p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulati on, DNA repair, and programmed cell death, Several lines of evidence sugges t 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 b y cleavage of caspase substrates and was preceded by mitochondrial cytochro me c release. The addition of the broad-spectrum caspase inhibitor benzylox ycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transdu ction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcystei ne, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduc ed with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was st ill active in the presence of zVAD-fmk, When Bar was immunodepleted from th e cytosolic extracts of p53 expressing cells before incubation with isolate d mitochondria, the in vitro cytochrome c release was abolished. Thus, we c ould demonstrate in cells and in vitro that p53 activates the apoptotic mac hinery through induction of the release of cytochrome c from the mitochondr ial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bar for this cytochrome c-releasing activity of p5 3 in Saos-2 cells.