Abstract
Abstract Background: ABT-737 is a BH3-mimetic that has a wide spectrum of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. Previously (J Nat Cancer Inst 100:580-595, 2008), we reported that Mcl-1, an anti-apoptotic Bcl-2 family protein that is not inhibited by ABT-737, decreased in response to ABT-737 in ABT-737-sensitive ALL cell lines while a reactionary Mcl-1 increase is observed in ABT-737-resistant cell lines. In the current study, we investigated the pathways of Mc-1 down-regulation in response to ABT-737 in ALL cell lines. Methods: To identify caspase cleavage sites and to determine whether the cleavage products are pro-apoptotic or anti-apoptotic, we transduced into the NALM-6 ALL cell line lentiviral vectors with several Mcl-1 variants tagged with V5 epitope, including wild type: Mcl-1 1-350 full length tagged with V5, point mutations at aspartate 127 and aspartate 157 to alanine: Mcl-1 D127A and Mcl-1 D157A, and C-terminus fragments of caspase cleavage, deleted: Mcl-1 β127 and Mcl-1 α157. Localization of Mcl-1 1-350 and Mcl-1 β157 in ALL cells, cytosol and mitochondria were fractionated from the cells expressing the proteins, and Mcl-1 and V5 were assayed in the fractions. Results: The decrease in Mcl-1 by ABT-737 in ABT-737-sensitive cell lines was via formation of an Mcl-1 fragment which is prevented by a caspase inhibitor (Boc-d-fmk), but not a proteosome inhibitor (MG-132), indicating that Mcl-1 cleavage is caspase-dependent. Of the two caspase-dependent Mcl-1 cleavage sites (D127 and D157), D157 is the major site of cleavage in response to ABT-737 treatment in ALL cells. Localization experiments revealed that full length Mcl-1 resides in both the cytosol and mitochondrial fractions, while the Mcl-1_158-350 fragment localizes mostly to mitochondria. Caspase 3- and caspase 9-activation in response to ABT-737 was higher in transduced cells expressing the Mcl-1 β157 fragment, suggesting that the fragment increases sensitivity to apoptotic stimuli. Pretreating cells with okadaic acid, a protein phosphatase inhibitor, resulted in greater fragmentation of Mcl-1 in resistant cell lines while the effect of okadaric acid was minimal in a ABT-737-sensitive cell line, suggesting that dephosphorylation makes Mcl-1 more susceptible to Mcl-1 cleavage in response to ABT-737. Conclusion: Mcl-1 is cleaved to Mcl-1 β157 by caspases in ALL cells that are treated with ABT-737, and the pro-apopoptotic activity of Mcl-1 β157 contributes to a vicious cycle promoting cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4647. doi:1538-7445.AM2012-4647
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