UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1.

UCN-01 (7-hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. It has been shown previously that UCN-01 abrogates DNA-damage-induced G(2) checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site previously identified for Chk2 (or/and Chk1) kinase. We found that in human colon carcinoma HCT116 cells, IR treatment enhanced Chk2 kinase activity, whereas Chk1 activity remained unchanged, which suggested that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN-01 inhibits Chk2 immunoprecipitated from HCT116 cells (IC(50), approximately 10 nM). In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G(2) arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G(2) checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was no p21(Waf1/Cip1) induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells.