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

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 lentivirusmediated 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.

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

“…Thymocytes harboring the Bim shRNAmiR demonstrated significantly less apoptosis in response to dexamethasone than the vector control as expected from downregulating a gene known to be crucial to glucocorticoid-mediated apoptosis. 12 FTOCs harboring both Tnfaip8 shRNAmiRs demonstrated comparable reductions to Bim in glucocorticoid-mediated thymocyte apoptosis relative to the control (Figure 4e). These data show that Tnfaip8 has a crucial role in glucocorticoid-induced thymocyte apoptosis.…”

“…10 Thymocytes from Bim À/À mice show significantly reduced sensitivity to glucocorticoid-induced apoptosis 11 and downregulation of Bim expression in a B-cell line by RNA interference (RNAi) greatly reduces sensitivity to glucocorticoid. 12 There is considerable debate regarding how endogenous glucocorticoid levels can influence thymic development and homeostasis. [1][2][3]13,14 Glucocorticoids are produced within the thymus by thymic epithelial cells, 15,16 and evidence exists to suggest they have a considerable effect not only on thymus homeostasis, but also on the nature of the developing T-cell receptor repertoire.…”

The BCL-2 protein family, BH3-mimetics and cancer therapy

“…[141][142][143][144][145] PUMA and BIM together account for most of the proapoptotic activity of glucocorticoids. 141,143,[146][147][148][149] BIM is also critical for taxane-induced cell killing. 150,151 Furthermore, BMF as well as BIM are critical for the killing of non-transformed lymphoid cells as well as certain lymphoma cells by inhibitors of histone deacetylases.…”

Regulatory phosphorylation of Bim: sorting out the ERK from the JNK

“…In the cellular context, this may be achievable with splice form-selective RNAi reagents. 57 However, in vivo, the real challenge will be to derive knock-in mice in which splice donor/acceptor sites or individual phosphorylation sites are mutated.…”