8-hydroxyguanine and Cell-Transformation--Neoplastic

8-Hydroxyguanine (oh8G) is a major base lesion produced by reactive oxygen species. oh8G in DNA causes G:C to T:A transversions and, thus, could be responsible for mutations that lead to carcinogenesis. A human DNA glycosylase/AP lyase encoded by the OGG1 gene has an activity to remove directly oh8G from DNA, and suppresses the mutagenic effect of oh8G. OGG1 protein has a helix-hairpin-helix-GPD motif as a domain for both DNA binding and catalysis, a nuclear localization signal, and a mitochondria targeting signal. Among multiple OGG1 isoforms, OGG1-type la is expressed predominantly in human cells and repairs chromosomal DNA in the nucleus. Inactivation of the OGG1 gene in yeast and mice leads to elevated spontaneous mutation frequency in the cells. The human OGG1 gene maps to chromosome 3p26.2, and allelic deletions of this region occur frequently in a variety of human cancers. Moreover, the OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among human populations. Repair activities of some mutated and polymorphic OGG1 proteins are lower than those of wild-type OGG1-type la-Ser326 protein and, thus, could be involved in human carcinogenesis.

Topics: Amino Acid Sequence; Animals; Carbon-Oxygen Lyases; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 3; Conserved Sequence; DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Exons; Genes; Guanine; Humans; Kinetics; Mice; Molecular Sequence Data; Mutagenesis; N-Glycosyl Hydrolases; Neoplasms; Oxidation-Reduction; Oxidative Stress; Polymorphism, Genetic; Protein Isoforms; Rats; Reactive Oxygen Species; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity

Nitric oxide (NO) and superoxide rapidly react to yield peroxynitrite. Peroxynitrite is a potent oxidant which reacts with proteins, lipids, and DNA. The present paper overviews the various DNA modifications induced by exposure to peroxynitrite or NO and superoxide concurrently, with special reference to the formation of 8-nitroguanine and 8-oxoguanine as well as the induction of DNA single strand breakage. In addition, we review the secondary processes that may follow the process of DNA damage, such as activation of the nuclear enzyme, poly(ADP-ribose) synthetase, apoptosis, and carcinogenesis.

Topics: Animals; Apoptosis; Cell Transformation, Neoplastic; DNA; DNA Damage; Guanine; Humans; Inflammation; Nitrates; Nitric Oxide; Oxidants; Poly(ADP-ribose) Polymerases; Superoxides

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and nonhomologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single-nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared with wild-type (WT) Pol λ-expressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer.. The Pol λ R438W mutation can serve as a biomarker to predict cancer risk and implicates that treatment with estrogen in individuals with this mutation may further increase their risk of breast cancer. Mol Cancer Res; 14(11); 1068-77. ©2016 AACR.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA Damage; DNA Polymerase beta; DNA Repair; Estrogens; Female; Genetic Predisposition to Disease; Germ-Line Mutation; Guanine; High-Throughput Nucleotide Sequencing; Humans; Polymorphism, Single Nucleotide

MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh-/- mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh-/- than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh-/- mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh-/- mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh-/- mice provides a good model for MAP.

Topics: Adenocarcinoma; Adenoma; Animals; Azoxymethane; Bone Marrow Cells; CD8-Positive T-Lymphocytes; Cell Transformation, Neoplastic; Colitis; Colon; Colorectal Neoplasms; Cytokines; Dextran Sulfate; Disease Models, Animal; DNA Glycosylases; Forkhead Transcription Factors; Guanine; Inflammation Mediators; Mice, Knockout; T-Lymphocytes, Regulatory; Time Factors

HBx is strongly associated with hepatocellular carcinoma development through transcription factor activation and reactive oxygen species (ROSs) production. However, the exact role of HBx during hepatocellular carcinogenesis is not fully understood. Recently, it was reported that C-terminal truncated HBx is associated with tumor metastasis. In the present study, we confirmed that the C-terminal region of HBx is required for ROS production and 8-oxoguanine (8-oxoG) formation, which is considered as a reliable biomarker of oxidative stress. These results suggest ROS production induced by the C-terminal region of HBx leads to mitochondrial DNA damage, which may play a role in HCC development.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; DNA Damage; DNA, Mitochondrial; Gene Expression Regulation; Guanine; Humans; Immunoenzyme Techniques; Liver Neoplasms; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins

