阻断泛素—蛋白酶体通路诱导白血病细胞凋亡及机制探讨

泛素-蛋白酶体系统是真核细胞内降解调节因子等各种短寿蛋白的重要途径，本实验就阻断泛素化通路诱发白血病细胞凋亡及机制进行了初步探讨，应用MTT法显示蛋白酶体抑制剂Z-LLL-CHO对多种白血病细胞系呈剂量依赖性的细胞杀伤作用，流式细胞仪检测提示细胞出现凋亡，并经DNA琼脂糖凝胶电泳和形态学观察得到进一步证实，通过对M-07e细胞裂解物做免疫印迹检测，发现在Z-LLL-CHO作用过程中Bcl-2蛋白被酶解及caspase-3酶原发生降解，色敏比色法检测显示caspase-3在凋亡过程中被激活，而同为bca-2高表达的白血病细胞系KG-1a对Z-LLL-CHO诱导凋亡不敏感，提示不同细胞对阻断泛素化通路敏感与否与Bcl-2蛋白表达量无关，而可能与细胞caspase-3的激活及bcl-2是否发发生酶解有关。

Induction of Apoptosis in Leukemic Cells by Inhibiting the Ubiquitin- Proteasome Pathway and Its Possible Mechanism

The ubiquitin proteasome pathway is the principal mechanism for the degradation of short lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined. Evaluated by MTT assay, treatment of leukemic cells with Z LLL CHO, a reversible proteasome inhibitor, induced cell death in a dose dependent manner. Appearance of the sub G 0/G 1 fraction of cell cycle observed in flow cytometry assay suggested the induction of apoptosis, which was further proved by typical DNA ladder and morphological study. Western blot displayed the cleavage of bcl 2 into a shortened 22 kD fragment and the decrease in the levels of caspase 3 precursor. A highly sensitive colorimetric assay was employed and the elevation of caspase 3 activity was detected in both cell lines after treatment with Z LLL CHO. By comparison, these results showed that the leukemic cell line M 07e and KG 1a, which both express bcl 2 at a relative high level, had different susceptibility to undergo apoptosis induced by Z LLL CHO, which possibly due to their different levels of expression and activation of caspase 3 precursor, as well as their different degree of bcl 2 cleavage after treated by Z LLL CHO.