title: Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis.
abstract: Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP(S)) and a long, 55-kDa form (c-FLIP(L)). The role of c-FLIP(S) as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIP(L) remains controversial. Although overexpression of transfected c-FLIP(L) inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIP(L) on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIP(L) in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIP(L) augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIP(L) functions primarily as an inhibitor of death receptor-mediated apoptosis.
authors.affiliation: Department of Molecular Oncology, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: Darcie A
authors.initials: DA
authors.lastname: Sharp
authors.forename: David A
authors.initials: DA
authors.lastname: Lawrence
authors.forename: Avi
authors.initials: A
authors.lastname: Ashkenazi
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053455:Caspase 10; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000502:physiology*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; C505530:CASP8 protein, human; D053455:Caspase 10; D053181:Caspase 8; D020169:Caspases; C506438:CASP10 protein, human