Caspase-7 is directly activated by the approximately 700-kDa apoptosome complex and is released as a stable XIAP-caspase-7 approximately 200-kDa complex.
MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.
Twiddy
Davina
D
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, UK.
Cohen
Gerald M
GM
Macfarlane
Marion
M
Cain
Kelvin
K
D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D001943:Breast Neoplasms / Q000473:pathology; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D002474:Cell-Free System; D045304:Cytochromes c / Q000494:pharmacology; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003838:Deoxyadenine Nucleotides; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D045304:Cytochromes c; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate