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 Bar was upregulated, it remained larg ely in the cytosol and there was no detectable translocation to the mitocho ndria. Bid was not activated as no cleavage could be detected. Thus, the ab sence of cytochrome c release may be due to the lack of Bar translocation t o 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 incr ease of Delta psi followed by a decrease. Antioxidants could inhibit the al terations of Delta psi, thereby preventing apoptosis. z-VAD-fmk was unable to abrogate Delta psi elevation but inhibited Delta psi decrease, indicatin g that Delta psi elevation and its decrease are two independent events. Bcl -2 may abolish elevation as well as decrease of Delta psi without interferi ng with ROS levels. Thus, the ROS-mediated disruption of Delta psi constitu tes a pivotal step in the apoptotic pathway of p53, and this pathway does n ot involve cytochrome c release.