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

The antimicrotubule anticancer drug, Taxol, suppresses microtubule dynamics , causes mitotic arrest, and induces caspase-3 cleavage and activity result ing in apoptosis of human AML HL-60 cells. Caspase-3 cleavage is triggered by the mitochondrial release and cytosolic accumulation of the electron tra nsfer protein, cytochrome c (cyt c). Taxol-induced G2/M transition is media ted by p34(cdc-2) (CDK1) which, if prematurely activated, may also trigger apoptosis. In the present studies following S-phase synchronization and rel ease, 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 exami ne 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 v itro 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 ki nase activity occurs earlier in HL-60/neo cells, there are no significant d ifferences in cyclin B1 accumulation, tyrosine dephosphorylation of CDK1, a nd 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 (s imilar to six-fold) of cyt c. The latter event was blocked by Bcl-x(L) over expression but not by inhibitors of caspase-3. Although the caspase inhibit ors and high Bcl-x(L) levels inhibited caspase-3 cleavage and activity, the y did not significantly affect Taxol-induced CDK1 activation or mitotic arr est. These findings indicate that Bcl-x(L) overexpression does not affect T axol-induced CDK1 activity leading to G2/M transition, which temporally pre cedes the cytosolic cyt c-mediated cleavage and activity of caspase-3 and a poptosis.