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In human leukemic cells clinically relevant concentrations of taxol have been demonstrated to induce the biochemical and morphologic hallmarks of apoptosis (Leukemia 1993;7:563-568). Since overexpression of the bcl-2 gene has been reported to retard apoptosis due to a variety of anticancer agents, we examined and compared taxol-induced intracellular microtubular bundling and apoptosis in pre-B human leukemia 697 cells and their counterparts which have been transfected with and overexpress cDNA derived from the bcl-2 gene. Treatment with 0.1 or 1.0 mumol/l taxol for 24 h resulted in internucleosomal DNA fragmentation and morphologic features of apoptosis in 697 cells, but not in 697/BCL-2 cells. However, indirect immunofluorescent staining with anti-tubulin antibody revealed that taxol treatment produces stable microtubule bundles resistant to calcium-mediated disassembly in 697, as well as 697/BCL-2 cells. In addition, taxol-induced microtubule bundling was associated with a marked accumulation of the two cell types in the G2/M phase of the cell cycle. Following exposure to taxol, when 697 cells were washed and kept in drug-free medium, they showed rapid onset of apoptosis followed by loss of cell viability and a decline in cell numbers. In contrast, identically treated 697/BCL-2 cells kept in drug-free medium remained in a growth arrested state, but showed little evidence of apoptosis for up to 4 days. They eventually demonstrated features of apoptotic cell death and loss of viability between 5 and 7 days. This was not accompanied by a decrease in p26BCL-2 levels. Anti-phosphotyrosine or anti-MAP kinase immunoblot analyses of proteins isolated from taxol-treated 697 and 697/BCL-2 cells failed to show any difference in tyrosine phosphorylation of cellular proteins. Therefore, our findings indicate that in 697/BCL-2 cells, high levels of p26BCL-2 significantly delay taxol-induced endonucleolytic internucleosomal DNA fragmentation and apoptosis, but do not affect taxol-induced microtubule bundling or cell cycle growth arrest. The delayed onset of taxol-induced DNA fragmentation and apoptosis in 697/BCL-2 cells without down-regulation of p26BCL-2 levels suggests that an alternative mechanism of taxol-mediated apoptosis might be triggered which is unimpeded by high p26BCL-2 levels, or taxol-induced prolongation of mitotic arrest may lead to the inactivation or inhibition of that mechanism by which p26BCL-2 is able to block apoptosis.

Taxol-resistant sublines of HL-60 myeloid leukemia cells (HL-60/TAX100 and HL-60/TAX1000) have been isolated in vitro by subculturing in progressively higher concentrations of taxol. HL-60/TAX100 and HL-60/TAX1000 cells are capable of continuous growth in the presence of 0.1 microM and 1.0 microM taxol, respectively, and the IC50 (50% growth inhibitory dose) values for taxol for the two sublines are 0.34 and 2.44 microM as compared to 3.1 nM for the parent HL-60 cells. HL-60/TAX100 and HL-60/TAX1000 cells display a variable degree of cross-resistance to taxotere, vincristine and doxorubicin, but are sensitive to the antimetabolite Ara-C. Both HL-60/TAX100 and HL-60/TAX1000 cells over-express MDR-1 m-RNA and the membrane efflux multidrug transporter P-glycoprotein (PGP), as determined by Western blot and immunofluorescence labeling with anti-PGP antibodies. Consequently, exposure of the taxol-resistant cells to [3H]taxol or daunomycin results in the accumulation of significantly lower levels of the two drugs. Co-treatment with cyclosporine (0.5 microgram/ml) or verapamil (10 microM) partially overcomes taxol resistance in HL-60/TAX1000 cells. Following treatment with clinically relevant concentration of taxol (1.0 microM for 24 h), HL-60 but not HL-60/TAX1000 cells display intracellular microtubular bundling, markedly enhanced accumulation of the cells in G2/M phase of cell-cycle and internucleosomal DNA fragmentation associated with apoptosis which is independent of bcl-2 gene expression. These taxol-resistant myeloid leukemia cells may serve as in vitro experimental models for examinating strategies which may have potential applicability for overcoming taxol resistance.