Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change.
It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.
Institute for Clinical Science, Exeter University, Noy Scott House, Wonford, Exeter, UK.
J M
JM
Garland
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Rudin
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases; D002417:Cattle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000502:physiology*; D003574:Cytochrome c Group / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D016298:Hematopoietic Cell Growth Factors / Q000037:antagonists & inhibitors*; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007942:Leukemia, Experimental / Q000473:pathology; D051379:Mice; D008928:Mitochondria / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases; D008954:Models, Biological; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects
D003574:Cytochrome c Group; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases