Ponnathpur V, Ibrado A M, Reed J C, Ray S, Huang Y, Self S, Bullock G, Nawabi A, Bhalla K
Division of Hematology/Oncology, Departments of Medicine and Pathology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Clin Cancer Res. 1995 Nov;1(11):1399-406.
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.
紫杉醇诱导微管蛋白聚合成稳定的微管以及细胞周期中期阻滞，已被证明会导致人白血病细胞出现核小体间DNA片段化和凋亡的形态学特征。最近的研究还表明，在过表达bcl-2基因产物p26BCL-2的细胞中，紫杉醇诱导的凋亡受到显著抑制，但紫杉醇诱导的微管束集或有丝分裂阻滞未受影响。在本研究中，我们检测了几种蛋白激酶活性调节剂对转染了bcl-2基因cDNA并在细胞内高表达p26BCL-2的人前B白血病697细胞（697/BCL-2细胞）中紫杉醇诱导的DNA片段化和凋亡的影响。用0.1 - 1.0 microM紫杉醇处理24小时，可使已转染新霉素抗性基因的对照697/neo细胞出现长时间的有丝分裂阻滞。这导致了凋亡相关的5至200 kb大小的大DNA片段以及核小体间DNA片段化。与佛波酯佛波醇二丁酸酯（PdBU）共同处理可显著减少紫杉醇诱导的核小体间和大DNA片段化，并抑制697/neo细胞的凋亡。相反，联合暴露于紫杉醇和星形孢菌素（ST；5或50 ng/ml），一种蛋白激酶C和其他激酶的强效抑制剂，可显著增加697/neo细胞的DNA片段化和凋亡。此外，在697/BCL-2细胞中，ST部分克服了高p26BCL-2水平对紫杉醇诱导凋亡的抑制作用。与酪氨酸激酶抑制剂染料木黄酮（30 microM）共同处理可显著抑制697/neo细胞中紫杉醇诱导的DNA片段化和凋亡。然而，值得注意的是，PdBU、ST和染料木黄酮对紫杉醇诱导的DNA片段化和凋亡的调节作用，并未对697/neo细胞中紫杉醇介导的有丝分裂阻滞产生显著影响。这些试剂也未影响697/neo或697/BCL-2细胞内的p26BCL-2水平。这些发现表明，紫杉醇诱导的凋亡可被影响蛋白激酶活性的试剂所调节，且这些作用并非通过调节紫杉醇诱导的有丝分裂阻滞或细胞内p26BCL-2水平的改变来介导。
