Thomas A, El Rouby S, Reed J C, Krajewski S, Silber R, Potmesil M, Newcomb E W
Department of Pathology, New York University School of Medicine, New York 10016, USA.
Oncogene. 1996 Mar 7;12(5):1055-62.
We investigated the relationship among chemosensitivity to drug-induced apoptosis in vitro, the presence of p53 gene mutations, and the expression of bcl-2 and bax proteins in B-cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. Apoptosis was induced with a camptothecin analogue, 9-amino-20(s)-camptothecin, or a purine analogue, fludarabine. Cell death was monitored by propidium iodide staining and FACS analysis. Drug-induced apoptosis in B-CLL cells was p53-independent. Immunoblot analysis of bcl-2 and bax expression revealed a correlation between drug-induced apoptosis and the ratio of endogenous levels of bcl-2 to bax proteins. B-CLL cells with none to low bcl-2/bax ratios were drug-sensitive as compared to cells with intermediate to high ratios that were drug-resistant (P = 0.015). Prior to drug treatment, bax protein migrated as a single species of 21 kDa. Following drug-induced apoptosis, anti-bax specific protein complexes of 36-42 kDa were up-regulated. Using two-dimensional gel electrophoresis, bax complexes were disrupted under reducing conditions to reveal homo- and heterodimers of 18 and 21 kDa suggesting that disulfide interactions were required for complex formation. The de novo appearance of the 18 kDa anti-bax specific protein together with its increased expression in drug-sensitive B-CLL B-cells undergoing cell death suggests a role for this protein in the regulation of apoptosis.
我们研究了B细胞慢性淋巴细胞白血病（B-CLL）患者B细胞中对药物诱导凋亡的化学敏感性、p53基因突变的存在以及bcl-2和bax蛋白表达之间的关系。用喜树碱类似物9-氨基-20（s）-喜树碱或嘌呤类似物氟达拉滨诱导凋亡。通过碘化丙啶染色和流式细胞术分析监测细胞死亡。B-CLL细胞中药物诱导的凋亡不依赖于p53。对bcl-2和bax表达的免疫印迹分析显示，药物诱导的凋亡与bcl-2和bax蛋白内源性水平的比值之间存在相关性。与具有中等至高比值且耐药的细胞相比，bcl-2/bax比值低至无的B-CLL细胞对药物敏感（P = 0.015）。在药物治疗前，bax蛋白以单一的21 kDa形式迁移。药物诱导凋亡后，36 - 42 kDa的抗bax特异性蛋白复合物上调。使用二维凝胶电泳，在还原条件下bax复合物被破坏，以揭示18 kDa和21 kDa的同源二聚体和异源二聚体，这表明二硫键相互作用是复合物形成所必需的。在药物敏感的B-CLL B细胞发生细胞死亡时，18 kDa抗bax特异性蛋白的从头出现及其表达增加，表明该蛋白在凋亡调节中起作用。
