1.The effects of HBx gene on the expression of DNA repair enzymes hOGG1 and hMYHalpha mRNA in HepG2 cells.
Bin, CHENG ; Xiaorong, GUO ; Yaochu, ZHENG ; Ying, WANG ; Chunyan, LIU ; Peiyuan, LI
Journal of Huazhong University of Science and Technology (Medical Sciences) 2009;29(2):187-92
To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the beta-actin as the interior control, real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHalpha in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1). The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P<0.05) and HepG2/pDNA3.1 (0.121+/-0.005) (P<0.05). However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains (P>0.05). The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05). It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHalpha mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.
DNA Glycosylases/genetics
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DNA Glycosylases/*metabolism
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Deoxyguanosine/analogs & derivatives
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Deoxyguanosine/metabolism
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Hep G2 Cells
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RNA, Messenger/genetics
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RNA, Messenger/metabolism
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Trans-Activators/*genetics
3.A study of the substitution effect of hOGG1 and hMTH1 in oxidative DNA damage with gene-deficient cell strains.
Yuebin KE ; Shuang WU ; Juan HUANG ; Jianhui YUAN ; Pingjian DENG ; Jinquan CHENG
Chinese Journal of Preventive Medicine 2014;48(3):197-202
OBJECTIVETo investigate the potential substitution effect of hOGG1 and hMTH1 on oxidative DNA damage, based on gene-deficient cell strains models.
METHODShOGG1 and hMTH1 gene deficient cell strains models were established by Human embryonic lung fibroblasts (HFL) cells. After HFL cells being exposed to 100 µmol/L H₂O₂ for 12 h, HPLC-EC detecting technique and RT-PCR method were adopted to analyze the genetic expression level of 8-oxo-dG (7, 8-dihydro-8-oxoguanine).
RESULTSThe gene-deficient cell strains models of hOGG1 and hMTH1 were obtained by infecting target cells with high titer of lentivirus. The mRNA expression level of hOGG1 was 0.09 ± 0.02, 91% lower than it in normal HFL cells, which was 1.00 ± 0.04. As the same, the mRNA expression level of hMTH1 (0.41 ± 0.04) also decreased by 60% compared with it in normal HFL cells (1.02 ± 0.06). After induced by 100 µmol/L H₂O₂ for 12 h, the genetic expression level of hMTH1 in hOGG1 gene-deficient cells (1.26 ± 0.18) increased 25% compared with it in control group (1.01 ± 0.07). Meanwhile, the genetic expression level of hOGG1 in hMTH1 gene-deficient cells (1.54 ± 0.25) also increased by 52%. The DNA 8-oxo-dG levels in hOGG1 gene-deficient cells (2.48 ± 0.54) was 3.1 times compared with it in the control group (0.80 ± 0.16), the difference showed statistical significance (P < 0.01). Whereas the 8-oxo-dG levels in hMTH1 gene-deficient cells (1.84 ± 0.46) was 2.3 times of it in the control group, the difference also showed statistical significance (P < 0.01).
CONCLUSIONBased on gene-deficient HFL cells models, a synergetic substitution effect on DNA damage and repair activity by both hOGG1 and hMTH1 were firstly discovered when induced by oxidation. The substitution effect of hOGG1 were stronger than that of hMTH1.
Cell Line ; DNA Damage ; DNA Glycosylases ; genetics ; DNA Repair ; DNA Repair Enzymes ; genetics ; Fibroblasts ; metabolism ; Humans ; Oxidative Stress ; genetics ; Phosphoric Monoester Hydrolases ; genetics
4.Effect of down-regulated hOGG1 gene expression on cytotoxicity and genotoxicity of hydroquinone.
Ya SHU ; Mei WU ; Chen CHEN ; Zun-zhen ZHANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2009;27(10):584-588
OBJECTIVETo explore the effects of down- regulated hOGG1 gene expression on cytotoxicity and genotoxicity of hydroquinone.
METHODSA549 cells and A549-R cells with down- regulated hOGG1 gene were treated with different concentrations of hydroquinone (0, 5, 10, 20, 40 and 80 μmol/L). The cellular sensitivity and contents of ROS were measured by MTT assay and fluorescence method, respectively. The chromosome damage was measured by micronucleus test. The DNA damage and repair were examined using comet assay in both cells.
RESULTSThe cell viability decreased with increasing concentration of hydroquinone. The IC₅₀ of hydroquinone was 160.49 and 228.42 μmol/L in hOGG1 deficient A549-R cell and in A549 cell respectively (P < 0.05). When the dose of hydroquinone reached 5 micromol/L and above, the contents of ROS and the rate of micronucleated cells in A549-R cells were significantly higher than in A549 (P < 0.05) cells. At the same time, the comet rate and OTM in A549-R cells were significantly higher compared with A549 cells at 5 micromol/L and above in a dose-response way (P < 0.05). Furthermore, in DNA repair assay, A549-R cells with down- regulated hOGG1 gene were more difficult to repair than A549 cells. In A549-R cells, the comet rate and OTM reduced significantly until after 2 h repair time and even after 3 h the DNA damage was not repaired completely.
