The effect of co-inhibition of Ku80 and DNA-PKcs on radiobiological function of HeLa cells

Objective:To study the changes in radiosensitivity and cell cycle distribution of HeLa cells after inhibition of one or several DNA double-strand break(DSB) repair proteins by small interfering RNA(siRNA) and LY294002.Methods:Ku80 silenced cells(HeLa/Ku80-siRNA) and control cells(HeLa/Neg-siRNA) were transfected with siRNA targeting DNA-dependent protein kinase catalytic subunit(DNA-PKcs) or pretreated with 50μmol/L LY294002,a chemically specific inhibitor of DNA-PKcs.After the cells received 6MV X-ray irradiation,the radiosensitivity of the cells was detected by clony formation assay and cell cycle distribution was analyzed by flow cytometry.Results:The survival fraction at 2 Gy(SF2) value was 0.08±0.01 for HeLa/Ku80-siRNA cells after being transfected with DNA-PKcs siRNA;the SF2 value reached 0.03±0.01 when HeLa/Ku80-siRNA cells were pretreated with LY294002.Both of them were significantly lower than that of untreated HeLa/Ku80-siRNA cells(0.20±0.05).The cells in all the groups were arrested in G2/M phase after irradiaton with 6 Gy X-ray.The G2/M arrest occurred slowly in the DNA-PKcs-siRNA-transfected HeLa/Neg-siRNA cells and LY294002-pretreated HeLa/Ku80-siRNA and HeLa/Neg-siRNA cells,which did not reach the peak at 72 h post-irradiation.The G2/M accumulation was maximal at 48 h post-irradiation in other cell lines.Conclusion:Based on 95% inhibition of Ku80 protein,DNA-PKcs or ataxia-telangiectasia mutant(ATM) gene could compensate the DSB repair function.Co-inhibition of these proteins led to increase in radiosensitivity of HeLa cells;Ku80,DNA-PKcs and ATM play different roles in G2/M arrest.