Wang, X. Q., Stanbridge, E. J., Lao, X. Y., Cai, Q., Fan, S. T. and Redpath, J. L. p53-Dependent Chk1 Phosphorylation is Required for Maintenance of Prolonged G₂ Arrest. Radiat. Res. 168, 706–715 (2007).

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 G₂ checkpoint in p53^−/− as opposed to p53 ^/ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G₂ 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 G₂ arrest. The G₂ arrest in p53 ^/ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G₂ 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 → G₂ 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 G₂ checkpoint in p53 ^/ cells.