Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis

Cytochrome c (cyto c) release from mitochondria is a critical event in apop tosis, By investigating the ordering of molecular events during genotoxic s tress-induced apoptosis, we found that ionizing radiation (IR) and etoposid e induced the release of cyto c from mitochondria in two distinct stages. T he early release of low levels of cyto c into the cytosol preceded the acti vation of caspase 9 and 3, but had no eff ect on ATP levels or mitochrondri al transmembrane potential (Delta psi(m)). In contrast, the late stage cyto c release resulted in a drastic loss of mitochondrial cyto c and was assoc iated with reduction of ATP levels and Delta psi(m). Moreover, caspases con tributed to the late cyto c release since the caspase inhibitor zVAD preven ted only the late but not the early-stage cyto c release. Recombinant caspa se 3 induced cyto c release from isolated mitochondria in the absence of cy tosolic factors. Bcl-2 but not Bid was cleaved during apoptosis after caspa se activation. This suggests that Bcl-2 cleavage might contribute to the la te cyto c release, which results in mitochondrial dysfunction manifested by the decrease of ATP and Delta psi(m). zVAD prevented the reduction of ATP, Delta psi(m), and nuclear condensation when added up to 8 h after IR, at t he time the caspases were highly activated but when the majority of cyto c was still maintained in the mitochondria. These findings link the feedback loop control of caspase-induced cyto c release with mitochondrial dysfuncti on manifested by ATP and Delta psi(m) decline.