CONCLUSIONOxidative damage may be one of the toxicological mechanisms of hydroquinone, and hOGG1 deficiency could increase sensitivity of A549-R cells to hydroquinone.
Cell Line, Tumor ; Cell Survival ; Comet Assay ; DNA Damage ; DNA Glycosylases ; genetics ; Down-Regulation ; Humans ; Hydroquinones ; toxicity ; Oxidative Stress
5.The relation of asbestosis to human 8-oxoguanine DNA glycosidase polymorphism and DNA damage in peripheral lymphocytes.
Jun-Xia GUO ; Xiao-Hong ZHAO ; Yong-Quan LIU
Chinese Journal of Preventive Medicine 2006;40(6):381-385
OBJECTIVETo explore the relation of asbestosis to human 8-oxoguanine DNA glycosidase (hOGG1) genotype and DNA damage, the investigation of hOGG1 polymorphism distribution and DNA strand breakages in peripheral lymphocytes was carried on in occupational population.
METHODSA total 101 asbestos-exposed workers and 141 controls were investigated. The DNA damage level was obtained by single cell gel electrophoresis (SCGE) and hOGG1 Ser326Cys polymorphism by PCR-RELP.
RESULTS(1) A significant increase in the exposed group was observed in comet scores at basal (34.8 +/- 16.8), H2O2-induced (136.7 +/- 36.0) and 4 hours after repair (51.0 +/- 18.7) as compared with the control group (P < 0.01). And the scores in H2O2-induced (147.0 +/- 30.8) and 4 hours after repair (56.9 +/- 21.4) were significantly higher in asbestosis workers than in non-asbestosis ones (125.7 +/- 38.2 and 44.9 +/- 15.4, P < 0.01). (2) There was no differences of the genotype distribution between the asbestos group and the control group (chi(2) = 0.22, P = 0.89). A significant difference in the distribution of this polymorphism (Ser/Ser, Ser/Cys, Cys/Cys) between asbestosis group (25.5%, 51.0%, 23.5%) and the non-asbestosis group (48.0%, 36.0%, 16.0%) was observed (chi(2) = 6.023, P < 0.05). The comet scores at H2O2-induced and 4 hours after repair were higher in asbestosis subjects than in non-asbestosis ones (P < 0.05). (3) After adjusting ages, sex, smoking and drinking status, the odds ratios of the Cys allele for asbestosis were 0.66 (95% CI = 0.38 - 1.13) in the exposed subjects.
CONCLUSIONThe results suggested that the asbestos occupational exposure might induce DNA damage and the augment on susceptivity of H2O2 oxidation and the fall of the capacity of repairing DNA damage might be one of the mechanisms to induce asbestosis among subjects with the Cys allele.
Aged ; Alleles ; Asbestosis ; blood ; epidemiology ; genetics ; China ; Comet Assay ; DNA Damage ; DNA Glycosylases ; genetics ; DNA Repair ; Female ; Genotype ; Humans ; Lymphocytes ; cytology ; Male ; Middle Aged ; Polymorphism, Genetic
6.Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli.
Yun Song LEE ; Myung Hee CHUNG
Experimental & Molecular Medicine 2003;35(2):106-112
8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
DNA Glycosylases/metabolism
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*DNA Repair
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DNA, Bacterial/*chemistry/*metabolism
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DNA-Formamidopyrimidine Glycosylase/metabolism
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Escherichia coli/*enzymology/*genetics
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Guanine/*analogs & derivatives/*metabolism
7.Inherited mutations of MUTYH and colorectal cancer.
Journal of Zhejiang University. Medical sciences 2007;36(4):406-411
MUTYH, one of base-excision repair enzymes, is associated with human genetic disorders. Inherited biallelic mutations in the human MUTYH gene are responsible for an autosomal recessive syndrome-adenomatous colorectal polyposis (MUTYH associated polyposis, MAP), which significantly increases the risk of colorectal cancer (CRC). In this article we review the relationship between BER and the oxidative damage to DNA, the functional overlap of BER with other repair proteins, the molecular mechanism of tumourigenesis in MAP, and delineate the MUTYH polyposis phenotype and its prevention.
Adenomatous Polyposis Coli
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complications
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enzymology
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genetics
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Colorectal Neoplasms
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enzymology
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etiology
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genetics
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DNA Damage
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DNA Glycosylases
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genetics
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Germ-Line Mutation
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Humans
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Mutation
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Risk Factors
8.Advance in research on the correlation between genotypes of susceptible mutations and clinical phenotype of familial adenomatous polyposis.
Shengliang HE ; Jing DU ; Fangqi LIU
Chinese Journal of Medical Genetics 2017;34(6):919-923
Familial adenomatous polyposis (FAP) is one of the most common hereditary colorectal cancers. Its intestinal and extra-intestinal manifestations are correlated with mutation sties of the APC gene. Potential gene modulation sites in patients who have typical clinical manifestations but with unidentified APC mutations are also discussed, which included MUTYH gene, AXIN gene and certain epigenetic changes. With the generalization of Precision Medicine, to offer individualized treatment and surveillance strategy based on the genotype-phenotype correlation will be of great value for FAP patients. This review focuses on the research advance in genotype - phenotype correlation studies of FAP patients.
