Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells

Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs-induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs-induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs-induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549-R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549-R and A549 cell lines treated with COF condensate (COFC). RT-PCR and Western blot results showed that the expression of hOGG1 in A549-R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549-R cell line was much lower than that in the A549 cell line, and more COFC-induced DNA damage was detected in the A549-R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549-R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF-induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1-mediated repair pathways, and oxidative DNA damage may be involved in COF-induced carcinogenesis.     

Proficient deoxyribonucleic acid repair of methylation damage in hamster ERCC-gene mutants

Three major pathways, nucleotide excision repair (NER), base excision repair (BER) and O⁶-methylguanine–DNA methyltransferase (MGMT), are responsible for the removal of most adducts to DNA and thus for the survival of cells influenced by deoxyribonucleic acid (DNA) adduct-forming chemicals. We have evaluated host cell reactivation and cell survival of wild type Chinese hamster ovary cells and of mutants in the NER-genes ERCC1, ERCC2, and ERCC4 after treatment with the methylating compounds dimethylsulfate and methylnitrosourea. No effect of the three genes could be demonstrated, i.e., survival and host cell reactivation after methylation damage in the mutants and the wild type cells were similar. Gene-specific repair experiments confirmed the proficient removal of methyl lesions. We conclude that the three nucleotide excision repair genes are immaterial to the repair of methylation damage. This suggests that NER does not play a role in the removal of methylation in mammalian cells and that BER and MGMT are responsible for the survival of such cells, when they are challenged with methylation of DNA.     

Lower levels of oxidative DNA damage and apoptosis in lymphocytes from patients undergoing surgery with propofol anesthesia

Propofol, which is widely used as an intravenous anesthetic, has a phenolic structure similar to that of α‐tocopherol with antioxidant properties that could prevent genotoxicity and cytotoxicity in lymphocytes of anesthetized patients. The aims of this study were to evaluate oxidative DNA damage and apoptosis in lymphocytes and the expression of DNA repair genes in blood cells from patients undergoing elective surgery under anesthesia with propofol. Twenty healthy adults of both genders (18–50 years old) who were scheduled for otorhinological surgery were enrolled in this study. Blood samples were collected before anesthesia induction (T₁‐baseline), 120 min after anesthesia induction (T₂), and on the first postoperative day (T₃). Oxidative DNA damage in peripheral lymphocytes was assessed using the comet assay. Lymphocytes were phenotyped as T helper or cytotoxic T cells, and apoptosis was evaluated using flow cytometry. The expression of DNA repair genes (hOGG1 and XRCC1) was assessed by quantitative polymerase chain reaction. A reduction in the level of oxidized purines in DNA (P < 0.01) was observed 120 min after anesthesia induction, and reduced apoptosis of T helper cells was observed 120 min after anesthesia induction and on the first postoperative day. Down‐regulation of hOGG1 and XRCC1 gene expression was observed on the first postoperative day. In conclusion, patients undergoing non‐invasive surgery under propofol anesthesia presented lower levels of oxidized purines and apoptosis of T helper lymphocytes. Furthermore, anesthesia with propofol did not directly influence the expression of the DNA repair genes hOGG1 and XRCC1 in blood cells. © Environ. Mol. Mutagen. 2012. Published 2011 Wiley Periodicals, Inc.     

DNA mismatch repair in lymphoblastoid cells from hereditary non-polyposis colorectal cancer (HNPCC) patients is normal under conditions of rapid cell division and increased mutational load

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have germ-line mutations in one of four DNA mismatch repair genes (hMSH2, hMLH1, hPMS1 and hPMS2). It is supposed that a single functional copy of these genes is sufficient for normal mismatch repair, but it is not certain that this is the case under abnormal conditions such as rapid cell division or an increased tendency to DNA replication errors (RERs). We have analysed mismatch repair by examining replication errors in immortalised lymphoblastoid cells derived from two HNPCC patients heterozygous for mismatch repair defects (one hMSH2 mutant and one hMLH1 mutant), and from control individuals. Three conditions of cell culture have been used: (i) relatively slow cell growth and division; (ii) relatively fast growth and division; and (iii) chronic perturbation of the intracellular dNTP pool to promote a increased frequency of replication errors. No significant differences in microsatellite instability were found between HNPCC patients and controls in any of these environments. Lymphoblastoid cells from hMSH2 and hMLH1 mutant/wild-type heterozygotes appear, therefore, to have normal levels of mismatch repair, even under conditions that increase the requirement for repair. The pool bias cultures from the HNPCC patients and controls did, however, show similar, increased frequencies of RERs, suggesting that the mismatch repair capacity of the cells had been overloaded, but that the number of normal HNPCC alleles was not the limiting factor.     

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Souza L R, Fonseca‐Silva T, Pereira C S, Santos E P, Lima L C, Carvalho H A, Gomez R S, Guimarães A L S & De Paula A M B
(2011) Histopathology 58 , 352–360
Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results:  Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions:  Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.     

