Lock R B, Thompson B S, Sullivan D M, Stribinskiene L
Henry Vogt Cancer Research Institute, J. Graham Brown Cancer Center, Department of Medicine, University of Louisville, KY 40292, USA.
Cancer Chemother Pharmacol. 1997;39(5):399-409. doi: 10.1007/s002800050590.
Protein kinase inhibitors have demonstrated potential for use in the therapy of human cancers, in particular leukemia. Staurosporine, a protein kinase inhibitor with broad specificity, enhances the cytotoxic effects of various antitumor agents with different modes of action. The topoisomerase II inhibitor, etoposide, has shown clinical activity against a wide range of tumor types.
The purpose of this study was to assess the effects of staurosporine on etoposide-induced cell death processes in a human tumor of epithelial origin.
Modulation of etoposide-induced apoptosis by staurosporine in HeLa cells was assessed by cell morphology, extraction of low molecular weight DNA, quantitation of DNA-protein complexes, and measurements of rates of DNA synthesis. The effects on cellular genes implicated in apoptosis were determined by Northern and Western blotting, along with assays of cyclin-dependent kinase activities.
Staurosporine exhibited a two- to three-fold potentiation of apoptosis caused by etoposide in HeLa cells when applied concurrently, or immediately following etoposide removal, but did not alter the quantity of DNA-protein complexes produced by etoposide. Etoposide-induced apoptosis, and its potentiation by staurosporine, were associated with reduced c-myc expression, and a moderate increase in p21WAF1/CIP1 mRNA and protein levels. Inhibitors of cyclic AMP-dependent protein kinase and protein kinase C, which exhibit greater specificity than staurosporine, were without effect on apoptosis caused by etoposide, whereas use of the tyrosine phosphatase inhibitor, vanadate, resulted in its abrogation. The potentiation of etoposide-induced apoptosis by staurosprine was associated with a significant increase in cyclin A-dependent kinase activity. In addition, etoposide caused substantial inhibition of DNA synthesis.
These results indicate that staurosporine potentiates apoptosis through events which occur downstream of DNA damage, and implicate unscheduled activation of cyclin A-dependent kinase during inhibition of DNA synthesis as a possible cause.
蛋白激酶抑制剂已显示出用于治疗人类癌症，特别是白血病的潜力。星形孢菌素是一种具有广泛特异性的蛋白激酶抑制剂，可增强各种具有不同作用方式的抗肿瘤药物的细胞毒性作用。拓扑异构酶II抑制剂依托泊苷已显示出对多种肿瘤类型具有临床活性。
本研究的目的是评估星形孢菌素对依托泊苷诱导的上皮来源的人类肿瘤细胞死亡过程的影响。
通过细胞形态学、低分子量DNA提取、DNA-蛋白质复合物定量以及DNA合成速率测量，评估星形孢菌素对HeLa细胞中依托泊苷诱导的凋亡的调节作用。通过Northern和Western印迹以及细胞周期蛋白依赖性激酶活性测定，确定对与凋亡相关的细胞基因的影响。
当同时应用或在去除依托泊苷后立即应用时，星形孢菌素在HeLa细胞中使依托泊苷引起的凋亡增强了两到三倍，但并未改变依托泊苷产生的DNA-蛋白质复合物的数量。依托泊苷诱导的凋亡及其被星形孢菌素增强，与c-myc表达降低以及p21WAF1/CIP1 mRNA和蛋白质水平适度增加有关。与星形孢菌素相比具有更高特异性的环磷酸腺苷依赖性蛋白激酶和蛋白激酶C抑制剂，对依托泊苷引起的凋亡没有影响，而使用酪氨酸磷酸酶抑制剂钒酸盐则导致其被废除。星形孢菌素对依托泊苷诱导的凋亡的增强与细胞周期蛋白A依赖性激酶活性的显著增加有关。此外，依托泊苷对DNA合成有实质性抑制作用。
这些结果表明，星形孢菌素通过DNA损伤下游发生的事件增强凋亡，并暗示在DNA合成抑制期间细胞周期蛋白A依赖性激酶的异常激活可能是一个原因。
