p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G, checkpoint in p53(-/-) as opposed to p53(+/+) cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G, checkpoint are unclear. Using normal human diploid p53(+/+) and p53(-/-) fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G, arrest. The G, arrest in p53(+/+) cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G, arrest in p53(-/-) cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53(+/+) and p53(-/-) cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53(+/+) cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation -> Cdc25A degradation -> inhibition of cyclin B1/Cdk1 activity ->) G, arrest is accordingly resistant to staurosporine and UCN-01 in p53(+/+) cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G, checkpoint in p53(+/+) cells. (c) 2007 by Radiation Research society.