Adenomatous Polyposis Coli
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genetics
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Axin Protein
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genetics
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DNA Glycosylases
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genetics
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Genes, APC
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Genetic Association Studies
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Genetic Predisposition to Disease
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Humans
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Mutation
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beta Catenin
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genetics
9.Effects of hydroquinone on expression of human 8-oxo-guanine DNA glycosylase mRNA in human A549 lung adenocarcinoma cell strains.
Ya SHU ; Yue-bin KE ; Ling WANG ; Zun-zhen ZHANG
Chinese Journal of Preventive Medicine 2009;43(5):428-433
OBJECTIVETo explore the effects of hydroquinone (HQ) on reactive oxygen species (ROS) generation, antioxydase activities and the expression of human 8-oxo-guanine DNA glycosylase (hOGG1) mRNA in human A549 lung adenocarcinoma cell strains.
METHODSA549 cells were treated with different concentrations of HQ. Cell survival was determined by methyl thiazolyl tetrazolium (MTT). Changes of ROS were detected by fluorescent probe. The contents of malonaldehyde and activities of antioxydase were determined through colorimetry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of hOGG1 mRNA.
RESULTSWith the increased concentration of HQ, the findings were as follows. (1) The absorbance value of A549 cell decreased. There was significant difference between 160 micromol/L (0.584+/-0.098) and 320 micromol/L (0.328+/-0.066) of HQ (q=5.56 and 9.07, P<0.05) with the control group (0.989+/-0.150), and the cell survival rate were less than 80%. (2) The ROS in A549 cell increased. 40 micromol/L (39.80+/-4.15) and 80 micromol/L (101.99+/-9.45) had statistical significance (q=10.74 and 30.32, P<0.05) with the control group (5.71+/-0.50). (3) It was found that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased and malonaldehyde (MDA) increased. Compared with the control group [(25.62+/-0.28) U/mg prot and (38.97+/-2.61) U/mg prot], the activities of SOD and GSH-Px had a significant decrease (q=12.17 and 8.78, P<0.05) in 80 micromol/L [(22.93+/-0.56) U/mg prot and (25.60+/-2.31) U/mg prot]. And MDA had a significant increase (q=10.90 and 15.49, P<0.05) in 40 micromol/L [(1.07+/-0.01) nmol/mg prot] and 80 micromol/L [(1.19+/-0.08) nmol/mg prot] as compared with the control group [(0.77+/-0.04) nmol/mg prot]. The decrease of SOD (r=-0.95, F=20.00, P=0.04) and GSH-Px activities (r=-0.99, F=115.48, P=0.01) and the increase of MDA contents (r=0.96, F=21.31, P=0.04) all had a dose-response relationship. (4) RT-PCR results showed that the expression of hOGG1 mRNA decreased. The significant difference was observed between the expression of hOGG1 mRNA in 80 micromol/L (0.478+/-0.017) (q=11.70, P<0.05) with the control group (0.715+/-0.038).
CONCLUSIONThis study suggests that HQ could induce oxidative damage and changes of the expression of hOGG1 mRNA in A549 cells.
Cell Line, Tumor ; DNA Glycosylases ; genetics ; Down-Regulation ; Gene Expression ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Hydroquinones ; toxicity ; RNA, Messenger ; genetics
10.Association of 8-hydroxyguanine glycosidase OGG1 Ser326Cys polymorphism with male infertility.
Jian-Zhong CHEN ; Sheng-Min WU ; Gui-Xiang JI ; Ai-Hua GU
National Journal of Andrology 2014;20(6):518-522
OBJECTIVETo explore the association of 8-hydroxyguanine glycosidase OGG1 Ser326Cys polymorphism with semen quality and the risk of male infertility.
METHODSThis case-control study included 620 idiopathic infertile patients and 385 normal fertile controls. We determined their genotypes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and analyzed their semen quality by computer-aided semen analysis (CASA).
RESULTSThe individuals with OGG1 326 Cys/Cys showed significantly lower sperm motility and concentration ([52.1 +/- 26.7]% and (3.75 +/- 0.91) x 10(6)/ml, ln transformed value) than the Ser/Ser carriers ([59.0 +/- 21.8] % and (4.12 +/- 0.88) x 10(6)/ml, ln transformed value) (P < 0.05). The risk of male infertility increased 69% in the OGG1 326Cys allele carriers as compared with the Ser carriers (OR = 1.69, 95% CI: 1.24 -2.31).
CONCLUSIONOGG1 326 Ser/Cys polymorphism might contribute to the risk of male infertility in the southern Chinese population.
Adult ; Case-Control Studies ; DNA Glycosylases ; genetics ; Genotype ; Humans ; Infertility, Male ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Semen Analysis ; Young Adult