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Expression of constitutively active Raf-1 in the mitochondria restores anti-apoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant

By Paolo Salomoni, Mariusz A. Wasik, Richard F. Riedel, Krzysztof Reiss, John K. Choi, Tomasz Skorski and Bruno Calabretta

Abstract

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176–426 in the BCR domain (p185�BCR) failed to protect interleukin 3–deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185�BCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185�BCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185�BCR cells transfected with a mitochondriatargeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185�BCR/M-Raf–transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185�BCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185�BCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemiainducing effects of BCR/ABL. Key words: oncogene • signal transduction • phosphorylation • apoptosis 1 Abbreviations used in this paper: AML, acute myelogenous leukemia; C

Topics: cytoplasmic, COX, cytochrome oxidase, CML, chronic myelogenous
Year: 1998
OAI identifier: oai:CiteSeerX.psu:10.1.1.322.557
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