The Limitations of the G1-S Checkpoint

It has been proposed that the G(1)-S checkpoint is the critical regulator of genomic stability, preventing the cell cycle progression of cells with a single DNA double-strand break. Using fluorescence-activated cell sorting analysis of asynchronous cells and microscopic analysis of asynchronous and synchronized cells, we show that full blockage of S-phase entry is only observed >4 hours after irradiation. The process is ataxia-telangiectasia mutated (ATM) dependent and Chk1/2 independent and can be activated throughout G(1) phase. By monitoring S-phase entry of irradiated synchronized cells, we show that the duration of arrest is dose dependent, with S-phase entry recommencing after arrest with kinetics similar to that observed in unirradiated cells. Thus, G(1)-S checkpoint arrest is not always permanent. Following exposure to higher doses (>= 2 Gy), G(1)-S arrest is inefficiently maintained, allowing progression of G(1)-phase cells into G(2) with elevated gamma H2AX foci and chromosome breaks. At early times after irradiation (<= 4 h), G(1)-S checkpoint arrest is not established but cells enter S phase at a reduced rate. This early slowing in S-phase entry is ATM and Chk2 dependent and detectable after 100 mGy, showing a novel and sensitive damage response. However, the time needed to establish G(1)-S checkpoint arrest provides a window when cells can progress to G(2) and form chromosome breaks. Our findings detail the efficacy of the G(1)-S checkpoint and define two significant limitations: At early times after IR, the activated checkpoint fails to efficiently prevent S-phase entry, and at later times, the checkpoint is inefficiently maintained. Cancer Res; 70(11); 4412-21. (C) 2010 AACR.