MECHANISM OF DITHIOCARBAMATE INHIBITION OF APOPTOSIS - THIOL OXIDATION BY DITHIOCARBAMATE DISULFIDES DIRECTLY INHIBITS PROCESSING OF THE CASPASE-3 PROENZYME

Dithiocarbamates (DCs) have been reported to be potent inhibitors of a poptosis in several different model systems, which suggests a target c ommon to the apoptotic machinery, Without further investigation, this has been assumed to reflect an antioxidant activity of the DCs. Howeve r, we have recently shown that DCs exert prooxidant effects on T cells [Nobel et al. (1995) J. Biol. Chem. 270, 26202-26208], which are depe ndent on their transfer of external copper into the cells and can be i nhibited by the inclusion of high-affinity external copper chelators i n the medium. Investigating antiapoptotic actions of DCs, we found tha t inclusion of a membrane-impermeable copper chelator severely comprom ised 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 agree ment with this we observed that DC disulfides were more potent inhibit ors 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 i n a cell-free model system of caspase-3 activation, Significantly, dit hiothreitol reduction of the DC disulfide abolished its inhibition of in vitro proenzyme processing, thereby demonstrating thiol-disulfide e xchange between the DC disulfide and a free thiol group on an activato r(s) of caspase-3, Since T cell apoptosis involves the generation of m ature caspase-3 and requires caspase-3-like activity, we propose that (1) DC disulfides are the active agents behind DC inhibition of apopto sis and (2) their site of action is the proteolytic activation of this enzyme. These findings also reveal the potential for other thiol-oxid izing toxicants to inhibit apoptosis by preventing the proteolytic act ivation of caspases.