Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein k...Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.展开更多
AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analys...AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analysis,sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells.Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR).Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins.In vivo 32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation,eIF2α phosphorylation,and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus.In a sucrose gradient,approxi-mately 30% of the overexpressed p67 was bound with ribosomes.p67 interacted with the kinase domain,butnot the dsRNA-binding domains of PKR.Only the glycosylated p67 was associated with the ribosome,and p67 did not compete with PKR for ribosome binding.In breast cancer cells,there was increased autophosphorylation of PKR but no phosphorylation of eIF2α,compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.展开更多
目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目...目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目前最常用的乙型肝炎病毒感染的体外实验模型,将细胞随机分为空白对照组、阳性对照组(3TC)、不同浓度的HH胶囊组.用CCK-8检测药物细胞毒性,酶联免疫法检测乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量聚合酶链反应法检测乙型肝炎病毒脱氧核糖核酸(HBVDNA);用Westernblot、荧光定量PCR方法分别检测细胞内抗病毒蛋白2'5'-OAS、PKR及其mRNA水平.结果:HH胶囊的TC50是2.11g/L、312.00mg/L、156.00mg/L、78.00mg/L、39.00mg/LHH胶囊均可以降低细胞外HBeAg(0.285±0.007,0.462±0.008,0.565±0.009,0.733±0.008vs1.334±0.007)和HbsAg(0.834±0.008,1.021±0.011,1.347±0.017,1.548±0.015vs2.593±0.008)水平,312.00mg/L、156.00mg/LHH胶囊均可以减低细胞内乙型肝炎病毒DNA[(3.423±0.110)×109copies/L,(3.640±0.082)×109copies/Lvs(6.857±0.060)×109copies/L]、细胞外乙型肝炎病毒DNA(6547±87、7710±62vs24300±200),312.00mg/LHH胶囊可以增加细胞内OASmRNA(0.885±0.038vs0.688±0.068)、PKRmRNA(0.139±0.06vs0.058±0.005)表达及其OAS、PKR蛋白水平.结论:HH胶囊具有良好的抗乙型肝炎病毒作用,推测可能与增加细胞内OAS、PKR及其mRNA水平有关.展开更多
文摘Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.
文摘AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analysis,sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells.Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR).Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins.In vivo 32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation,eIF2α phosphorylation,and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus.In a sucrose gradient,approxi-mately 30% of the overexpressed p67 was bound with ribosomes.p67 interacted with the kinase domain,butnot the dsRNA-binding domains of PKR.Only the glycosylated p67 was associated with the ribosome,and p67 did not compete with PKR for ribosome binding.In breast cancer cells,there was increased autophosphorylation of PKR but no phosphorylation of eIF2α,compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.
文摘目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目前最常用的乙型肝炎病毒感染的体外实验模型,将细胞随机分为空白对照组、阳性对照组(3TC)、不同浓度的HH胶囊组.用CCK-8检测药物细胞毒性,酶联免疫法检测乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量聚合酶链反应法检测乙型肝炎病毒脱氧核糖核酸(HBVDNA);用Westernblot、荧光定量PCR方法分别检测细胞内抗病毒蛋白2'5'-OAS、PKR及其mRNA水平.结果:HH胶囊的TC50是2.11g/L、312.00mg/L、156.00mg/L、78.00mg/L、39.00mg/LHH胶囊均可以降低细胞外HBeAg(0.285±0.007,0.462±0.008,0.565±0.009,0.733±0.008vs1.334±0.007)和HbsAg(0.834±0.008,1.021±0.011,1.347±0.017,1.548±0.015vs2.593±0.008)水平,312.00mg/L、156.00mg/LHH胶囊均可以减低细胞内乙型肝炎病毒DNA[(3.423±0.110)×109copies/L,(3.640±0.082)×109copies/Lvs(6.857±0.060)×109copies/L]、细胞外乙型肝炎病毒DNA(6547±87、7710±62vs24300±200),312.00mg/LHH胶囊可以增加细胞内OASmRNA(0.885±0.038vs0.688±0.068)、PKRmRNA(0.139±0.06vs0.058±0.005)表达及其OAS、PKR蛋白水平.结论:HH胶囊具有良好的抗乙型肝炎病毒作用,推测可能与增加细胞内OAS、PKR及其mRNA水平有关.
