Caspase-9 activation by the apoptosome is not required for fas-mediated apoptosis in type II Jurkat cells.
Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x(L) were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x(L)-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.
Shawgo
Mary E
ME
Department of Pharmacology, Toxicology & Therapeutics and the Kansas University Cancer Center, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Shelton
Shary N
SN
Robertson
John D
JD
D017209:Apoptosis; D053478:Apoptosomes / Q000378:metabolism*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism*; D045305:Cytochromes c' / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*
C506592:APAF1 protein, human; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; C494811:BCL2L1 protein, human; D045305:Cytochromes c'; D019253:Proto-Oncogene Proteins c-bcl-2; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051020:bcl-X Protein; D019014:fas Receptor; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9