ABSTRACT
Objective@#To explore the functions of DNA-PKcs in cellular low dose hyper-radiosensitivity.@*Methods@#Colony-formation assay was used to detect the survival fractions of M059K and M059J cell lines treated by X-ray irradiation. Micronucleus assay and γ-H2AX foci assay were used to measure the radiation-induced DNA damage. Western blot was used to detect the relative expression levels of phospho-Chk1, total Chk1, phospho-Chk2 and total Chk2 of M059K and M059J cells.@*Results@#The hyper-radiosensitivity was observed in M059K cells irradiated with X-ray of doses lower than 1 Gy. DNA damage levels did not show HRS/IRR in the cell lines we used. pChk1/Chk1 in M059K cells was significantly increased during 20 min to 60 min after 0.2 Gy X-ray irradiation (t=14.157, 13.661, 14.177, 11.317, 14.512, P<0.05); pChk2/Chk2 in M059K cells was markedly increased during 20 min to 50 min after 0.2 Gy X-ray irradiation (t=13.182, 13.868, 14.155, 14.477, P<0.05).@*Conclusions@#M059K cells show the phenomenon of low dose hyper-radiosensitivity, which may be related to activation of proteins in G2/M phase checkpoints regulated by DNA-PKcs.
ABSTRACT
The low dose hyper-radiosensitivity (HRS) in human lung cancer cell line A549 was in-vestigated,the changes of ATM kinase,cell cycle and apoptosis of cells at different doses of radiation were observed,and the possible mechanisms were discussed.A549 cells in logarithmic growth phase were irradiated with 60Co γ-rays at doses of 0-2 Gy.Together with flow cytometry for precise cell sorting,cell survival fraction was measured by means of conventional colony-formation assay.The expression of ATM1981Ser-P protein was examined by Western blot 1 h after radiation.Apoptosis was detected by Hoechst 33258 fluorescent staining,and Annexin V-FITC/PI staining flow cytometry 24 h after radiation.Cell cycle distribution was observed by flow cytometly 6,12 and 24 h after ra-diation.The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy,followed by an increase at >0.2 Gy,and reached the peak at 0.5 Gy,with little further increase as the dose exceeded 0.5 Gy.Twenty-four h after radiation,partial cells presented the characteristic mor-phological changes of apoptosis,and the cell apoptosis curve was coincident with the survival curve.As compared with control group,the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation (P>0.05).After exposure to 0.3,0.4 and 0.5 Cry radiation,G2/M phase arrest occurred 6 and 12 h after radiation (P<0.05),and the ratio of G2/M phase cells was decreased 24 h after radiation (P<0.05).It was concluded that A549 cells displayed the phenomenon of HRS/IRR.The mode of cell death was mainly apoptosis.The activity of ATM and cell cycle change may take an important role in HRS/IRR.
ABSTRACT
Objective To study the low dose hyper-radiosensitivity in human lung cancer cell line A549,and its possible mechanisms.Methods Exponentially growing A549 cells were irradiated with 60Co γ-rays at doses of 0-2 Gy.Together with flow cytometry for precise cell sorting,cell survival fraction was measured by mean of conventional colony-formation assay.ATM1981 Ser-P protein expression was examined by Western blot.Apoptosis was identified by Hoechst 33258 fluorescent staining,and Annexin V-FITC and propidium iodide staining flow cytometry.Cell cycle distribution was observed by flow cytometry.Results There was an excessive cell killing per unit dose when the doses were below about 0.3 Gy,and the cells exhibited more resistant response at the doses between 0.3 and 0.5 Gy,the cell survival fraction was decreased as the doses over 0.5 Gy.The expression of ATM1981Ser-P protein was first observed at 0.2 Gy,followed by an increase over 0.2 Gy,and reached the peak at 0.5 Gy(compared with 0.2 Gy group,t=7.96,P＜0.05),with no further increase as the doses at 1.0 and 2.0 Gy(t=0.69,0.55,P＞0.05).24 hours after irradiation,part cells presented the characteristic morpholos4cal change of apoptosis,and the apoptosis curve was coincident with the dose-survival curve.Compared with the control group,the cell cycle had no change post-irradiation to 0.1 and 0.2 Gy.G2/M phase arrest was manifested at 6 and 12 hours post-irradiation to 0.3,0.4 and 0.5 Gy(t=2.87,2.88,4.92 and 3.70,3.12,8.11,P＜0.05),and the ratio of G2/M phase was decreased at 24 hours post-irradiation(t=3.87,4.77,3.01,P＜0.05).Conclusions A549 cells displays the phenomenon of hyper-radiosensitivity(HRS)/induced radioresistance(IRR).The model of cell death induced by low dose irradiation is mainly apoptosis.The activity of ATM and cell cycle change might play an important role in HRS/IRR.