Apoptosisis activated during the early phase of reperfusion after liver ischemia and after liver transplantation in animals. However, the molecular basis of ischemia-induced cell death remains poorly understood. In this study we show that hepatocytes from ischemic liver lobes undergo apoptosis after reperfusion. In vivo pretreatment of rats with a specific inhibitor of caspases abrogates the apoptotic response in ischemic liver lobes. Inhibition of apoptosis can be accounted for by total inhibition of caspase activation as assessed in an enzymatic assay and by specific affinity labeling. Treatment with a caspase inhibitor fully protects rats from death induced by ischemia/reperfusion. These findings indicate that liver injury after ischemia/reperfusion can be prevented by inhibition of caspases. Thus, caspase inhibitors may have important therapeutic implications in liver ischemic diseases and after liver transplantation.

Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKC, PKC⑀, and PKC␣, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKC and PKC⑀ rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.In several cell lines, apoptosis is antagonized by growth factors and hormones and, more generally, by stimuli that promote cell survival. Interleukin 3 and insulin-like growth factor 1 exert their antiapoptotic effect through activation of phosphatidylinositol 3-kinase, which, in turn, leads to activation of the serine/threonine protein kinase B (PKB/Akt), which promotes cell survival by phosphorylating BAD at Ser 136 (1-6). Interestingly, interleukin 3, through activation of a mitochondrial membrane based protein kinase A, also stimulates phosphorylation of BAD at Ser 112 (6, 7 (8,9). Brain-derived neurotrophic factor exerts its antiapoptotic effect in a mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase pathway (8). The mechanism of action of TPA remains unclear, although MAPKdependent (10 -13) and -independent pathways have been described (9). TPA is a tumor promoter that binds and activates members of a family of serine/threonine protein kinases termed protein kinase C (PKC). PKC comprised at least 12 isotypes that have been classified into three groups according to their structure and cofactor requirement: (a) conventional PKCs (PKC␣, PKC␤I, PKC␤II, and PKC␥) are diacylglycerol-and calcium-dependent, (b) novel PKCs (PKC␦, PKC⑀, PKC, PKC, and PKC) are diacylglycerol-dependent but calciumindependent, and (c) atypical PKCs (PKC, PKC, and PKC) are not activated by phorbol esters but can bind diacylglycerol (14, 15). Overexpression of PKC⑀, PKC␣, or PKC increases the resistance of cells to apoptosis, and PKC inhibitors are known to sensitize cells to apoptosis (16 -20). Additionally, Fas ligation-induced apoptosis in Jurkat T cells resulted in a blockade of cellular PKC activity, suggesting a link between the two events (21). Although involvement of PKC in the suppression of apoptosis has been demonstrated recently, the mechanisms by which PKC promotes cell survival remain to be elucidated. EXPERIMENTAL PROCEDURESReporter Plasmids, Tr...

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Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identi®cation of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.