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 (Delta psi). p53 induced ROS generation, which then caused a transient increase of Delta psi followed by a decrease. Antioxidants could inhibit the alterations of Delta psi, thereby preventing apoptosis. z-VAD-fmk was unable to abrogate Delta psi elevation but inhibited Delta psi decrease, indicating that Delta psi elevation and its decrease are two independent events. Bcl-2 may abolish elevation as well as decrease of Delta psi without interfering with ROS levels. Thus, the ROS-mediated disruption of Delta psi constitutes a pivotal step in the apoptotic pathway of p53, and this pathway does not involve cytochrome c release.