p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G 2 arrest

Abstract

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 2 checkpoint in p53 -/- as opposed to p53 +/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G 2 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 γ-radiation-induced G 2 arrest. The G 2 arrest in p53 +/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G 2 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 2 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 2 checkpoint in p53 +/+ cells. © 2007 by Radiation Research Society.