Smac-mediated sensitization of human B-lymphoma cells to staurosporine- and lactacystin-triggered apoptosis is apoptosome-dependent.
Second mitochondrial activator of caspase (Smac)-derived peptides have previously been shown to facilitate apoptosis of various types of cancer cells. However, it remains unclear whether the effects of such Smac agonists are dependent on apoptotic protease-activating factor-1 (Apaf-1), a key component of the apoptosome. Here, we explored the role of Apaf-1 through overexpression of this protein in the B-lymphoma cell line Raji that is defective for cytosolic Apaf-1 expression. Enforced expression of Apaf-1 rendered Raji cells sensitive to staurosporine as well as to the proteasome inhibitor, lactacystin. Importantly, co-treatment with Smac peptides resulted in a threefold higher degree of apoptosis in Apaf-1-expressing Raji cells, but not in mock-transfected cells. Smac peptides also potentiated apoptosis of the DG-75 cell line following liberation of endogenous Apaf-1 from the plasma membrane, but were ineffective when added alone. Furthermore, we observed high levels of expression in several B-lymphoma cell lines of cellular inhibitor of apoptosis protein-2 (cIAP2), and immunodepletion of cIAP2 (a target of Smac) was found to sensitize Apaf-1-overexpressing Raji cells to cytochrome c-dependent caspase activation. Collectively, these results demonstrate the importance of Apaf-1 in Smac-mediated potentiation of apoptosis of B-lymphoma-derived cells.
Sun
Y
Y
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Ottosson
A
A
Pervaiz
S
S
Fadeel
B
B
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D053478:Apoptosomes / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000502:physiology; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000032:analysis; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000473:pathology*; D021962:Membrane Microdomains / Q000502:physiology; D024101:Mitochondrial Proteins / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases
C506592:APAF1 protein, human; D051017:Apoptosis Regulatory Proteins; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; C067713:lactacystin; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D044767:Ubiquitin-Protein Ligases; D020169:Caspases; D019311:Staurosporine; D000111:Acetylcysteine