G₂ checkpoint kinase inhibitors exert their radiosensitizing effects prior to the G₂/M transition

Chemical inhibitors of the G₂ checkpoint can sensitize p53-defective cancer cells to DNA damage and several are in preclinical or clinical development. These compounds are commonly thought to increase killing at the G₂/M transition by forcing cells to divide with unrepaired DNA. We examined the effects of the ATM/ATR inhibitor caffeine and the Chk1 inhibitor isogranulatimide on the clonogenic survival of two p53-defective cell lines, MCF7-mp53 and HCT-116 p53^-/- cells, when added at different times after exposure to ionizing radiation. Exposure 16-24 hrs after irradiation, when G₂ arrest is maximal, forced premature entry into mitosis but increased clonogenic survival. Radiosensitization occurred mostly upon exposure between 2 and 16 hrs after irradiation, correlating with S-phase traversal. These results suggest that inhibition of the S phase activities of ATM/ATR and Chk1 may be more relevant to radiosensitization of p53-defective cells than G₂ checkpoint abrogation and that careful scheduling of combination treatments might be required for synergistic antitumor effects in vivo.