Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis

Abstract

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK ؊ cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporinetreated 143B.TK ؊ osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK ؊ cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

“…Therefore, flow cytometry detected nondying cells as a homogeneous population with high fluorescence. Our results differ from those shown previously (21,22) where mitochondrial outer membrane of cells undergoing apoptosis induced either by staurosporine or anti-Fas were more sensitive to permeabilization by digitonin than control untreated cells. In these two studies, however, the authors used 5-10 times more digitonin (0.005%) than we used here (0.0005%-0.001%).…”

Granzyme F induces a novel death pathway characterized by Bid-independent cytochrome c release without caspase activation

“…Respiratory rates are designated as nmol O 2 /min. 39 DNA fragmentation and nicking assay. Treated Yac-1 cells were harvested and resuspended in a DNA extraction buffer (100 mM Tris-Cl, pH 8.0, 5 mM EDTA, 0.2 M NaCl, 0.5% SDS and 0.2 mg/ml proteinase K) for 2 h at 521C.…”

Mitochondrial Regulation of Caspase Activation by Cytochrome Oxidase and Tetramethylphenylenediamine via Cytosolic Cytochrome c Redox State

“…TMPD Prevents Staurosporine-induced Caspase Activation in Cells-Staurosporine is known to cause mitochondria-mediated apoptosis in cells (15,16). To test whether TMPD can block cytochrome c-mediated caspase activation in cells, we treated J774 macrophage cells with staurosporine in the presence/absence of TMPD for 4 h and then measured the activity of caspases in cytosolic extracts.…”