The MUTYH gene encodes a DNA glycosylase that can initiate the excision repair of adenine mispaired with 8-hydroxyguanine (8OHG) and is responsible for a susceptibility to multiple colorectal adenomas and carcinomas. To determine whether the MUTYH gene is involved in gastric carcinogenesis, we first examined the expression level of MUTYH in gastric cancer. The reduced expression of MUTYH mRNA transcript was detected in both gastric cancer cell lines and primary gastric cancers using qRT-PCR analysis. Immunohistochemical analysis also showed a significant reduction in MUTYH protein expression in gastric cancer, compared with non-cancerous gastric epithelium (immunohistochemical score, 175.5 ± 43.0 versus 281.5 ± 24.8; p < 0.0001). Among the gastric cancers, the MUTYH expression level was significantly associated with the histopathology (p < 0.0001) and the pT stage (p < 0.001). The outcome of patients with gastric cancer exhibiting low MUTYH expression was significantly worse than the outcome of patients with gastric cancer exhibiting high MUTYH expression (p = 0.0007, log-rank test) and a multivariate analysis revealed that reduced MUTYH expression was an independent predictor of a poor survival outcome among the gastric cancer patients (hazard ratio, 1.865; 95% confidence interval, 1.028-3.529; p = 0.0401). We next compared the functional effects of MUTYH on gastric cancer cells, based on their MUTYH expression levels. MUTYH-over-expressing stable clones of the gastric cancer cell line AGS showed: (a) higher DNA cleavage activity towards adenine:8OHG mispair-containing substrates; (b) higher suppressive activity against mutations caused by 8OHG in a supF forward mutation assay; and (c) higher suppressive activity for cellular proliferation than empty vector-transfected AGS clones. These results suggested that MUTYH is a suppressor of mutations caused by 8OHG in gastric cells and that its reduced expression is associated with a poor prognosis in gastric cancer.

Topics: Aged; Biomarkers, Tumor; Cell Transformation, Neoplastic; DNA Glycosylases; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Guanine; Humans; Male; Middle Aged; Mutation; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Tumor Cells, Cultured

To examine the effects of alcohol consumption on cancer risk, we measured oxidative DNA damage and its repair activity in the livers and esophagi of rats fed with ethanol. Using our previously designed protocol for feeding rats with a high concentration of ethanol, we examined the effects of ethanol consumption on 8-oxo-Gua generation and repair activity in the livers and esophagi of rats. We found that a high concentration of ethanol accompanied with a vitamin-depleted diet increased 8-oxo-Gua and its repair activity. 8-Oxo-Gua is known to induce point mutations, leading to carcinogenesis. Therefore, these results suggested that a high concentration of ethanol and an irregular diet increased liver and esophageal cancer risk. On the other hand, we showed that a low concentration of ethanol decreased 8-oxo-Gua and its repair activity in the livers of mice treated with a carcinogen. Taken together, the effects of ethanol consumption on cancer risk depend on the ethanol concentration and the diet pattern.

Topics: Alcohol Drinking; Animals; Carcinogens; Cell Transformation, Neoplastic; Diet; DNA Damage; Esophageal Neoplasms; Esophagus; Ethanol; Guanine; Liver; Liver Neoplasms; Mice; Oxidative Stress; Rats; Rats, Sprague-Dawley

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

Topics: Adenocarcinoma; Animals; Cell Proliferation; Cell Transformation, Neoplastic; DNA Damage; Genes, ras; Guanine; Mice; Mutation; Nitric Oxide Synthase Type II; Oxidative Stress

Excessive exposure to UV radiation is a major risk factor for developing skin cancer. UV-induced reactive oxygen species (ROS) cause accumulation of DNA damage products such as 8-oxoguanine (8-oxoG) in the skin. We have previously shown that mice lacking the repair enzyme 8-oxoguanine glycosylase (Ogg1 knockout mice) are highly susceptible to skin cancer after long-term UVB exposure. To investigate the genes involved, we performed gene profiling of Ogg1 knockout mouse skin after UVB exposure. Among the up-regulated genes in UVB-treated Ogg1 knockout mice, inflammatory response pathway-related genes were most affected. The Vcan gene, which encodes the large extracellular matrix proteoglycan versican, was continuously up-regulated in UVB-treated Ogg1 knockout mice, suggesting that versican is a mediator of skin cancer development. We examined the expression pattern of versican in skin tumors from wild-type mice and UVB-treated Ogg1 knockout mice, and also analyzed 157 sun-related human skin tumors. Versican was strongly expressed in malignant skin tumors in both mice and humans, and especially in Ogg1 knockout mice. Additionally, infiltrating neutrophils strongly colocalized with versican in UVB-treated Ogg1 knockout mouse skin. These data demonstrate that inflammatory responses, particularly neutrophil infiltration and versican up-regulation, are closely involved in UVB/ROS-induced skin tumorigenesis.

Topics: Animals; Cell Transformation, Neoplastic; Dermatitis; DNA Damage; DNA Glycosylases; Down-Regulation; Gene Expression Profiling; Guanine; Humans; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Skin; Skin Neoplasms; Ultraviolet Rays; Up-Regulation; Versicans

Alcohol consumption is known to have opposing effects on carcinogenesis: promotion and prevention. In this study, we examined the effects of 12% ethanol on oxidative DNA damage accumulation and its repair in mouse livers treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), a well-known hepatic carcinogen. We previously reported that 3'-MeDAB increased 8-hydroxyguanine (8-OH-Gua) accumulation and its repair activity, accompanied by the fragmentation of 8-oxoguanine DNA glycosylase 1 (OGG1), the main repair enzyme of 8-OH-Gua. The present results showed that 12% ethanol intake attenuated the 8-OH-Gua accumulation, but not the fragmentation of OGG1 induced by 3'-MeDAB. Additionally, no significant changes in oxidative status, as monitored by lipid peroxidation (LPO), were observed among the 3'-MeDAB-treated mouse livers with/without alcohol administration. These findings suggested that 12% ethanol consumption may reduce the risk of 3'-MeDAB-induced carcinogenesis by decreasing 8-OH-Gua accumulation.

