Apoptosis by a cytosolic extract from Fas-activated cells.

Abstract

Fas is a type I membrane protein and its activation by binding of the Fas ligand or an agonistic anti‐Fas antibody induces apoptosis in Fas‐bearing cells. In this report we prepared lysates from cells treated with anti‐Fas antibody. The lysates induced apoptotic morphological changes in nuclei from normal mouse liver, accompanied by DNA degradation. The apoptosis‐inducing activity was quickly generated in cells by anti‐Fas antibody and was found in the soluble cytosolic fraction. Induction of the activity in cells was inhibited by a tetrapeptide, acetyl‐Tyr‐Val‐Ala‐Asp‐chloromethylketone, a specific inhibitor of interleukin‐1 beta converting enzyme. Addition of COS cell lysates containing Bcl‐2 to the assay significantly inhibited the apoptotic process, indicating that the in vitro process reflected apoptosis that occurs in intact cells.

Activation of CPP32-Like Caspases Contributes to Neuronal Apoptosis and Neurological Dysfunction after Traumatic Brain Injury

“…Then homogenates were layered over 10 ml of the buffer containing 2.3 M sucrose in a Beckman SW28 centrif uge tube and centrif uged at 22,000 rpm for 90 min at 4°C. The pellet was resuspended in homogenization buffer containing 50% glycerol at a concentration of 3-7 ϫ 10 7 nuclei /ml and stored at Ϫ85°C (Enari et al, 1995).…”

“…To examine further the induction of post-traumatic apoptosis, we used a recently developed model of reconstitution of this process in vitro (Lazebnik et al, 1993;Enari et al, 1995;. Protein extracts isolated from traumatic rat cortex were able to induce the internucleosomal fragmentation of DNA in Figure 1.…”

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

“…Electron microscopy was performed on osmiumtetroxide/ lead citrate-stained ultrathin sections as described (Marchetti et al, 1996a). Determination of DEVDase activity and PARP cleavage Cytosols (4610 6 cells/100 ml in CFS bu er: 220 mM mannitol; 68 mM sucrose, 2 mM NaCl, 2.5 mM PO 4 H 2 K, 0.5 mM EGTA, 2 mM MgCl 2 , 5 mM pyruvate, 0.1 mM phenylmethyl sulfonyl¯uoride [PMSF], 1 mM dithiotreitol, 10 mM HEPES-NaOH, pH 7.4 bu er; supplemented with additional protease inhibitors: 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 5 mg/ml antipain, 1 mg/ml chymopapain) were prepared by ®ve freeze/thaw cycles in liquid nitrogen and at 378C, followed by centrifugation (1.5610 5 g, 48C, 1 h) as described (Enari et al, 1995). The capacity of cytosols to cleave the CPP32 recognition site DEVD was determined using Ac-DEVD-amino-4-methylcoumarin (10 mM; Bachem, Basel, Switzerland) as¯uorogenic substrate (Nicholson et al, 1995).…”

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis

“…Bcl-2 localizes in multiple membrane compartments, including the nuclear envelope, endoplasmic reticulum and mitochondrial membranes (Hockenbery et al, 1990;Monaghan et al, 1992;Jacobson et al, 1993;Krajewski et al, 1993;Akao et al, 1994), raising the possibility that Bcl-2 also acts downstream of caspases. Indeed an anti-cell death role for Bcl-2 exerted downstream of active ICE family proteases has been suggested by the observations that (1) using in vitro apoptosis system, apoptotic DNA ladder formation is prevented by a Bcl-2-containing cell lysate, even after an e ector(s) downstream of caspases is activated, as judged by the failure of caspase inhibitors to prevent apoptotic changes in naked nuclei (Enari et al, 1995) and (2) apoptosis induced by transfection with caspase genes which encode precursor forms of proteases is prevented by overexpressed Bcl-2 (Miura et al, 1993;Wang et al, 1994). Using microinjection procedures, we show here that Bcl-2 and Bcl-x L exert their anti-cell death activity only upstream and/or within but not downstream of the caspase cascade.…”