Mechanism of Dithiocarbamate Inhibition of Apoptosis: Thiol Oxidation

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Chem. Res. Toxicol. 1997, 10, 636-643

Articles Mechanism of Dithiocarbamate Inhibition of Apoptosis: Thiol Oxidation by Dithiocarbamate Disulfides Directly Inhibits Processing of the Caspase-3 Proenzyme C. Stefan I. Nobel, David H. Burgess, Boris Zhivotovsky, Mark J. Burkitt,† Sten Orrenius,* and Andrew F. G. Slater Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden, and Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, U.K. Received January 16, 1997X

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.

Introduction As the molecular mechanisms behind apoptosis are unraveled, it has become apparent that the activity of a family of aspartic acid site-specific cysteine proteases, which are related to both mammalian interleukin-1βconverting enzyme (ICE)1 and the nematode ced-3 gene product, is conserved and that they are critical mediators of this type of cell death (1, 2). Recently, the recommendation for a unified nomenclature has been made (3) using the root name “caspase” reflecting two catalytic properties of these enzymes (c for the catalytic cysteine and aspase for their ability to cleave after an aspartic acid residue). We herein adopt this nomenclature. * Address correspondence to this author: tel, +46 8 7287590; fax, +46 8 329041; e-mail, [email protected]. † Rowett Research Institute. X Abstract published in Advance ACS Abstracts, May 1, 1997. 1 Abbreviations: AMC, amino-4-methylcoumarin; BCPS, bathocuproine disulfonate; caspases, proteases of the ICE (interleukin-1βconverting enzyme) protease family; caspase-3, CPP32/apopain; caspase8, MACH/FLICE, Mch5; caspase-10, Mch4; caspase-6, Mch2; DCs, dithiocarbamates; DEDC, diethyldithiocarbamate; CD95, Fas/APO-1; DSF, disulfiram; DTT, dithiothreitol; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PDTC, pyrrolidine dithiocarbamate; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

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In healthy mammalian cells, the caspase proteins typically exist as a proform (pro-caspase) which becomes proteolytically activated in response to cell-type-specific stimuli (2-5). Recent evidence from in vitro studies suggest the existence of an apoptotic proteolytic cascade where certain caspases are able to activate others (4, 5). In the case of CD95 (Fas/APO-1)-mediated apoptosis, one recently discovered protein, pro-caspase-8, has been demonstrated to bind directly to the activated CD95 receptor complex (6, 7) and become a catalytically active caspase-8. Supporting its role as an initiator of a CD95mediated caspase cascade is the recent finding that caspase-8 is able to activate several other caspases in vitro (5). Apart from caspase-8, only caspase-3 (CPP32) has so far been demonstrated to be critical for CD95mediated apoptosis (8). Caspase-3 is also implicated in apoptosis induced by a variety of other agents (2, 9, 10) as, e.g., etoposide-mediated apoptosis (10, 11). Consequently, caspase-3 (or caspase-3-like enzymes) seems to be part of a common apoptotic machinery in mammalian cells. However, at the moment it is not known how caspase-3 is activated in vivo. All caspases contain an active site cysteine nucleophile and are therefore inhibited in the presence of thiol-alkylating agents, such as © 1997 American Chemical Society

Inhibition of Pro-caspase-3 Processing by DCs

iodoacetamide or N-ethylmaleimide (2). The activity of caspases is optimal in a reducing environment (millimolar dithiothreitol, DTT, is routinely included in experimental assay buffers), strongly suggesting that active site thiol oxidation will also be inhibitory. Some pro-caspase processing may involve the activity of other caspases, and the effect of protein thiol-modifying agents (for example, various thiol-oxidizing toxicants) is predicted to prevent this proteolytic step (as well as overall caspase activity) and the detection of subsequent apoptotic events. Dithiocarbamates (DCs) comprise a broad class of molecules possessing a R1R2NC(S)SR3 functional group, which gives them the ability to react with sulfhydryl groups, as well as to chelate metals (reviewed in refs 12 and 24). These two chemical properties also underlie many of the observed biological effects of DCs (12). Their cytocidal effects have allowed them to be widely used in agriculture as insecticides, herbicides, and fungicides, while their medical uses include chemotherapy of various pathogens, antidotes against metal poisoning, alcohol deterrence (under the tradename Antabus or Aversan), and even the experimental therapy of HIV infection (1214). DCs have been shown to be potent inhibitors of apoptosis (15-17), while we and others have reported that they also possess the ability to induce apoptosis under some circumstances (18-20). Although DCs are reported to possess both prooxidant and antioxidant effects (21-24), until now it has been assumed that an antioxidant function underlies their inhibitory effect on apoptosis (15-17). However, in previous studies with thymocytes, we found that DCs act as copper ionophores to induce an oxidative stress (18) and observed that this correlates with a short-term reduction in the incidence of apoptotic cell death. Oxidative stress induced by increasing concentrations of the superoxide anion interferes with apoptosis signaled through the CD95 receptor (25), while a mild ascorbateinduced oxidative stress slows the rate of thymocyte apoptosis (26). Both findings suggest the existence of a redox-sensitive step in the apoptotic machinery. We now report that DCs need to be oxidized to their respective disulfides to inhibit T lymphocyte apoptosis, which in cell culture is probably achieved by an interaction with serum-associated copper. Inhibition of apoptosis by the DC disulfide is associated with a reduced proteolytic activation of the critical lymphocyte pro-caspase-3. Using a recently developed cell-free model system (27), we show that inhibition of this activation is achieved through the formation of a mixed disulfide with an activator(s) of procaspase-3. Our findings exclude an antioxidant explanation for the antiapoptotic action of DCs in lymphocytes and reveal the potential for other thiol-oxidizing toxicants to inhibit apoptosis by preventing the proteolytic activation of caspases.

