Inhibition of Glucocorticoid-induced Apoptosis by Targeting the Major Splice Variants of BIM mRNA with Small Interfering RNA and Short Hairpin RNA

Marc T Abrams,Noreen M Robertson,Kyonggeun Yoon,Eric Wickstrom

doi:10.1074/jbc.m411767200

Abstract

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.

Highlights

The pro-apoptotic Bcl-2 relative Bim has been shown to be up-regulated by GC in GC-sensitive lymphocytes, suggesting that Bim may function as an early sensor for intrinsic apoptosis [7, 9, 27]
To determine whether there is a correlation between Bim induction and GC sensitivity in human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cells, we monitored a panel of human pre-B ALL cell lines for viability in response to the potent GC triamcinolone acetonide after 48- and 72-h exposures (Fig. 1, A and B)
We have presented data demonstrating that GC-mediated induction of Bim is a critical signaling event in the GC-induced apoptosis of human pre-B ALL cells

Summary

Introduction

Microarray profiling has shown that a short GC exposure induces or represses the transcription of Ͼ100 genes by a factor of 3-fold or greater in multiple models of GC-induced apoptosis [7,8,9] Among these genes are universal regulators of intrinsic apoptosis such as BCL2 and BIM, a gene encoding several splice variants of the related Bcl-2 homology (BH) region 3 domain-containing protein Bim [7, 9]. BH3-only proteins are transcriptionally activated, post-translationally modified, or released from sequestration in response to death stimuli and promote apoptosis by interacting with Bcl-2 family members that contain multiple BH domains (BH1–BH4) (reviewed in Ref. 11). Lowering BIM mRNA by RNAi causes partial protection against apoptosis induced by the drugs paclitaxel [19] and imitinib [22], as well as apoptosis induced by forced suspension culture (anoikis) [20, 21]

Methods

Results

Conclusion