p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release.
The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.
Schuler
M
M
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.
Bossy-Wetzel
E
E
Goldstein
J C
JC
Fitzgerald
P
P
Green
D R
DR
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis; D020169:Caspases / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D016158:Genes, p53 / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D020169:Caspases; D000111:Acetylcysteine