Imatinib regulates the alternative pre-mRNA splicing of Bcl-x in K562 cells

Abstract

Background: The alternative splicing of Bcl-x generates the proapoptotic Bcl-xs protein and the antiapoptotic variant Bcl-xl. Previous studies have demonstrated that some chemotherapeutic agents such as emetine, staurosporine, and epigallocatechin gallate (EGCG) in combination with ibuprofen significantly altered the ratio of the Bcl-x variants Bcl-xs/Bcl-xl in various cell lines, suggesting Bcl-x splicing might be affected by the exogenous stimuli. Objective: We investigated the regulative role of imatinib in the alternative pre-mRNA splicing of Bcl-x in K562 cells and the related mechanism. Methods: Cell proliferation was measured using WST assay kit. Cell apoptosis was assayed using an Annexin V–FITC Apoptosis Detection Kit. RT-PCR and western blot assay was used to analyze the mRNA and protein level of alternative splicing of exon 2 in the Bcl-x gene respectively. Results: Imatinib regulated the alternative splicing in the Bcl-x gene in the K562 cells. In addition, we found that hydroxyurea, another agent for the therapy of CML, could enhance the effect of imatinib on the ratio of the Bcl-xl/Bcl-xs. Moreover, the induction of alternative splicing was correlated with protein phosphatase 1 (PP1). Alternatively, pretreatment with calyculin efficiently blocked imatinib-induced alternative splcing in the K562 cells compared with okadaic acid, which showed an important role of PP1 in regulating imatinib-induced splicing. Conclusion: Imatinib regulates the alternative splicing of Bcl-x in K562 cells, which may be associated with the activation of PP1. Keywords: Alternative splcing, Bcl-x, chronic myelogenous leukemia, imatinib, protein phosphatase 1