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© The Rockefeller University Press, 0022-1007/1998/6/1995/ $5.00
The Journal of Experimental Medicine, Volume 187, Number 12, June 15, 1998 1995-2007

Articles

Expression of Constitutively Active Raf-1 in the Mitochondria Restores Antiapoptotic and Leukemogenic Potential of a Transformation-deficient BCR/ABL Mutant

Paolo Salomoni*,{ddagger}, Mariusz A. Wasik§, Richard F. Riedel*, Krzysztof Reiss*, John K. Choi§, Tomasz Skorski*, and Bruno Calabretta*

From the * Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; the {ddagger} Department of Biomedical Sciences, University of Modena, 41100 Modena, Italy; and the § Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19102

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{Delta}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{Delta}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{Delta}BCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185{Delta}BCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185{Delta}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{Delta}BCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185{Delta}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 leukemia-inducing effects of BCR/ABL.

Key Words: oncogene • signal transduction • phosphorylation • apoptosis

Address correspondence to Bruno Calabretta, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th St., Rm. 630, Philadelphia, PA 19107. Phone: 215-503-4522; Fax: 215-923-0249.

P. Salomoni was supported in part by a postdoctoral fellowship from the University of Modena Medical School, Modena, Italy. T. Skorski was supported in part by R29 CA70815. This work was supported in part by National Institutes of Health and American Cancer Society grants to B. Calabretta.

1 Abbreviations used in this paper: AML, acute myelogenous leukemia; C, cytoplasmic; COX, cytochrome oxidase; CML, chronic myelogenous leukemia; DTT, dithiothreitol; HM, heavy membrane; LM, light membrane; M, mitochondria-targeted; MAP, mitogen-activated protein; MAPK, MAP kinase; MBP, myelin basic protein.


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