Multiple Kinetics of Mitochondrial CytochromecRelease in Drug-Induced Apoptosis

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-α, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-α and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochromec-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochromec release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.