title: Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release.
abstract: Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
authors.affiliation: IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA. lfritz@idun.com
authors.forename: F
authors.initials: F
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Srinivasan
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: R C
authors.initials: RC
authors.lastname: Armstrong
authors.forename: K J
authors.initials: KJ
authors.lastname: Tomaselli
authors.forename: L C
authors.initials: LC
authors.lastname: Fritz
mesh_terms: D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000502:physiology; D003574:Cytochrome c Group / Q000008:administration & dosage*; D016207:Cytokines / Q000494:pharmacology; D006801:Humans; D008845:Microinjections; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein
chemical_list: C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D016207:Cytokines; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases