Thiol redox state in apoptosis : physiological and toxicant modulation

Abstract

Apoptosis is a physiological type of cell death used to regulate the number of cells during development and in adult organs. However, apoptosis can also be inappropriately activated or inhibited under pathological conditions. One of the critical mechanisms of apoptosis is the activity of cysteine proteases belonging to the caspase family. The present study was designed to investigate the role of oxidative stress in apoptosis and how the apoptotic death program might be regulated by redox-active toxicants.

We identified markers for oxidative stress in apoptotic rat thymocytes, assayed by the depletion of reduced glutathione (GSH), protein thiols, and a-tocopherol as compared to healthy control cells. Supporting a biological role for these events, we observed that different antioxidants could inhibit rat thymocyte apoptosis induced by a variety of non-oxidant stimuli. This suggests that in rat thymocytes the apoptotic program induces oxidative stress which might be critical for the process.

We continued these studies in a human leukemic T cell line (Jurkat T cells), where it was found that apoptosis induced by anti-CD95 (Fas/APO-l) antibody (anti-CD95 Ab) dramatically depleted intracellular GSH, which agrees with the above findings. Interestingly, glutathione was found to be depleted through efflux of the reduced form of the molecule. The apoptotic GSH efflux was dependent on caspase activity and could be blocked by inhibitors specific for the hepatocyte canalicular GSH transporter.

Investigating the role for apoptotic GSH efflux, it was found that preventing the GSH depletion did not inhibit CD95-mediated DNA fragmentation. Furthermore, we found that anti-CD95 Ab-treatment mediated caspase-dependent K+ efflux and caspase-dependent cell shrinkage in Jurkat T cells, which both also seemed to be independent on GSH efflux. Preliminary results indicate that none of the known K+-channels or K+-co-transporters were responsible for the CD95-mediated K+ efflux. However, Na+ / K+-ATPase activity of Jurkat T cells was inhibited by the anti CD95 Ab, suggesting that passive diffusion of K across the cell membrane might be responsible. Preliminary results show that inhibition of Na+/K+-ATPase might also be involved in apoptotic cell shrinkage.

While screening antioxidants as inhibitors of apoptosis, it was found that pyrrolidine dithiocarbamate (PDTC) was the most potent inhibitor of thymocyte apoptosis then available. Surprisingly, at the same time, PDTC induced an oxidative stress, which was dependent upon its ability to transport extracellular copper into the cells. In longer incubations, PDTC was clearly toxic, inducing apoptosis or necrosis depending on the dose.

Investigating the potent anti-apoptotic effect by dithiocarbamates (DCs), we found that it probably was the DC disulfides which mediated the inhibitory effect. The inhibition of CD95 mediated apoptosis by the DC disulfide disulfiram (DSF) was correlated to the inhibition of the processing of pro-caspase-3. This was confirmed with an in vitro activation model of this proenzyme. DSF was unable to inhibit the in vitro activation of pro-caspase-3 if dithiothreitol was included, suggesting thiol-disulfide exchange between the DC disulfide and its target(s).

Since processing of pro-caspases may need activity of other caspases, studies on the interaction between active caspases and DSF were performed in vitro. DSF was found to be a potent inhibitor of both purified caspase-3 and caspase-1. The inhibition seemed to be competitive and was shown to be accomplished through thiol-disulfide exchange. This suggests that the anti-apoptotic effect of DC disulfides occurs by inhibition of some yet unidentified caspase, that is responsible for the activation of a caspase cascade subsequent to CD95 receptor clustering.