ACTIVATION OF P34(CDC2) COINCIDENT WITH TAXOL-INDUCED APOPTOSIS

Toxicity elicited by the antitumor compound taxol has been linked to i rreversible tubulin polymerization, cell cycle block at mitosis, and c ell death from apoptosis. We have used pulsed drug exposure of synchro nized populations to identify two points, one in transition from G(0) to G(1) and the other at G(2)/M of cell cycle, that are most sensitive to taxol-induced cell killing. By analyzing these lesions separately, we have differentiated events related to mitotic block from those tha t may contribute to apoptosis. The taxol lesion forms rapidly and stab ly in transition or mitotic cells, because secondary washes to remove residual drug will decrease cytotoxicity except for cells in these pop ulations. Both G(2)/M cells and G(0)/G(1) transition cells synchronous ly initiated apoptotic DNA fragmentation within 20 h of pulsed taxol t reatment, indicating that a sustained mitotic block is not requisite t o initiate cell death. Apoptosis was inhibited by cyclohexamide and by 2-aminopurine and sodium orthovanadate; thus, cell cycle progression appeared requisite for cells death. Taxol treatment of G(0)/G(1) or G( 2)/M cells clearly leads to a block of mitosis followed by a perturbat ion of tyrosine phosphoprotein regulation; however, protein tyrosine p hosphorylation correlated with miotic block rather than time after dru g exposure. Conversely, p34(cdc2) kinase activation does not occur at mitotic block but rather 20 h after drug exposure and coincident with DNA fragmentation. Taken together, these results suggest that mitotic block may not be a sufficient signal for taxol-induced apoptosis and t hat the taxol lesion initiates apoptosis via a phosphoregulation pathw ay possibly involving the p34(cdc2) kinase.