p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2.
Downstream mediators of p53 in apoptosis induction remain to be elucidated. We report that p53-induced apoptosis occurred in the absence of cytochrome c release into the cytosol. Although Bax was upregulated, it remained largely in the cytosol and there was no detectable translocation to the mitochondria. Bid was not activated as no cleavage could be detected. Thus, the absence of cytochrome c release may be due to the lack of Bax translocation to mitochondria and/or Bid inactivation. Nevertheless, p53-induced apoptosis is still caspase dependent because it could be abolished by z-VAD-fmk. To search for alternative downstream targets of p53, we detected production of reactive oxygen species (ROS) as well as mitochondrial membrane potential (Deltapsi). p53 induced ROS generation, which then caused a transient increase of Deltapsi followed by a decrease. Antioxidants could inhibit the alterations of Deltapsi, thereby preventing apoptosis. z-VAD-fmk was unable to abrogate Deltapsi elevation but inhibited Deltapsi decrease, indicating that Deltapsi elevation and its decrease are two independent events. Bcl-2 may abolish elevation as well as decrease of Deltapsi without interfering with ROS levels. Thus, the ROS-mediated disruption of Deltapsi constitutes a pivotal step in the apoptotic pathway of p53, and this pathway does not involve cytochrome c release.
Li
P F
PF
Department of Cardiology, Franz Volhard Klinik, Universitätskliuikum Charité, Humboldt University, Wiltbergstrasse 50, 13125 Berlin, Germany.
Dietz
R
R
von Harsdorf
R
R
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein; D002232:Carbocyanines / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; D000975:Antioxidants; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002232:Carbocyanines; D002352:Carrier Proteins; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D020169:Caspases