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Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes

Hájek, Petr and Villani, Gaetano and Attardi, Giuseppe (2001) Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes. Journal of Biological Chemistry, 276 (1). pp. 606-615. ISSN 0021-9258. doi:10.1074/jbc.M007871200.

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In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ~30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ~40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane.

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Additional Information:© 2001 by the American Society for Biochemistry and Molecular Biology. Received for publication, August 29, 2000. Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007871200 We are very grateful to Anne Chomyn, Elisabetta Ferraro, and Jordi Asin-Cayuela for helpful discussion, and Benneta Keeley, Arger Drew, and Rosario Zedan for expert technical assistance. This work was supported by United States Public Health Service Grant GM11726 (to G. A.) and by 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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Deposited On:06 Jun 2006
Last Modified:08 Nov 2021 19:55

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