Cysteine proteases of the interleukin 1 beta Converting Enzyme (ICE)/CED-3 family have been implicated in the effector process of apoptosis in several systems, including Fas-mediated apoptosis. We have recently isolated and partially characterized a protease present in extracts from anti-Fas antibody treated Jurkat T cells that promotes apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and Orrenius, S. (1995) FEBS Lett. 364, 139-142). We now show that this protease cleaves poly-(ADP-ribose) polymerase (PARP) with high efficiency and specificity. Both PARP proteolysis and the proapoptotic effects of the protease are inhibited by nanomolar concentrations of a selective inhibitor of apopain (CPP32), while an inhibitor of IL-1 beta converting enzyme is much less effective, requiring micromolar concentrations for the inhibition of the isolated protease. Kinetic analysis of the isolated protease reveals kinetic constants similar to those reported for apopain. The isolated protease is recognized by antibodies specific for CPP32/apopain but not by an anti-ICE antibody. Furthermore, a selective inhibitor of apopain prevents Fas-induced apoptosis in intact Jurkat T cells. We therefore conclude that CPP32/apopain is activated in Fas-induced apoptosis.

Although human JURKAT T lymphocytes induced to undergo apoptosis with anti-Fas/APO-1 antibody were observed to rapidly lose reduced glutathione (GSH), increased concentrations of oxidized products were not detectable. Unexpectedly, the reduced tripeptide was instead quantitatively recovered in the incubation medium of the cells. As GSH loss was blocked by bromosulfophthalein and dibromosulfophthalein, known inhibitors of hepatocyte GSH transport, a specific export rather than nonspecific leakiness through plasma membranes is proposed to be responsible. Apoptosis was delayed when GSH-diethylesters were used to elevate intracellular GSH, although the high capacity of the activated efflux system quickly negated the benefit of this treatment. Stimulation of GSH efflux provides a novel mechanism whereby Fas/APO-1 ligation can deplete GSH. We speculate that it enhances the oxidative tonus of a responding cell without requiring an increase in the production of reactive oxygen species.In the current paradigm for apoptotic cell death, stimulation of the proteolytic activity of a family of interleukin 1-␤-converting enzyme (ICE) 1 -like proteases initiates several distinct events within a cell (1-3). These include chromatin fragmentation, plasma membrane blebbing, and an overall cell shrinkage, eventually culminating in either the phagocytosis or lysis of the apoptotic cell. There have also been numerous reports that apoptotic cells accumulate oxidized proteins and lipids and are presumably therefore exposed to some degree of oxidative stress (4). In addition, antioxidants have been widely reported to protect against, or delay, many different forms of apoptosis (5). However, several recent reports of apparently normal apoptosis occurring in very low oxygen environments indicate that reactive oxygen species (ROS) are unlikely to be essential mediators of this type of cell death (6 -8).Human JURKAT T lymphocytes are rapidly induced to undergo apoptosis after exposure to anti-Fas/APO-1 antibody (9 -11). One of the earliest detectable events downstream of ligandreceptor binding is activation after 15 min of the ICE-like protease CPP-32/apopain (12, 13). Plasma membrane blebbing and degradation of the cytoskeleton commence soon after, while chromatin breakdown is first detectable at 45 min with internucleosomal DNA fragments beginning to accumulate after approximately 90 min (14). Gross alterations in plasma membrane permeability develop several hours later. Although it has been reported that these changes occur with similar kinetics independently of ambient oxygen tension (6), the possibility remains that oxygen-independent modification of intracellular redox states are involved in these events. To further understand this controversial area, the glutathione metabolism of JURKAT cells exposed to anti-Fas/APO-1 antibody was investigated. Consistent with results in thymocytes (15, 16), T lymphocytes undergoing apoptosis became dramatically depleted of their reduced glutathione (GSH) coincident with the onset of chromatin fragmen...

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