The unscheduled mitotic entry either with damaged DNA or with incompletely replicated genome causes mitotic catastrophe. Both Chk1 and p53, two critical components in the DNA damage checkpoint pathways, function independently to prevent inappropriate mitotic entry by inducing cell cycle arrest. In p53-deficient cells, prevention from the entry into mitosis in response to DNA damage relies solely on Chk1-mediated checkpoint and thus the inhibition of Chk1 could selectively sensitize p53-deficient cells to mitotic catastrophe. However, the mechanism underlying DNA damage-elicited mitotic catastrophe remains unclear. Using paired cell lines in which the derivative line contains a p53 shRNA to knock down the wild-type p53 expressed in the parental line, we show that a potent and cell-active small molecule inhibitor of Chk1 (Chk1i) preferentially enhanced mitotic entry and cell death in p53-deficient cells after exposure to doxorubicin, a DNA topoisomerase II inhibitor which causes DNA breaks. Intriguingly, p53-kd derivatives treated with doxorubicin and Chk1i exhibited an induction of the mitotic marker Histone H3 phosphorylation without activation of the spindle checkpoint, which is in contrast to the robust mitotic arrest and activation of the spindle checkpoint seen with the microtubule inhibitor paclitaxel. Furthermore, the addition of Chk1i significantly augments the induction of caspase-3 activity only in p53-kd cells over the background of weak caspase-3 induction by single agent doxorubicin in both p53-kd and wt cells. Finally, suppression of caspase activities by the pan-caspase inhibitor z-VAD significantly reduced the doxorubicin and Chk1i combination-induced mitotic death in p53-kd cells, suggesting that DNA damage-induced mitotic catastrophe is indeed a form of caspase-dependent apoptosis.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA