p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2

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