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
The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression leve...The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~unol/L）, and NVP-BKM120 （1 ~tmol/L） or NVP-BKM120 （1 pmol/L） plus gefitinib （1 ~tmol/L） obviously inhibited the activation of Akt, $6 and 4E-BP1 as compared with control group, but single use of gefitinib （1 pmol/L） exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H 1975 cells to gefitinib.展开更多
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.
基金supported by grants from the Wu Jieping Medical Foundation of China(No.320-6700-09069)the Hubei Provincial Natural Science Foundation of China(No.2010CDB07702)Novartis International AG
文摘The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~unol/L）, and NVP-BKM120 （1 ~tmol/L） or NVP-BKM120 （1 pmol/L） plus gefitinib （1 ~tmol/L） obviously inhibited the activation of Akt, $6 and 4E-BP1 as compared with control group, but single use of gefitinib （1 pmol/L） exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H 1975 cells to gefitinib.
