Rate-limiting step preceding cytochrome c release in cells primed for Fas-mediated apoptosis revealed by analysis of cellular mosaicism of respiratory changes.
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 approximately 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 approximately 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.
Hájek
P
P
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Villani
G
G
Attardi
G
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D019069:Cell Respiration / Q000187:drug effects; D002469:Cell Separation; D003574:Cytochrome c Group / Q000378:metabolism* / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009030:Mosaicism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D003574:Cytochrome c Group; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D019802:Succinic Acid; D010089:Oxidoreductases, N-Demethylating; C022726:tetramethylphenylenediamine oxidase; D003576:Electron Transport Complex IV; D004072:Digitonin; D010100:Oxygen