(39) MULTIPLE KINETICS OF MITOCHONDRIAL CYTOCHROME C RELEASE IN APOPTOSIS

J.H.M. Prehn¹, C.M. Luetjens¹, D. Koegel¹, C. Reimertz¹, A. Renz², M. Poppe¹, K. Schulze-Osthoff² and A-L. Nieminen^3
1Interdisciplinary Center for Clinical Research 
²Division of Immunology and Cell Biology, Westphalian Wilhelms-University, D-48149 Muenster, Germany 
³Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 

We investigated cytochrome c release kinetics in response to three apoptotic stimuli in MCF-7/Casp-3 cells stably transfected with green fluorescent protein-tagged cytochrome c. Subcellular fractionation experiments revealed that tumor necrosis factor-alpha, staurosporine and valinomycin all induced cytochrome c release from mitochondria and a significant increase in caspase-3-like protease activity in the cultures. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c from mitochondria. Confocal images did not reveal a release starting point within cells, and indicated that mitochondria opened and closed their outer membrane in a coordinated manner. In contrast, valinomycin-induced cytochrome c release was not coordinated among individual mitochondria and occurred slowly over several hours. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space prior to the loss of plasma membrane integrity. Our data demonstrate the existence of multiple kinetics of mitochondrial cytochrome c release in apoptosis. Rapidity of release and secretion out of the cell may modulate the cellular response to cytoplasmic cytochrome c.

Supported by IZKF Universitaet Muenster (BMBF 01 KS 9604/0).

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