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.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
4.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
5.Acetoaminophen-induced accumulation of 8-oxodeoxyguanosine through reduction of Ogg1 DNA repair enzyme in C6 glioma cells.
Jie WAN ; Myung Ae BAE ; Byoung Joon SONG
Experimental & Molecular Medicine 2004;36(1):71-77
Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome P450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.
Acetaminophen/*metabolism
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Analgesics, Non-Narcotic/*metabolism
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Animals
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Cell Line, Tumor
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DNA/metabolism
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DNA Damage
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DNA Glycosylases/*metabolism
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DNA Repair
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Deoxyguanosine/chemistry/*metabolism
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Glioma/*metabolism
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Glutathione/metabolism
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Humans
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Rats
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Reactive Nitrogen Species/metabolism
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Reactive Oxygen Species/metabolism
6.Effect of hOGG1 expression level on oxidative DNA damage among workers exposed to nickel in stainless steel production environment.
YanLi LI ; Jianya SUN ; Huizhen SHANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2014;32(8):578-581
OBJECTIVETo study the excision repair capacity of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) for 8-OH-dG and the oxidative DNA damage among workers exposed to nickel in stainless steel production environment.
METHODSA total of 231 workers exposed to nickel in a stainless steel production enterprise were recruited as nickel exposure group, and another 75 water pump workers in that enterprise were recruited as control group. The workplace occupational hazard factors were determined. Double-antigen sandwich ELISA was used to determine urinary 8-OH-dG level; RT-PCR was used to determine hOGG1 mRNA level. Pearson correlation was used to analyze the correlation between urinary 8-OH-dG level and hOGG1 mRNA level.
RESULTSLevel of 8-OH-dG was compared between different types of nickel-exposed workers and control workers; rolling mill workers showed no significant difference from the control group (P > 0.05), while steel making workers and steel slag disposing workers showed significant differences from the control group (P < 0.05). Level of 8-OH-dG was also compared between nickel-exposed workers with different working years and control workers; nickel-exposed workers with 0∼5 and 6∼10 working years showed no significant differences from the control group (P > 0.05), while other exposed workers showed significant differences from the control group (P < 0.05). Different types of nickel-exposed workers all showed significant differences from the control group in hOGG1 mRNA level (P < 0.05). Nickel-exposed workers with 0∼5 working years showed no significant difference from the control group in hOGG1 mRNA level (P > 0.05), while other exposed workers showed significant differences from the control group (P < 0.05). Pearson correlation analysis showed that urinary 8-OH-dG level was positively correlated with hOGG1 mRNA level (r = 0.993) in different types of nickel-exposed workers, and the correlation was significant at α = 0.01 (P < 0.05); urinary 8-OH-dG level also showed a positive correlation with hOGG1 mRNA level in nickel-exposed workers with different working years (r = 0.968), and the correlation was significant at α = 0.01 (P < 0.05).
CONCLUSIONExposure to nickel increases oxidative DNA damage among steel workers, and hOGG1 shows active excision repair capacity for 8-OH-dG.
Adult ; DNA Damage ; DNA Glycosylases ; metabolism ; DNA Repair ; Humans ; Male ; Metallurgy ; Middle Aged ; Nickel ; adverse effects ; Occupational Exposure ; adverse effects ; Stainless Steel ; Young Adult
7.Effect of adenoviral N-methylpurine DNA glycosylase overexpression on chemosensitivity of human osteosarcoma cells.
Dong WANG ; Zhao-yang ZHONG ; Qin-hong ZHANG ; Zeng-peng LI ; Mark R KELLEY
Chinese Journal of Pathology 2006;35(6):352-356
OBJECTIVEThe overexpression of N-methylpurine DNA glycosylase (MPG) may imbalance the DNA base excision repair (BER) to sensitize tumor cells to current DNA damage chemotherapy. In an effort to improve the efficacy of cancer chemotherapy, we have constructed adenoviral vector of MPG, to study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents.
METHODSThe adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blot, and HEX labeled oligonucleotide-based assay respectively. The cell survival/proliferation was measured using MTS, SRB, and [(3)H] thymidine incorporation assay. Apoptosis cell death was assayed by flow cytometry after treatment using phycoerythin (PE)-conjugated Annexin V and 7-amino-actinomycin (7-AAD).
RESULTSA 10 MOI of recombinant nonreplicating adenovirus was found to infect more than 90% of HOS cells within 24 hours by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The MPG overexpression HOS cells were significantly more sensitive to the DNA damage agents, including MMS, MNNG, and TMZ, with changes in the IC50 of 6.0, 4.5, and 2.5 fold respectively.
CONCLUSIONSThese data establish transient MPG overexpression as a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent chemotherapy.