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5′ untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.     

Essential role of β‐human 8‐oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair

8‐Oxoguanine (8‐OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8‐OG is primarily removed by human 8‐OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated α‐ and β‐hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two‐hybrid screening assays, we found that β‐ but not α‐hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide‐induced oxidative stress, suggesting that β‐ but not α‐hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the β‐ but not α‐ hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of β‐hOGG1 for mtDNA repair. β‐hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that β‐hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define β‐hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function. Mol. Mutagen. 2013. © 2012 Wiley Periodicals, Inc.     

Deregulated expression of DNA polymerase β is involved in the progression of genomic instability

Deregulated expression of DNA polymerase beta (pol β) has been implicated in genomic instability that leads to tumorigenesis, yet the mechanisms underlying the pol β‐mediated genetic instability remain elusive. In this study, we investigated the roles of deregulated expression of pol β in spontaneous and xenobiotic‐induced genetic instability using mouse embryonic fibroblasts (MEFs) that express distinct pol β levels (wild‐type, null, and overexpression) as a model system. Three genetic instability endpoints, DNA strand breaks, chromosome breakage, and gene mutation, were examined under various expression levels of pol β by comet assay, micronuclei test, and hprt mutation assay. Our results demonstrate that neither pol β deficiency nor pol β overexpression is sufficient for accumulation of spontaneous DNA damage that promotes a hyperproliferation phenotype. However, pol β null cells exhibit increased sensitivity to exogenous DNA damaging agents with increased genomic instability compared with pol β wild‐type and overexpression cells. This finding suggests that a pol β deficiency may underlie genomic instability induced by exogenous DNA damaging agents. Interestingly, pol β overexpression cells exhibit less chromosomal or DNA damage, but display a higher hprt mutation frequency upon methyl methanesulfonate exposure compared with the other two cell types. Our results therefore indicate that an excessive amount of pol β may promote genomic instability, presumably through an error‐prone repair response, although it enhances overall BER capacity for induced DNA damage. Environ. Mol. Mutagen. 2012. © 2012Wiley Periodicals, Inc.     

Roles of Chk2/CHEK2 in guarding against environmentally induced DNA damage and replication-stress

Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can “collapse” to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal “back-up” roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.     

Hyphal differentiation induced via a DNA damage checkpoint-dependent pathway engaged in crosstalk with nutrient stress signaling in Schizosaccharomyces japonicus

DNA damage response includes DNA repair, nucleotide metabolism and even a control of cell fates including differentiation, cell death pathway or some combination of these. The responses to DNA damage differ from species to species. Here we aim to delineate the checkpoint pathway in the dimorphic fission yeast Schizosaccharomyces japonicus, where DNA damage can trigger a differentiation pathway that is a switch from a bidirectional yeast growth mode to an apical hyphal growth mode, and the switching is regulated via a checkpoint kinase, Chk1. This Chk1-dependent switch to hyphal growth is activated with even low doses of agents that damage DNA; therefore, we reasoned that this switch may depend on other genes orthologous to the components of the classical Sz. pombe Chk1-dependent DNA checkpoint pathway. As an initial test of this hypothesis, we assessed the effects of mutations in Sz. japonicus orthologs of Sz. pombe checkpoint genes on this switch from bidirectional to hyphal growth. The same set of DNA checkpoint genes was confirmed in Sz. japonicus. We tested the effect of each DNA checkpoint mutants on hyphal differentiation by DNA damage. We found that the Sz. japonicus hyphal differentiation pathway was dependent on Sz. japonicus orthologs of Sz. pombe checkpoint genes—_SP rad3, _SP rad26, _SP rad9, _SP rad1, _SP rad24, _SP rad25, _SP crb2, and _SP chk1—that function in the DNA damage checkpoint pathway, but was not dependent on orthologs of two Sz. pombe genes—_SP cds1 or _SP mrc1—that function in the DNA replication checkpoint pathway. These findings indicated that although the role of each component of the DNA damage checkpoint and DNA replication checkpoint is mostly same between the two fission yeasts, the DNA damage checkpoint was the only pathway that governed DNA damage-dependent hyphal growth. We also examined whether DNA damage checkpoint signaling engaged in functional crosstalk with other hyphal differentiation pathways because hyphal differentiation can also be triggered by nutritional stress. Here, we discovered genetic interactions that indicated that the cAMP pathway engaged in crosstalk with Chk1-dependent signaling.     

