亚细胞蛋白质组学技术分析MG132诱导HL-60细胞G_2/M期阻滞的分子机制

Proteomic Analysis of G_2/M Arrest of HL-60 Cells Induced by MG132

背景与目的:蛋白酶体抑制剂能够诱导多种肿瘤细胞凋亡,是一种有应用前景的抗肿瘤制剂。本研究通过对蛋白酶体抑制剂——MG132诱导G2/M期阻滞的HL-60细胞核蛋白进行蛋白质组学分析,探索参与HL-60细胞G2/M期阻滞调控的相关蛋白。方法:流式细胞术分析MG132处理的HL-60细胞周期,选取合适的时间点,提取细胞核,光镜和Westernblot检测细胞核纯度,裂解制样,通过二维电泳和MALDI-TOF-TOF串联质谱分析,鉴定表达显著差异的细胞核蛋白。结果:HL-60细胞在被2.5μmol/LMG132诱导凋亡之前8h,有明显的G2/M期阻滞发生;提取阻滞发生时和阻滞发生前的HL-60细胞核蛋白,二维电泳分析获得23个差异表达的蛋白点,质谱鉴定了8个核蛋白。结论:质谱鉴定的eIF5A、mRNA前体剪接因子等蛋白可能参与HL-60细胞的G2/M周期阻滞调控,为研究蛋白酶体抑制剂诱导白血病细胞周期阻滞及凋亡的分子机制提供了一些线索。

BACKGROUND ＆ OBJECTIVE. Proteasome inhibitor, which can induce apoptosis in various tumor cells, is a kind of potential antitumor drug. This study was to identify the proteins involved in G2/M arrest of leukemia cell line HL-60 exposed to proteasome inhibitor MG132 by proteomic techniques. METHODS： Flow cytometry was used to examine cell cycle of HL-60 cells exposed to 2.5 μmol/L MG132. Nuclear extracts of HL-60 cells were prepared, and the purity was detected by light microscopy and Western blot, and the differentially expressed protein spots were dertermined by two-dimensional gel electrophoresis and identified with MALDI-TOF-TOF/MS. RESULTS.. There was a distinct G2/M phase arrest before the apoptosis of HL-60 cells induced by 2.5 μmol/L MG132. Twentythree differentially expressed protein spots were found between MG132- treated and control HL-60 cells; 8 nuclear proteins were identified by MALDI- TOF-TOF/MS analysis. CONCLUSIONS： The detected proteins, such as elF5A and splicing factor, may be involved in regulation of G2/M arrest of HL-60 cells. These findings will be helpful for revealing molecular mechanisms of proteasome inhibitor-induced G2/M phase arrest and apoptosis of leukemia cell line.