Adenoviridae ; enzymology ; Antineoplastic Agents ; metabolism ; pharmacology ; Cell Line, Tumor ; DNA Glycosylases ; genetics ; metabolism ; pharmacology ; Gene Expression Regulation, Neoplastic ; Humans ; Osteosarcoma ; pathology
8.Exercise training in hypoxia prevents hypoxia induced mitochondrial DNA oxidative damage in skeletal muscle.
Hai BO ; ; Ling LI ; Fu-Qiang DUAN ; Jiang ZHU
Acta Physiologica Sinica 2014;66(5):597-604
This study was undertaken to investigate the effect of exercise training on mitochondrial DNA (mtDNA) oxidative damage and 8-oxoguanine DNA glycosylase-1 (OGG1) expression in skeletal muscle of rats under continuous exposure to hypoxia. Male Sprague-Dawley rats were randomly divided into 4 groups (n = 8): normoxia control group (NC), normoxia training group (NT), hypoxia control group (HC), and hypoxia training group (HT). The hypoxia-treated animals were housed in normobaric hypoxic tent containing 11.3% oxygen for consecutive 4 weeks. The exercise-trained animals were exercised on a motor-driven rodent treadmill at a speed of 15 m/min, 5% grade for 60 min/day, 5 days per week for 4 weeks. The results showed that, compared with NC group, hypoxia attenuated complex I, II, IV and ATP synthase activities of the electron transport chain, and the level of mitochondrial membrane potential in HC group (P < 0.05 or P < 0.01). Moreover, hypoxia decreased mitochondrial OGG1, MnSOD, and GPx activities (P < 0.05 or P < 0.01), whereas elevated reactive oxygen species (ROS) generation and the level of 8-oxo-deoxyguanosine (8-oxodG) in mtDNA (P < 0.01). Furthermore, hypoxia attenuated muscle and mitochondrial [NAD⁺]/ [NADH] ratio, and SIRT3 protein expression (P < 0.05 or P < 0.01). Compared with HC group, exercise training in hypoxia elevated complex I, II, IV and ATP synthase activities, and the level of mitochondrial membrane potential in HT group (P < 0.05 or P < 0.01). Moreover, exercise training in hypoxia increased MnSOD and GPx activities and mitochondrial OGG1 level (P < 0.01), whereas decreased ROS generation and the level of 8-oxodG in mtDNA (P < 0.01). Furthermore, exercise training in hypoxia increased muscle and mitochondrial [NAD⁺]/[NADH] ratio, as well as SIRT3 protein expression (P < 0.05 or P < 0.01). These findings suggest that exercise training in hypoxia can decrease hypoxia-induced mtDNA oxidative damage in the skeletal muscle through up-regulating exercise-induced mitochondrial OGG1 and antioxidant enzymes. Exercise training in hypoxia may improve hypoxia tolerance in skeletal muscle mitochondria via elevating [NAD⁺]/[NADH] ratio and SIRT3 expression.
Animals
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DNA Glycosylases
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metabolism
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DNA, Mitochondrial
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chemistry
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Glutathione Peroxidase
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metabolism
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Guanine
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analogs & derivatives
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metabolism
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Hypoxia
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physiopathology
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Male
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Mitochondria, Muscle
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pathology
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Muscle, Skeletal
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metabolism
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Oxidative Stress
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Physical Conditioning, Animal
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Rats
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Rats, Sprague-Dawley
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Reactive Oxygen Species
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metabolism
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Superoxide Dismutase
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metabolism
9.Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia.
Long-Xiu YANG ; Xiao ZHANG ; Gang ZHAO
Chinese Medical Journal 2016;129(16):1955-1962
BACKGROUNDGinsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia.
METHODSNEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay.
RESULTSWe found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO.
CONCLUSIONSOur results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEIL1 and NEIL3 expressions.
Animals ; Blotting, Western ; Brain Ischemia ; drug therapy ; enzymology ; DNA Damage ; drug effects ; DNA Glycosylases ; genetics ; metabolism ; Ginsenosides ; therapeutic use ; Infarction, Middle Cerebral Artery ; drug therapy ; enzymology ; Male ; N-Glycosyl Hydrolases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley
10.Expression of hOGG1 protein during differentiation of HL-60 cells.
Yun Song LEE ; Kyeong Hoon LEE ; Myung Hee CHUNG
Experimental & Molecular Medicine 2003;35(2):98-105
Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D3. Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.
Blotting, Western
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*Cell Differentiation
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Culture Media, Serum-Free/pharmacology
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DNA Glycosylases/*metabolism
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Enzyme Activation
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*Gene Expression Regulation, Enzymologic/drug effects
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Granulocytes/cytology/drug effects/metabolism
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HL-60 Cells
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Human
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Monocytes/cytology/drug effects/metabolism