Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis.
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 staurosporine-treated 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.
Duan
Shili
S
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Hajek
Petr
P
Lin
Catherine
C
Shin
Soo Kyung
SK
Attardi
Giuseppe
G
Chomyn
Anne
A
D017209:Apoptosis / Q000187:drug effects*; D002463:Cell Membrane Permeability; D003574:Cytochrome c Group / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010101:Oxygen Consumption / Q000187:drug effects; D010770:Phosphotransferases / Q000502:physiology; D018272:Porins / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D050994:Voltage-Dependent Anion Channels
D003574:Cytochrome c Group; D018272:Porins; D019253:Proto-Oncogene Proteins c-bcl-2; D050994:Voltage-Dependent Anion Channels; D010770:Phosphotransferases; D011493:Protein Kinase C; D019311:Staurosporine; D004072:Digitonin