicity and sustained immunosuppression.

Formamidopyrimidine adducts are detected using the comet assay in human cells treated with reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most lung-specific of the carcinogens present in tobacco smoke. Its bioactivation in cells leads to a small amount of methylation or pyridyloxobutylation DNA damage. Considering its great sensitivity, the comet assay seems a technique of choice to investigate NNK-related damage. Several strategies were used to impart some specificity to the assay: (1) using analogs that produce a limited variety of DNA lesions, as they mimic either the methylation or the pyridyloxobutylation pathway; (2) using cells with different bioactivation abilities; (3) using alkali conversion and/or enzymes specific for cleaving particular classes of damage; (4) using different lysis conditions to convert a specific class of DNA lesions into enzyme-sensitive lesions. We determined that several NNK-associated lesions can be detected with some specificity with the comet assay. For the methylation pathway, they are AP sites and the more frequent formamidopyrimidine (fapy) adducts. These fapy adducts correspond to N7-methylguanines generated in the cells that were ring-opened during the assay by the lysis solution at pH 10. For the pyridyloxobutylation pathway, alkylphosphotriesters and a roughly equal frequency of fapy sites were detected. By analogy to the methylation damage, these fapy adducts are thought to be the ring-opened form of N7-pyridyloxobutylguanines (N7-pobG). N7-pobG are unstable and this constitutes the first indirect demonstration of their formation in cells. But contrary to N7-m-fapy, the lysis time or pH did not influence the frequency of N7-pob-fapy adducts detected, suggesting that they already exist in the cells and are not related to the experimental conditions. These N7-pob-fapy have a strong mutagenic potential and we think that the comet assay, in spite of its limitations, is a good way to study them considering their low frequency and the inherent instability of the adduct from which they originate.

A Methylation-Dependent Electrostatic Switch Controls DNA Repair and Transcriptional Activation by E. coli Ada

The transcriptional activity of many sequence-specific DNA binding proteins is directly regulated by posttranslational covalent modification. Although this form of regulation was first described nearly two decades ago, it remains poorly understood at a mechanistic level. The prototype for a transcription factor controlled by posttranslational modification is E. coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Ada repairs methyl phosphotriester lesions in DNA by transferring the aberrant methyl group to one of its own cysteine residues; this site-specific methylation enhances tremendously the DNA binding activity of the protein, thereby enabling it to activate a methylation-resistance regulon. Here, we report solution and X-ray structures of the Cys-methylated chemosensor domain of Ada bound to DNA. The structures reveal that both phosphotriester repair and methylation-dependent transcriptional activation function through a zinc- and methylation-dependent electrostatic switch.

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Mismatch repair-dependent transcriptome changes in human cells treated with the methylating agent N-methyl-n'-nitro-N-nitrosoguanidine.

DNA mismatch repair (MMR) plays a key role in the cytotoxic response of human cells to methylating agents, however, the cascade of events leading to cell cycle arrest and cell death has yet to be characterized. We studied the role of MMR in the transcriptional response to DNA methylation damage in two human cellular models: (a). the lymphoblastoid cell line TK6 and its derivative MT1, which is mutated in the MMR gene hMSH6; and (b). the epithelial cell line 293T Lalpha in which the expression of the MMR gene hMLH1 can be tightly regulated and p53 is inactivated. Upon N-methyl-N'-nitro-N-nitrosoguanidine treatment, only cells with functional MMR were killed, but the type of cytotoxic response differed. In TK6 cells, S-phase arrest and apoptosis were accompanied by a dramatic change in gene expression, notably, an up-regulation of several genes encoding growth inhibitors and proapoptotic factors both p53 dependent and independent. In contrast, the MMR-dependent transcriptional response in 293T Lalpha cells was substantially less pronounced than in TK6 cells, despite an efficient induction of a G(2)-M checkpoint and nonapoptotic cell death. Thus, we demonstrate that in human cells of different origin, MMR-mediated killing by methylating agents occurs through different pathways and regardless of the p53 status. Moreover, once DNA methylation damage has been processed by the MMR system, tumor cells might be committed to die, although one or more of their signaling pathways are impaired.

Involvement of DNA polymerase β in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase β (β-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H 2O 2). This is surprising because β-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, β-pol null cells become hypersensitive to H 2O 2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage β-pol null cell extracts. Therefore, in early-passage wild-type and β-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H 2O 2. Expression of β-pol protein in the late-passage null cells almost completely reverses the H 2O 2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H 2O 2 as expected for β-pol-mediated single-nucleotide BER; however in β-pol null cells, MX has no effect. The data indicate a role(s) of β-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.

Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated lambda phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by gamma irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts

To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. Maximal depletion of ATase in tumour, to 2.52 +/- 0.23% of pretreatment levels, occurred 4-8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 +/- 1.39 days with temozolomide 100 mg/kg 4-hourly x versus 23.2 +/- 1.43 days with temozolomide 100 mg/kg once daily x 5; P < 0.0001) at the expense of increased toxicity (17.4 +/- 1.55% animal weight loss versus 10.6 +/- 1.27%. respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA 06-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule.

Detection of methylation damage in DNA of gastric cancer tissues using 32P postlabelling assay.

Gastric cancer is the most common cancer in Korea. The causes are still unknown but it has been speculated that gastric cancer is associated with consumption of foods rich in nitrates/nitrites or a high dietary intake of salt or pickled food. In the present study, we studied the level of alkylated DNA adducts formed in gastric cancer tissues in comparison with that in normal gastric mucosa. DNA was extracted from surgically removed gastric cancer tissues and patient‐matched normal gastric mucosa. The level of N7‐methyldeoxyguanosine was measured by 32P‐postlabelling assay after high performance liquid chromatography (HPLC) enrichment. We found that the level of N7‐methyldeoxyguanosine of gastric cancerous tissues was significantly higher than that of normal gastric mucosa (P=0.01685).

Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes methylation but not UV- or ionizing radiation-induced DNA damage.

Interaction between long patch mismatch repair (MMR) and persistent DNA O6-methylguanine or 6-thioguanine (6-TG) is implicated in the cytotoxicity of methylating agents and 6-TG, respectively. Human cells with defective MMR tolerate DNA methylation damage and are cross-resistant to 6-TG. To determine whether MMR contributes to the lethal effects of persistent UV-induced DNA lesions, MMR deficiency was introduced into nucleotide excision repair (NER)-defective XP12RO cells. The doubly repair-defective cells, designated XP12ROB4, did not express detectable hMSH2 protein. They had the mutator phenotype, N-methyl-N-nitrosourea and 6-TG resistance typical of MMR-defective cells. Active MMR was not required for the cytotoxicity of UV light, and the hMSH2 defect did not detectably alter the survival of XP12ROB4. The level of spontaneous or UV-induced SCE was also similar in XP12RO and XP12ROB4, indicating that hMSH2 is not required for this recombination process. The combined deficiency in MMR and NER did not confer a significant degree of tolerance to ionizing radiation, and the survival of XP12RO and XP12ROB4 after gamma-radiation was similar. Although it recognizes and processes some persistent damaged or modified DNA base pairs, MMR is unlikely to serve as a general sensor of DNA damage.