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

ABSTRACT

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/Ku8O-siRNA) and control cells (HeLa/ Neg-siRNA) were transfected with siRNA targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) or pretreated with 50 μmol/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-transfec ted 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.