title: Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein.
abstract: Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).
authors.affiliation: Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Stegh
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Barnhart
authors.forename: Jorg
authors.initials: J
authors.lastname: Volkland
authors.forename: Alicia
authors.initials: A
authors.lastname: Algeciras-Schimnich
authors.forename: Ning
authors.initials: N
authors.lastname: Ke
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
authors.forename: Marcus E
authors.initials: ME
authors.lastname: Peter
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D011233:Precipitin Tests; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
chemical_list: C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9