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Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis

Duan, Shili and Hájek, Petr and Lin, Catherine and Shin, Soo Kyung and Attardi, Giuseppe and Chomyn, Anne (2003) Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis. Journal of Biological Chemistry, 278 (2). pp. 1346-1353. ISSN 0021-9258. doi:10.1074/jbc.M209269200.

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We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK- cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK- osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK- cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.

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Additional Information:© 2003 The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, September 10, 2002, and in revised form, October 18, 2002. Originally published In Press as doi:10.1074/jbc.M209269200 on October 25, 2002. We are grateful to Drs. Stanley Korsmeyer and David Hockenbery for the gift of the Bcl2-alpha cDNA and to Sandoz Research Institute for the gift of cyclosporine A. We are grateful also to Dr. Svetlana Lyapina for advice on calcium phosphate-mediated transfection of cells, to Dr. Elisabetta Ferraro for suggesting the experiment with Bis-I, and to Dr. Gaetano Villani for helpful discussions. We also thank Benneta Keeley, Arger Drew, Rosie Zedan, Elisa Chan, and Huamei Xu for excellent technical assistance. This work was supported by National Institutes of Health Grant GM-11726 (to G.A.) and a Gosney Fellowship (to P.H.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Caltech Gosney FellowshipUNSPECIFIED
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ID Code:12264
Deposited By: Archive Administrator
Deposited On:31 Oct 2008 04:58
Last Modified:08 Nov 2021 22:27

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