Topics: Alcohol Drinking; Animals; Carcinogens; Cell Transformation, Neoplastic; DNA Damage; DNA Repair; Ethanol; Guanine; Liver; Male; Methyldimethylaminoazobenzene; Mice; Oxidative Stress

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell-Free System; Cyclin-Dependent Kinase Inhibitor p16; DNA; DNA Glycosylases; DNA Polymerase beta; DNA Repair; DNA Replication; Guanine; Humans; Mice; Mutation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Reactive Oxygen Species; Replication Protein A; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Topics: Acrolein; Adenine; Animals; Antibodies, Monoclonal; Cell Transformation, Neoplastic; Chromosome Mapping; DNA; DNA Glycosylases; Gene Expression; Gene Library; Genes, Neoplasm; Genome; Guanine; Immunoprecipitation; Kidney; Kidney Neoplasms; Male; Mice; Mice, Knockout; Oxidation-Reduction; Oxidative Stress

The OGG1 and MYH DNA glycosylases prevent the accumulation of DNA 8-hydroxyguanine. In Myh(-/-) mice, there was no time-dependent accumulation of DNA 8-hydroxyguanine in brain, small intestine, lung, spleen, or kidney. Liver was an exception to this general pattern. Inactivation of both MYH and OGG1 caused an age-associated accumulation of DNA 8-hydroxyguanine in lung and small intestine. The effects of abrogated OGG1 and MYH on hepatic DNA 8-hydroxyguanine levels were additive. Because there is an increased incidence of lung and small intestine cancer in Myh(-/-)/Ogg1(-/-) mice, these findings support a causal role for unrepaired oxidized DNA bases in cancer development.

Topics: Animals; Cell Transformation, Neoplastic; DNA; DNA Glycosylases; Female; Guanine; Intestine, Small; Lung; Male; Mice; Mice, Knockout; Neoplasms, Experimental; Oxidation-Reduction

We measured concentrations and ratios of mutagenic (8-OH) lesions to putatively nonmutagenic formamidopyrimidine (Fapy) lesions of adenine (Ade) and guanine (Gua) to elucidate radical (.OH)-induced changes in DNA of normal, normal from cancer, and cancer tissues of the prostate. The relationship between the lesions was expressed using the mathematical model log(10)[(8-OH-Ade + 8-OH-Gua)/(FapyAde + FapyGua)]. Logistic regression analysis of the log ratios for DNA of normal and cancer tissues discriminated between the two tissue groups with high sensitivity and specificity. Correlation analysis of log ratios for normal prostates revealed a highly significant increase in the proportion of mutagenic base lesions with age. Data from correlation analysis of the log ratios for normal tissues from cancer were consistent with an age-dependent, dose-response relationship. The slopes for both correlations intersected at approximately 61 years, an age when prostate cancer incidence is known to rise sharply. The age-related increase in the proportion of.OH-induced mutagenic base lesions is likely a significant factor in prostate cancer development.

Topics: Adenine; Age Factors; Cell Transformation, Neoplastic; DNA; DNA Damage; DNA, Neoplasm; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Hydroxyl Radical; Logistic Models; Male; Middle Aged; Models, Biological; Prostate; Prostatic Neoplasms; Pyrimidines

To determine the type of mutation induced by 8-hydroxyguanine in a mammalian system, we examined the mutations induced by a synthetic c-Ha-ras protooncogene containing 8-hydroxyguanine in the second position of codon 12 (GGC) in NIH3T3 cells. Transfection of this gene significantly increased the number of transformed foci. The c-Ha-ras gene present in these foci was analyzed by the polymerase chain reaction-restriction enzyme method. Interestingly, sequence analysis revealed random mutations at the modified site (G----T, G----A, and G----C) as well as mutations of the adjacent G on the 5'-side of 8-hydroxyguanine (G----A and G----T).

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Cell Transformation, Neoplastic; Codon; DNA Mutational Analysis; Genes, ras; Guanine; Mice; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Transfection

DNA of invasive ductal carcinomas from five women was analyzed for structural alterations in the purine nucleotides using gas chromatography-mass spectrometry with selected ion monitoring. The results were compared to those for a normal DNA control. The carcinoma DNA showed dramatically higher concentrations of the base modifications 8-hydroxy-guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyadenine. For example, the concentration of total identified base modifications represented a more than 9-fold increase over the control value. Base modifications of this type, which arise from radical-induced hydroxylation and cleavage reactions of the purine ring, likely play a major role in initiation and probably contribute to the further transformation of neoplastic cells in cancer of the female breast.

Topics: Adenine; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Chromatography, Gas; DNA; Female; Guanine; Humans; Hydroxylation; Mass Spectrometry; Pyrimidines