Radio-adaptive response,individual radio-sensitivity and correlation of base excision repair gene polymorphism (hOGG1, APE1, XRCC1, and LIGASE1) in human peripheral blood mononuclear cells exposed to gamma radiation

Radio-adaptive response (RAR) is a biological mechanism, where cells primed with a low dose exhibit reduced DNA damage with a high challenging dose. Single nucleotide polymorphisms (SNPs) of DNA repair genes including base excision repair (BER) pathway are known to be associated with radio-sensitivity but involvement in RAR is not yet understood. In the present study, attempt was made to correlate genotype frequencies of four BER SNPs [hOGG1(Ser326Cys), XRCC1(Arg399Gln), APE1(Asp148Glu) and LIGASE1(A/C)] with DNA damage, repair and mRNA expression level among 20 healthy donors (12 adaptive and 8 nonadaptive). Our results revealed that LIGASE1 (p = .002) showed significant correlation with DNA damage and mRNA expression level with increasing dose. hOGG1 (Ser326Cys), XRCC1 (Arg399Gln) and LIGASE1(A/C) polymorphisms showed significant difference with DNA damage (%T) and mRNA expression profile in primed cells among adaptive donors. In conclusion, BER gene polymorphisms play important role in identifying donors with radio-sensitivity and RAR in human cells.     

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells

The mechanisms by which arsenic‐induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT‐PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose‐dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome‐wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long‐lasting arsenic‐induced genomic instability that manifests in several ways, including aberrant chromosomal effects. Environ. Mol. Mutagen. 57:137–150, 2016. © 2015 Wiley Periodicals, Inc.     

A novel genetic screen identifies checkpoint-defective alleles of Schizosaccharomyces pombe chk1

The protein kinase Chk1 is required in the fission yeast Schizosaccharomyces pombe for delaying cell cycle progression in response to DNA damage. Chk1 becomes phosphorylated when DNA is damaged by a variety of agents, including the anti-tumor drug camptothecin. To further characterize the behavior of Chk1 in response to DNA damage, we used PCR-based mutagenesis of the chk1 gene coupled with in vivo gap repair to generate mutant alleles. Of 44 chk1 mutants recovered, six encode full-length proteins that confer a DNA damage-sensitive phenotype. All of the alleles render cells checkpoint-defective, but confer subtle differences in sensitivity to camptothecin or UV light. Mutant alleles were sequenced and served to identify regions of the protein that are critical for checkpoint function. Received: 7 August 2000 / Accepted: 21 September 2000     

The hOGG1 Ser326Cys polymorphism is not associated with sporadic Parkinson's disease

Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by progressive and profound loss of dopaminergic neurons in the substantia nigra (SN) resulting in resting tremor, rigidity, bradykinesia, and postural instability. The primary cause of the disease is still unknown, but mitochondrial dysfunction and oxidative stress have been implicated in the neurodegenerative process. Oxoguanine DNA glycosylase (OGG1) removes oxidized guanine (8-oxo-G) from the DNA, thus reducing the mutagenic potential of this modified base. Increased 8-oxo-G levels and up-regulation of OGG1 have been detected in the SN of PD brains. Moreover, studies performed in OGG1 knockout mice revealed the importance of this enzyme in protecting dopaminergic neurons against the accumulation of oxidative DNA damage. A common Ser326Cys polymorphism is known in the human gene encoding OGG1 (hOGG1), and the mutant Cys326 variant has been associated with reduced glycosylase activity. In the present study we screened 139 sporadic PD patients and 211 healthy matched controls for the presence of the hOGG1 Ser326Cys polymorphism. The Cys326 allele frequency was similar between the groups (0.20 in PD patients and 0.19 in controls; p = 0.817), and no difference in genotype frequencies was observed. Moreover, the hOGG1 Ser326Cys polymorphism was not associated with disease age at onset (p = 0.791). Overall, present results suggest that the hOGG1 Ser326Cys polymorphism is not associated with sporadic PD.     

Importance of DNA damage checkpoints in the pathogenesis of human cancers

All forms of life on earth must cope with constant exposure to DNA-damaging agents that may promote cancer development. As a biological barrier, known as DNA damage response (DDR), cells are provided with both DNA repair mechanisms and highly conserved cell cycle checkpoints. The latter are responsible for the control of cell cycle phase progression with ATM, ATR, Chk1, and Chk2 as the main signaling molecules, thus dealing with both endogenous and exogenous sources of DNA damage. As cell cycle checkpoint and also DNA repair genes, such as BRCA1 and BRCA2, are frequently mutated, we here discuss their fundamental roles in the pathogenesis of human cancers. Importantly, as current evidence also suggests a role of MAPK's (mitogen activated protein kinases) in cell cycle checkpoint control, we describe in this review both the ATR/ATM-Chk1/Chk2 signaling pathways as well as the regulation of cell cycle checkpoints by MAPK's as molecular mechanisms in DDR, and how their dysfunction is related to cancer development. Moreover, since damage to DNA might be the common underlying mechanism for the positive outcome of chemotherapy, we also discuss targeting anticancer treatments on cell cycle checkpoints as an important issue emerging in drug discovery.