Transforming growth factor-beta(1) induces apoptosis in rat FaO hepatoma cells via cytochrome c release and oligomerization of Apaf-1 to form a approximately 700-kd apoptosome caspase-processing complex.
In mammalian cells, non receptor-mediated apoptosis occurs via the cytochrome c-dependent assembly of a approximately 700-kd apoptotic protease-activating factor 1 (Apaf-1)/caspase-9 containing apoptosome complex. This initiates the postmitochondrial-mediated effector caspase cascade. We now show that receptor mediated transforming growth factor beta(1) (TGF-beta(1))-induced apoptosis in rat hepatoma cells is accompanied by processing and activation of caspases-2, -3, -7, and -8. Furthermore, we show that caspase activation is mediated via the release of cytochrome c and the oligomerization of Apaf-1 into an approximately 700-kd apoptosome complex. Similarly, in vitro activation of hepatoma cell lysates with 2'-deoxyadenosine 5'-triphosphate (dATP) results in the formation of the approximately 700-kd apoptosome complex, which recruits and processes caspases-3 and -7. Z-VAD.FMK [benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone], the pan-caspase inhibitor totally inhibits dATP-stimulated caspase activation but does not block the assembly of the large Apaf-1 containing apoptosome complex. However, the recruitment and subsequent processing of caspases-3 and -7 to the apoptosome is blocked. Similarly, in intact cells, although Z-VAD.FMK blocked TGF-beta(1)-induced apoptosis, it did not prevent the oligomerization of Apaf-1 into the apoptosome. However, recruitment and processing of caspases-3 and -7 were prevented by Z-VAD.FMK. These data show that TGF-beta(1) induces apoptosis via release of cytochrome c and activation of the Apaf-1 apoptosome complex, which initiates the caspase cascade.
Freathy
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MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester, UK.
Brown
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Roberts
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Cain
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1; D020169:Caspases / Q000378:metabolism*; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000378:metabolism*; D034882:Lamins; D008114:Liver Neoplasms, Experimental / Q000473:pathology*; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
C506594:Apaf1 protein, rat; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D034882:Lamins; D009687:Nuclear Proteins; D011506:Proteins; D016212:Transforming Growth Factor beta; D020169:Caspases