Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells

Abstract

The antimicrotubule anticancer drug, Taxol, suppresses microtubule dynamics, causes mitotic arrest, and induces caspase-3 cleavage and activity resulting in apoptosis of human AML HL-60 cells. Caspase-3 cleavage is triggered by the mitochondrial release and cytosolic accumulation of the electron transfer protein, cytochrome c (cyt c). Taxol-induced G2/M transition is mediated by p34^cdc-2 (CDK1) which, if prematurely activated, may also trigger apoptosis. In the present studies following S-phase synchronization and release, HL-60 cells with enforced expression of the bcl-x_L (HL-60/Bcl-x_L) and/or neomycin resistance gene (HL-60/neo) were exposed to Taxol to examine CDK1-related cell-cycle events and the cyt c-triggered molecular cascade of apoptosis. At various time-intervals after Taxol treatment, immunoblot analyses of cyclin B1 and CDK1 levels were performed. In addition, the in vitro histone H1 kinase activity of immunoprecipitated CDK1 and its tyrosine phosphorylation status (by anti-phosphotyrosine immunoblot analysis) were determined. Data presented here show that, while Taxol-induced peak CDK1 kinase activity occurs earlier in HL-60/neo cells, there are no significant differences in cyclin B1 accumulation, tyrosine dephosphorylation of CDK1, and mitotic arrest of Taxol-treated HL-60/neo vs HL-60/Bcl-x_L cells. Taxol-induced CDK1 activation and mitosis preceded the cytosolic accumulation (six-fold) of cyt c. The latter event was blocked by Bcl-x_L overexpression but not by inhibitors of caspase-3. Although the caspase inhibitors and high Bcl-x_L levels inhibited caspase-3 cleavage and activity, they did not significantly affect Taxol-induced CDK1 activation or mitotic arrest. These findings indicate that Bcl-x_L overexpression does not affect Taxol-induced CDK1 activity leading to G2/M transition, which temporally precedes the cytosolic cyt c-mediated cleavage and activity of caspase-3 and apoptosis.