Mechanism of Dithiocarbamate Inhibition of Apoptosis:  Thiol Oxidation by Dithiocarbamate Disulfides Directly Inhibits Processing of the Caspase-3 Proenzyme

Abstract

Dithiocarbamates (DCs) have been reported to be potent inhibitors of apoptosis in several different model systems, which suggests a target common to the apoptotic machinery. Without further investigation, this has been assumed to reflect an antioxidant activity of the DCs. However, we have recently shown that DCs exert prooxidant effects on T cells [Nobel et al. (1995) J. Biol. Chem. 270, 26202-26208], which are dependent on their transfer of external copper into the cells and can be inhibited by the inclusion of high-affinity external copper chelators in the medium. Investigating antiapoptotic actions of DCs, we found that inclusion of a membrane-impermeable copper chelator severely compromised the inhibitory activity of reduced DCs. Since copper can promote DC oxidation to the respective DC disulfides, the inhibitory effect on lymphocyte apoptosis might be mediated by the DC disulfides. In agreement with this we observed that DC disulfides were more potent inhibitors of T cell apoptosis than their reduced counterparts. Inhibition of apoptosis by DC disulfides correlated with the inhibition of caspase-3 proenzyme processing and activation. Similar results were obtained in a cell-free model system of caspase-3 activation. Significantly, dithiothreitol reduction of the DC disulfide abolished its inhibition of in vitro proenzyme processing, thereby demonstrating thiol-disulfide exchange between the DC disulfide and a free thiol group on an activator(s) of caspase-3. Since T cell apoptosis involves the generation of mature caspase-3 and requires caspase-3-like activity, we propose that (1) DC disulfides are the active agents behind DC inhibition of apoptosis and (2) their site of action is the proteolytic activation of this enzyme. These findings also reveal the potential for other thiol-oxidizing toxicants to inhibit apoptosis by preventing the proteolytic activation of caspases.

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

“…An internal apoptosis pathway activated via the cleavage of caspase 9 and 3 is well documented (74,75). Furthermore, previous studies also demonstrated that the mechanism behind caspase 3 activation and apoptosis (76)(77)(78) led to morphological and biochemical changes and the modification of key structural and regulatory proteins by caspase 3 (79,80). During this process, the chromatin becomes highly condensed and fragmented to form micronuclei called apoptotic bodies, in a process referred to as nuclear blebbing (81,82).…”

Oxidative stress induces caspase-independent retinal apoptosis in vitro

“…It has been suggested that a critical cysteine group in the active site of the caspases makes them sensitive to thiol oxidation. 41 In order to investigate the means of caspase inactivation in cell lysates from Fas treated Jurkat cells incubated with peroxide, we determined the ability of the thiol reducing agent dithiothreitol (DTT) to restore caspase activity. As Figure 4B demonstrates, incubation of hydrogen peroxide (200 mM) treated lysates from anti-Fas treated Jurkat cells with 100 and 200 mM DTT restores caspase activity.…”

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

“…Whereas cell death under conditions in which the caspase family cannot function has been reported to resemble necrosis rather than apoptosis in several studies (25)(26)(27)(28), a growing body of evidence that apoptotic cell death induced by oxidative stress occurs independently of caspase activation has been accumulated (29 -32). In fact, cells have shown apoptosis-like morphological changes, chromatin condensation and/or DNA fragmentation without caspase activation in these papers, suggesting that these distinctive characteristics of apoptosis take place in a caspase-independent manner.…”