Abstract 5161: Activation of Raf-1 through the depletion of RKIP by Bcr-Abl promotes CML cell proliferation

Abstract

Abstract Activation of Raf-1 through the depletion of RKIP by Bcr-Abl promotes CML cell proliferation [Background and Aims] RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of RKIP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. Moreover, in CML cells transfected with the RKIP siRNA, it is shown that the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to the untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of Erk1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through Erk1/2 phosphorylation. Moreover, induction of RKIP expression might regulate the proliferation of CML stem/progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5161.