DNA damage-induced cell cycle checkpoints as a target for cancer therapy

Cells respond to DNA damage by activating signal transduction pathways that cause cell cycle arrest at checkpoints in G1, S, or G 2 phases of the cell cycle; this allows time for repair of damaged DNA. The G1 checkpoint is dependent upon the function of the p53 tumor suppressor, whereas S and G2 checkpoints are activated independently of p53. Prior studies showed that p53-defective cells that arrest in S and G2, but not p53 wild-type cells, can be sensitized to DNA damage by the checkpoint inhibitor caffeine, which causes premature cell cycle progression and lethal mitosis. This laboratory previously demonstrated that UCN-01, an inhibitor of Chk1/2 activity, also abrogates G2 arrest and sensitizes p53-defective cells to DNA damage. The G2 checkpoint involves the inhibition of the Cdc25C phosphatase by the Chk1 and Chk2 kinases; however, the mechanism of the S phase checkpoint has remained unclear. Here, we examined the mechanism of S phase arrest induced by the topoisomerase I inhibitor SN38 in p53-defective breast cancer cells and p53 wild-type breast cells. It was found that Cdc25C is not the critical target of Chk1/2 in S phase; rather, the S phase checkpoint functions through the Chk1-Cdc25A-cyclin E/Cdk2 pathway. UCN-01 induced S and G2 progression in the p53-defective MDA-MB-231 cells at low nanomolar concentrations, whereas higher concentrations caused lethal mitosis without cell cycle progression, likely due to inhibition of C-TAK 1. Other novel, structurally related checkpoint inhibitors ICP-1 and Go6976 also abrogated arrest, but did not appear to inhibit C-TAK1. These analogs differ from UCN-01 in that their potency was not inhibited by human serum proteins. In p53 wild-type cells, S phase arrest was maintained in the presence of UCN-01, by p53-mediated transcriptional activation of target genes such as p21. G2 arrest was also maintained in p53 wild-type cells, by p53-mediated repression of genes such as cyclin B. These studies provide insight into the mechanism of the p53-independent S phase checkpoint, and the role of p53 in protecting cells against checkpoint inhibition. These results will facilitate the development of checkpoint inhibitors as anticancer drugs.