Caffeine,which specifically inhibits ATM/ATR kinases,efficiently abrogates the ionizing radiation(IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR.Mechanisms for the effect of caffeine ...Caffeine,which specifically inhibits ATM/ATR kinases,efficiently abrogates the ionizing radiation(IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR.Mechanisms for the effect of caffeine remain to be elucidated.As a target of ATM/ATR kinases,BRCA1 becomes activated and phosphorylated in response to IR.Thus,in this work,we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process.For these purposes,the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine.The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524,which was followed by an override of G2 arrest by caffeine.In addition,the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine.These data suggest that BRCA1 may be a potential target of caffeine.BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.展开更多
To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subj...To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subjected to irradiation at the dosage ranging from 0.05-2 Gy.Clonogenic survival was measured by using fluorescence-activated cell sorting (FACS) plating technique.Percentage of cells in M-phase after low doses of X-irradiation was evaluated by phospho-histone H3-FITC/PI and Western blotting was used to detect protein expression of Chk2 and phospo-Chk2.DNA DSBs repair efficiency was also measured by induction and persistence of γ-H2AX.The results showed that the killing ability of irradiation with A549 cells increased at low conditioning dose below 0.3 Gy.Within the dose of 0.3 to 0.5 Gy,A549 cells showed a certain extent of radiation resistance.And when the dose was more than 0.5 Gy,survival fraction exhibited a negative correlation with the dosage.There was no difference between the 0.1 or 0.2 Gy dosage groups and the un-irradiated group in terms of the percentage of cells in M phase.But in the high dosage group (0.3-1.0 Gy),the percentage of cells in M phase was decreased markedly.In addition,the percentage of cells in M phase began to decrease two hours after irradiation.One hour after irradiation,there was no conspicuous activation of Chk2 kinase in 0.1 or 0.2 Gy group,but when the irradiation dose reached 0.3 Gy or higher,Chk2 kinase started to be activated and the activation level showed no significant difference among high dosage groups (0.4,0.5,1.0 Gy).Within 1 to 6 h,the DNA DSBs repair efficiency was decreased at 0.2 Gy but increased at 0.5 Gy and 1.0 Gy,which was in line with Chk2 activation.We are led to conclude that the mechanism of HRS/IRR in A549 cell line was probably due to early G2/M checkpoint arrest and enhanced DNA DSBs repair.In this regard,Chk2 activation plays a key role in G2/M checkpoint activation.展开更多
基金supported by the National Basic Research Program of China (Grant No. 2010CB834202)the National Natural Science Foundation of China (Grant Nos. 10835011and 10805064)+1 种基金the Scientific Technology Research Projects of Gansu Province (Grant Nos. 0806RJYA020 and 099RJYA015)Western Talent Program of Chinese Academy of Sciences (Grant No. 0860260XBO)
文摘Caffeine,which specifically inhibits ATM/ATR kinases,efficiently abrogates the ionizing radiation(IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR.Mechanisms for the effect of caffeine remain to be elucidated.As a target of ATM/ATR kinases,BRCA1 becomes activated and phosphorylated in response to IR.Thus,in this work,we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process.For these purposes,the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine.The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524,which was followed by an override of G2 arrest by caffeine.In addition,the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine.These data suggest that BRCA1 may be a potential target of caffeine.BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.
基金supported by a grant from a Key ScientificResearch Program of Hubei Provincial Authorities (No.JX2A11)
文摘To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subjected to irradiation at the dosage ranging from 0.05-2 Gy.Clonogenic survival was measured by using fluorescence-activated cell sorting (FACS) plating technique.Percentage of cells in M-phase after low doses of X-irradiation was evaluated by phospho-histone H3-FITC/PI and Western blotting was used to detect protein expression of Chk2 and phospo-Chk2.DNA DSBs repair efficiency was also measured by induction and persistence of γ-H2AX.The results showed that the killing ability of irradiation with A549 cells increased at low conditioning dose below 0.3 Gy.Within the dose of 0.3 to 0.5 Gy,A549 cells showed a certain extent of radiation resistance.And when the dose was more than 0.5 Gy,survival fraction exhibited a negative correlation with the dosage.There was no difference between the 0.1 or 0.2 Gy dosage groups and the un-irradiated group in terms of the percentage of cells in M phase.But in the high dosage group (0.3-1.0 Gy),the percentage of cells in M phase was decreased markedly.In addition,the percentage of cells in M phase began to decrease two hours after irradiation.One hour after irradiation,there was no conspicuous activation of Chk2 kinase in 0.1 or 0.2 Gy group,but when the irradiation dose reached 0.3 Gy or higher,Chk2 kinase started to be activated and the activation level showed no significant difference among high dosage groups (0.4,0.5,1.0 Gy).Within 1 to 6 h,the DNA DSBs repair efficiency was decreased at 0.2 Gy but increased at 0.5 Gy and 1.0 Gy,which was in line with Chk2 activation.We are led to conclude that the mechanism of HRS/IRR in A549 cell line was probably due to early G2/M checkpoint arrest and enhanced DNA DSBs repair.In this regard,Chk2 activation plays a key role in G2/M checkpoint activation.
