Radiosensitivity, apoptosis and repair of DNA double-strand breaks in radiation-sensitive Chinese hamster ovary cell mutants treated at different dose rates.

The relationship of cell survival to induction and repair of DNA double-strand breaks (DSBs), as assessed by the neutral comet assay, was studied in two pairs of CHO cell lines, repair-deficient mutants xrs-5-11 and V3, and their respective parent lines K-1 and AA8, treated at two dose rates, 10.5 and 0.09 Gy/min. A marked difference in cell survival after irradiation was found between each pair of lines. For initial DNA damage, there was a significant difference between each pair of lines after the low-dose-rate treatment but not with high-dose-rate irradiation. Initial DSBs were dependent on dose at both dose rates. When residual damage at 2 h after irradiation was assessed, the 5-11 cells showed significantly more damage than K-1 cells after both high- and low-dose-rate irradiation. The V3 cells also showed more residual damage than the AA8 cells, but this difference was significant only after high-dose-rate irradiation. The results indicate that low-dose-rate irradiation can differentiate better between the DSB rejoining capacity of the sensitive and resistant cells and are consistent with the idea that it is the slow component of repair of DSBs which is different between them. Further studies with 5-11 and K-1 cells showed that radiation-induced apoptosis was dependent on dose, with a higher fraction of apoptotic cells in 5-11 than in K-1 cells after exposure to a given high-dose-rate radiation dose. However, the levels and time course of induction of apoptosis were similar for doses which gave equal levels of clonogenic survival. Radiation treatment was found to delay the progression of 5-11 and K-1 cells through the cell cycle to the same extent, with accumulation of cells in S phase and G2 phase, at 4 and 12 h after irradiation, respectively. There was no evidence for a G1-phase arrest. Western blotting revealed that there were higher levels of p53 and Waf1 protein in nonirradiated 5-11 than in K-1 cells, and that a dose of 5 Gy of high-dose-rate radiation up-regulated the expression of p53 and Waf1 protein to similar levels in both cell lines. There was no change in levels of the Gadd45 and Bcl2 proteins in either cell line after irradiation. These results suggest that the accumulation of p53 and Waf1 protein does not cause early G1-phase arrest in these cells.