Imatinib Regulates The Alternative MRNA Splicing Of Bcl-X In K562

ObjectiveThe alternative splicing of pre-mRNA is fundamental mechanism of differential gene expression in terms of giving rise to functionally distinct proteins from single gene, according to the developmental and environmental stimulus states in multicellular organisms.Bcl-x,a member of Bcl-2 family, plays an important role in cell proliferation, differentiation and apoptosis. Alternative splicing of Bcl-x generates several mRNA variants, in particular the two transcripts coding for either a long (Bcl-xl) or short(Bcl-xs) form of the protein. Bcl-xl variant inhibits cell death while Bcl-xs antagonizes the anti-apoptotic action of both Bcl-2 and Bcl-xl.However, it remains unclear how Bcl-x pre-mRNA splicing is regulated by extracellular factors controlling cell growth, apoptosis, and differentiation. In this study,we explored the effect of imatinib on the alternative splicing of Bcl-x gene and also investigated the effect of synergy of imatinib with hydroxyurea on the alternative splicing of Bcl-x in K562 cells.Furthermore, we investigate the hypothesis that their splicing action is mediated via protein phosphatase activation.Method1.Proliferation and apoptosis assay:WST assay for proliferation of K562 cells (n=3) Cell proliferation inhibitory rate (%)=1-Dtest/ODcontrol×100%; After treated with drugs,K562 cells wer stained with both annexin V and propidium iodide and subsequently, analazed by double fluorescence FACS.2. Drug treatment:A total of 2×105 K562 cells were plated in 35-mm2 wells. Drugs were diluted according to instructions from the manufacturers. To explore whether imatinib-induced splicing is mediated via protein phosphatase-1, K562 cells were pretreated with Calyculin A (5nM) or okadaic acid (lOnM) for 2 hours. It is possible that imatinib differentially affects the stability of the Bcl-x mRNAs,to address this question, DRB with a series concentration was added 1 h before addition of imatinib. Imatinib and hydroxyurea were added with indicated concentrations..An equal volume of DMSO was added to control wells. 3. The ratio of Bcl-xs/Bcl-xl was analyzed by RT-PCR:After 24h of incubation with 1μM of imatinib, extracting the total mRNA, and reverse-transcribed to cDNA, and using the specific primer to conduct PCR reaction. The ratio of Bcl-xs/Bcl-xl was analyzed by Quantity One 4.6.24. The immunoreactive protein levels of the gene products of Bcl-x after treatement with imatinib was analyzed by Western blot:K562 cells were treated with imaitnib for 24h, and total protein were extracted and analyzed by Western blot.5. The data were expressed as means±standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the differences among groups by SPSS 11.0 software (SPSS inc. Chicago, USA). P<0.05 was considered statistically significant.Results1. The WST assay indicated that hydroxyurea, imatinib and their combination all could inhibit proliferation of k562 cells. And the inhibitory ratio of the co-treatment group is higher than both imatinib and hydroxyurea group. (P<0.01). The FAC results suggested the inhibitory ratio of the combinated group is higher than both imatinib and hydroxyurea group.(P<0.01),which is consistent with its anti-proliferative effects.2. According to the Quantity One 4.6.2, the ratio of Bcl-xs/Bcl-xl between imatinib group(1Mm,2μM and 5μM) and control group showed statistic significance.(P<0.01). And there was a increase in the ratio of Bcl-xs/Bcl-xl from 0.5 to 5μM with imatinib respectively The results revealed that combined treatment caused a significant increase in the ratio of Bcl-xs/Bcl-xl while treatment with imatinib or hydroxyurea alone modestly increase the ratio of Bcl-xs/Bcl-xl.(P<0.01)3. After pretreated K562 with the transcriptional elongation inhibitor DRB, the results suggest DRB antagonized the shift towards Bcl-xs, and the higher concentration DRB is, the smaller ratio of Bcl-xs/Bcl-xl is.4. After pretreated K562 cells with calyculin A or 1 okadaic acid followed, the results suggest calyculin A completely blocked imatinib-induced alternative splicing in contrast to okadaic acid. ConclusionImatinib made the shift in splicing from Bcl-xl to Bcl-xs in K562 cells and the effects of imatinib on the pre-mRNA processing are dose-dependent.Additional,it is PP1 that mediates the effects of imatinib on the alternative splicing of Bcl-x,furthermore, compared to the treatment with imatinib or hydroxyurea alone, combined treatment made the shift more significantly.