UCN-01 abrogates G(2) arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase

We have previously demonstrated that UCN-01, a potent protein kinase inhibi tor currently in phase I clinical trials for cancer treatment, abrogates G( 2) arrest following DNA damage. Here we used murine FT210 cells, which cont ain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G(2) arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the nonpermissive temperature for Cdc2 function. Failure to abrogate G(2) arrest was not due to UCN-01-in activation at the elevated temperature because parental FM3A cells, which h ave wild-type Cdc2, were sensitive to UCN-01-induced G(2) checkpoint abroga tion. Having established that UCN-01 acted through Cdc2, we next assessed U CN-01's effect on the Cdc2-inhibitory kinase, WeelHu, and the Cdc2-activati ng phosphatase, Cdc25C, We found that WeelHu was indeed inactivated in UCN0 1-treated cells, possibly just prior to Cdc2 activation and entry of DNA-da maged cells into mitosis, This inhibition appeared, however, to be a conseq uence of a further upstream action since in vitro studies revealed purified WeelHu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activat ion) of Cdc25C in DNA-damaged cells, Our results suggest that UCN-01 abroga tes G(2) checkpoint function through inhibition of a kinase residing upstre am of Cdc2, WeelHu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.