Transforming growth factor-β induces apoptosis in rat FaO hepatoma cells via cytochrome c Release and oligomerization of Apaf-1 to form a ˜700-kd apoptosome caspase-processing complex

Abstract

In mammalian cells, non receptor-mediated apoptosis occurs via the cytochrome c-dependent assembly of a ϳ700-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 ␤ 1 (TGF-␤ 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 ϳ700-kd apoptosome complex. Similarly, in vitro activation of hepatoma cell lysates with 2-deoxyadenosine 5-triphosphate (dATP) results in the formation of the ϳ700-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-␤ 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-␤ 1 induces apoptosis via release of cytochrome c and activation of the Apaf-1 apoptosome complex, which initiates the caspase cascade. (HEPATOLOGY 2000;32:750-760.)

Proteasome inhibitor-induced apoptosis of B-chronic lymphocytic leukaemia cells involves cytochrome c release and caspase activation, accompanied by formation of an ∼700 kDa Apaf-1 containing apoptosome complex

“…19,56 After dATP activation, we also observed formation of a larger ෂ1.4 MDa Apaf-1 apoptosome complex with reduced caspase processing activity. The lower activity of the larger complex appears to be due to inappropriate oligomerization of Apaf-1.…”

Activation of caspases occurs downstream from radical oxygen species production, Bcl-xL down-regulation, and early cytochrome C release in apoptosis induced by transforming growth factor β in rat fetal hepatocytes

“…Fetal hepatocytes were treated with TGF-␤1 (2 ng/mL) for the indicated periods; then, cells were scraped and lysed and Western blot analysis was performed as described in Materials and Methods. As it has been previously described in rat hepatocytes 12 or in rat hepatoma cells, 15 Time course analysis of caspase-3 and caspase-8 activities. Cells were incubated with TGF-␤1 (2 ng/mL) for different periods of time and lysed, and caspase-3 and -8 activities were assayed by using the fluorescent substrates Ac-DEVD-AMC and Ac-IETD-AFC, respectively, as described in Materials and Methods.…”

“…13,14 More recently, Freathy et al have shown that caspase activation by TGF-␤ in rat FaO hepatoma cells is mediated via the release of cytochrome c and the oligomerization of Apaf-1 into a 700-kd apoptosome complex. 15 In previous works from our group we found that treatment of fetal rat hepatocytes with TGF-␤1 is followed by apoptotic cell death. 16 TGF-␤1 mediates radical oxygen species (ROS) production 17,18 that precedes the loss of mitochondrial transmembrane potential (⌬ m ), the release of cytochrome c, and the activation of caspase-3.…”

Characterization of the Transforming Growth Factor-β1-induced Apoptotic Transcriptome in FaO Hepatoma Cells

“…The formation of the Apaf-1-containing apoptosome complex is central to the activation of the caspase cascade in mitochondrion-mediated cell death, and we have recently shown that TGF-␤ 1 -induced apoptosis in FaO hepatoma cells induces cytochrome c release and assembly of the ϳ700-kDa apoptosome complex, which then activates the effector caspases (caspase-3 and caspase-7) (28). Cytochrome c release during TGF-␤ 1 -induced apoptosis has subsequently been confirmed in fetal hepatocytes (29).…”