Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis

We investigated cytochrome c release kinetics in response to three apoptosi s-inducing agents (tumor necrosis factor-a, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent p rotein (EGFP)-tagged cytochrome c. All three agents induced significant cas pase activation in the cultures determined by monitoring the cleavage of fl uorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cel ls, albeit the valinomycin-induced activation was less pronounced. Time-lap se confocal microscopy showed that tumor necrosis factor-alpha and staurosp orine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release o ccurred slowly over several hours. Unlike staurosporine, the valinomycin-in duced cytochrome c release was not associated with translocation of the pro apoptotic Bax protein to the mitochondria, and was not accompanied by co-re lease of the proapoptotic Smac protein. Immunoprecipitation experiments rev ealed that cytochrome c was also released out of the cell into the extracel lular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apo ptosis.