Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA
Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA
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DOI：
10.1074/jbc.m411767200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Wickstrom, E
中科院分区：
文献类型：
--
作者：
Abrams, MT;Robertson, NM;Wickstrom, E
糖皮质激素（GC）可诱导淋巴细胞凋亡，是治疗白血病的有效药物。激活的糖皮质激素受体启动转录程序，导致 caspase 激活和细胞死亡，但 GC 诱导的细胞凋亡中的关键信号传导中间体在很大程度上仍不清楚。我们观察到，在一组人类前体 B 细胞 (pre-B) 急性淋巴细胞白血病 (ALL) 细胞系中，Bcl-2 相关 Bim 的三种主要蛋白产物（BimEL、BimS 和 BimL）的 GC 诱导与 GC 敏感性相关。为了检验 Bim 促进 GC 诱导的细胞凋亡的假设，我们通过 RNA 干扰降低了高度 GC 敏感性的 pre-B ALL 细胞中的 BIM mRNA 水平和 Bim 蛋白水平。通过合成小干扰 RNA (siRNA) 双链体的电穿孔或慢病毒介导的短发夹 RNA 的稳定表达来减少 Bim 蛋白，可抑制 caspase-3 的激活并增加 GC 暴露后的细胞活力。我们还观察到 GC 抗性程度与 siRNA 沉默效力相关。仅减少 BimEL 或 BimEL 和 BimL（但不减少 BimS）的 siRNA 双链体表现出比沉默所有三种主要亚型的有效 siRNA 更低的 GC 抗性，这意味着所有三种 Bim 蛋白的诱导都有助于细胞死亡。最后，Bim 沉默引起的 GC 诱导的细胞凋亡的调节与 Bcl-2 表达水平无关，否定了 Bim 与 Bcl-2 的比例调节细胞凋亡的假设。这些结果证明 GC 诱导 Bim 是 pre-B ALL 细胞完全凋亡反应的必要事件。
Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.