Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death.

Abstract

SummaryMice injected with anti-Fas antibody die within a few hours with total liver destruction due to massive apoptosis of hepatocytes. We show that this is preceded and accompanied by the sequential activation of cysteine proteases of the interleukin 1[3-converting enzyme (ICE) and CPP32 types in the cytosol of the hepatocytes, and that proCPP32 cleavage and enzymatic activity can be prevented by intravenous injections of the tripeptide N-benzyloxycarbonyl-Val-AlaAsp-fluoromethylketone (Z-VAD.fmk), an inhibitor of ICE-like proteases. Four Z-VAD.fmk injections at 1-hour intervals abolished all signs of liver damage after anti-Fas antibody injection and resulted in 100% long-range recovery, without residual tissue damage, from a condition otherwise uniformly fatal within <3 hours. This treatment was effective even when delayed unnl some liver DNA degradation was already detectable. Injections of the tetrapeptide Ac-YVAD.cmk, more specific for the ICE-like subfamily of cysteine proteases but less cell permeable, also gave protection, but at higher doses and when injections started before that of anti-Fas antibody. These observations afford a way of temporarily modulating a number of apoptotic processes in vivo and may have important therapeutic implications in some human diseases. There is now conclusive evidence that the process of apoptosis or programmed cell death (PCD) results from the activation of members of a new family of cysteine proteases with a specificity of cleavage for aspartate in the P1 position. The decisive importance of this mechanism as an effector of PCD was revealed by the discovery that the ced-3 gene of Caenorhabditis elegans, which is required for cell death occurring during the normal development of this nematode (1), encodes for a protein related to the mammalian IL-l[3-converting enzyme (ICE) (2), an aspartate-specific cysteine protease, and that, in certain conditions, overexpression of ICE itself in mammalian cells can lead to apoptosis (3). Several other members of this protease family have now been identified and are presently subdivided into three subfamilies, each containing variants: the ICE, CPP32 (also called YAMA, apopain, or priCE), and Ich-1 (or Nedd-2) subfamilies; all these enzymes are synthesized as inactive proenzymes requiring cleavage at specific Asp residues to be transformed into active proteases: thus, at least some of these proteases can activate each other in the form of a proteolytic cascade and/or may undergo, once activated, autoprocessing, allowing the potentially lethal amplification of a minor imtial proteolytic process (for review, see reference 4). Short peptides corresponding to the cleavage site of some of these cysteine proteases have been used as inhibitors, with the Tyr-Val-Ala-Asp (YVAD) and the Asp-Glu-Val-Asp (DEVD) motifs being rather specific inhibitors of the ICE and CPP32 subfamilies, respectively (5). Provided their extracellular concentration is high enough, addition of these or related inhibitory peptides to cell cultures prevents many form...

Lysosomes and Fas-mediated liver cell death

“…According to previous biochemical and morphological observations [17][18][19] and our observations, it is probable that the first process corresponds to apoptosis. In Jurkat cells, the second process goes with a rupture of plasma membrane as illustrated by the release of cytosolic enzymes into the medium.…”

“…Experiments were first performed by injecting 10 µg of aFas into animals. Under these conditions, a rapid and massive apoptosis of the liver cells takes place as assessed by morphological and biochemical observations [17][18][19] and causes the death of most animals after 1-2 h. Our results show that, in that case, aFas injection causes a large and rapid increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase without modifying the proportion of unsedimentable activity of lysosomal enzymes. If the amount of aFas injected is lower (5 µg), the development of apoptosis is slower, the peak of DEVDase activity and of unsedimentable sulfite cytochrome c reductase Abbreviations used: Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; aFas, anti-Fas antibody; DPPIII, dipeptidyl peptidase III.…”

“…Such an injection causes a massive apoptosis of hepatocytes as assessed by biochemical and morphological criteria [17][18][19] and the death of the animals 90-120 min after injection. To monitor liver apoptosis, we examined the chronological changes of DEVDase activity and of unsedimentable sulfite cytochrome c reductase taking place after aFas injection.…”

Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice

“…These approaches include: 1) inhibition of coupling between extracellular death factors (e.g., Fas ligand and TNF-␣) and their receptors, using a soluble receptor 17,49 or monoclonal antibodies 50 ; and, currently, 2) inhibition of intracellular apoptotic signals, using a specific inhibitor for caspase proteases. 51 We describe here a distinct strategy for the treatment of fulminant hepatic failure, using HGF, a physiological hepatotrophic factor. Use of HGF to treat subjects with fulminant hepatic failure seems to have definite advantages, and HGF has multiple biological activities that are beneficial in the liver and other organs.…”

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…To analyze the relations between lipid accumulation and caspase activity, human monocytic U937 cells were cultured in the absence or in the presence of 7KC (40 lg/ml) associated or not with the broad-spectrum caspase inhibitor z-VAD-fmk (200 lM) (18), or with the caspase-2 inhibitor z-VDVAD-fmk (100 lM) (19). The effects of these inhibitors on 7KC-induced lipid accumulation was measured after staining with NR which allows to distinguish neutral and polar lipids (8,9) both by flow cytometry (FCM), and by spectral analysis (20) by means of confocal laser scanning microscopy (CLSM).…”