Effects of modulators of protein kinases on taxol-induced apoptosis of human leukemic cells possessing disparate levels of p26BCL-2 protein.

Taxol-induced polymerization of tubulin into stable microtubules and cell cycle metaphase arrest have been demonstrated to result in internucleosomal DNA fragmentation and morphological features of apoptosis in human leukemia cells. Recent studies have also shown that Taxol-induced apoptosis, but not Taxol-induced microtubular bundling or mitotic arrest, is significantly inhibited in cells that overexpress the bcl-2 gene product p26BCL-2. In the present studies we examined the effects of several modulators of activities of protein kinases on Taxol-induced DNA fragmentation and apoptosis in human pre-B leukemia 697 cells transfected with the cDNA of the bcl-2 gene and expressing high intracellular levels of p26BCL-2 (697/BCL-2 cells). Treatment with 0.1-1.0 microM MTaxol for 24 h produced prolonged mitotic arrest of control 697/neo cells, which had been transfected with the neomycin resistance gene. This resulted in apoptosis-associated large DNA fragments ranging between 5 and 200 kb and internucleosomal DNA fragmentation. Cotreatment with the phorbol ester phorbol dibutyrate (PdBU) significantly reduced Taxol-induced internucleosomal and large DNA fragmentation and inhibited apoptosis of 697/neo cells. In contrast, a combined exposure to Taxol and staurosporine (ST; 5 or 50 ng/ml), a potent inhibitor of protein kinase C and other kinases, significantly increased DNA fragmentation and apoptosis of 697/neo cells. Additionally, in 697/BCL-2 cells, ST partially overcame the suppressive effects of high p26BCL-2 levels on Taxol-induced apoptosis. Cotreatment with the tyrosine kinase inhibitor Genistein (30 microM) markedly inhibited Taxol-induced DNA fragmentation and apoptosis of 697/neo cells. However, it is noteworthy that the modulations of Taxol-induced DNA fragmentation and apoptosis by PdBU, ST, and Genistein occurred without significant effects on Taxol-mediated mitotic arrest of 697/neo cells. These agents also did not affect intracellular p26BCL-2 levels in 697/neo or 697/BCL-2 cells. These findings indicate that Taxol-induced apoptosis can be modulated by agents that affect the activities of protein kinases, and these effects are not mediated by modulations of Taxol-induced mitotic arrest or by alterations of intracellular p26BCL-2 levels.