Activation of p34cdc2 coincident with taxol-induced apoptosis.
Toxicity elicited by the antitumor compound taxol has been linked to irreversible tubulin polymerization, cell cycle block at mitosis, and cell death from apoptosis. We have used pulsed drug exposure of synchronized populations to identify two points, one in transition from G0 to G1 and the other at G2/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 that may contribute to apoptosis. The taxol lesion forms rapidly and stably in transition or mitotic cells, because secondary washes to remove residual drug will decrease cytotoxicity except for cells in these populations. Both G2/M cells and G0/G1 transition cells synchronously initiated apoptotic DNA fragmentation within 20 h of pulsed taxol treatment, indicating that a sustained mitotic block is not requisite to initiate cell death. Apoptosis was inhibited by cyclohexamide and by 2-aminopurine and sodium orthovanadate; thus, cell cycle progression appeared requisite for cell death. Taxol treatment of G0/G1 or G2/M cells clearly leads to a block of mitosis followed by a perturbation of tyrosine phosphoprotein regulation; however, protein tyrosine phosphorylation correlated with mitotic block rather than time after drug exposure. Conversely, p34cdc2 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 that the taxol lesion initiates apoptosis via a phosphoregulation pathway possibly involving the p34cdc2 kinase.
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
K L
KL
Donaldson
G L
GL
Goolsby
P A
PA
Kiener
A F
AF
Wahl
D017209:Apoptosis / Q000187:drug effects*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D002453:Cell Cycle / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D004249:DNA Damage; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D013997:Time Factors
D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D017239:Paclitaxel