Experimental Procedures Materials. Pyrrolidine dithiocarbamate (PTDC), diethyldithiocarbamate (DEDC), and tetramethylthiuram disulfide (thiram) were purchased from Aldrich (Milwaukee, WI); tetraethylthiuram disulfide (disulfiram, DSF), bathocuproine disulfonate (BCPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and etoposide were from Sigma (Stockholm, Sweden); anti-CD95 (Fas/APO-1) monoclonal IgM antibody clone CH-11 was from Medical Biological Laboratories (Nagoya, Japan), the peptide substrate DEVD-amino-4-methylcoumarin (DEVD-AMC) from Bachem (Heidelberg, Germany), and monobromobimane from Calbiochem (Cambridge, MD).

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 637 Cell Culture. Rat thymocytes were prepared from 70-100 g male Sprague-Dawley rats (28) and incubated at 5 × 106 cells/ mL in RPMI supplemented with 2% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 µg/mL gentimicin. Jurkat T cell clone E6, initially obtained from the American Cell Type Culture Collection (ATCC, Rockville, MD), was cultured in RPMI supplemented with phenol red, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. In several experiments the Jurkat cells were cultured in media containing only 2% FBS to reduce the potential for interaction between DCs and serum proteins. DNA Fragmentation. Fragmentation of chromatin-associated DNA was assayed both quantitatively with diphenylamine reagent (29) and after agarose gel electrophoresis (30). ATP Determination. Cellular ATP was determined with a luciferase/lucigenin kit (Orbit, Finland) according to the manufacturers instructions. After determining the optimal extraction conditions, ATP was released from Jurkat cells with 2.5% trichloroacetic acid. GSH Determination. Glutathione (GSH) was determined after monobromobimane derivatization and subsequent HPLC separation (31). SDS-PAGE and Western Blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Biorad’s (Sundbyberg, Sweden) minigel system as instructed. Proteins were then transferred to a nitrocellulose membrane using a conventional electrophoretic Western blotting technique. Caspase-3 was detected with a polyclonal IgG antibody directed against the p17 subunit of the mature protein (a generous gift from Dr. Donald Nicholson, Merck Frost Centre for Therapeutic Research, Pointe-Claire Dorval, Quebec, Canada) and visualized using the ECL method (Amersham Inc., Amersham, U.K.). Caspase Protease Activity Assay. Proteolytic activity against the synthetic fluorogenic substrate DEVD-AMC was assayed in lysates from whole Jurkat T cells frozen in liquid nitrogen at a density of 80 × 106 cells/mL. The lysates were broken by freeze-thaw and then diluted with 2 volumes of reaction buffer (100 mM Hepes, pH 7.25/10% sucrose/0.1% CHAPS/0.001% NP-40). DEVD-AMC (40 µM) was added, and release of the fluorogenic group was detected in a Fluoroskan II plate reader (excitation 355 nm, emission 455 nm; Labsystems, Stockholm, Sweden). Assays were conducted at 37 °C in a 96-well plate, and fluorescence was measured every 70 s during a 30 min time period. Data from triplicate samples were then analyzed by linear regression. In Vitro Caspase-3 Activation Assay. Activation of caspase-3 was also assayed in organelle-free Jurkat cell S-100 cytosolic extracts prepared as described earlier (27). Extracts were mixed with 400 nM cytochrome c and 0.9 mM dATP and then incubated for 30 min at 30 °C to induce processing of the caspase-3 proenzyme. The activated cytosols were then either directly subjected to SDS-PAGE analysis or diluted in 5 volumes of reaction buffer and assayed for specific protease activity as described in the previous section.

Results DCs Inhibit T Lymphocyte Apoptosis. PDTC has been reported to significantly inhibit apoptosis in several different cell types (15-18). An example involving thymocytes induced to undergo apoptosis in response to the topoisomerase II inhibitor etoposide is presented in Figure 1, top. The related compound, DEDC, was similarly found to inhibit apoptotic DNA fragmentation in this model system (Table 1). We next investigated whether DCs would also inhibit CD95-mediated apoptosis, a system previously described as being insensitive to antioxidant inhibition (32, 33). To our surprise, the antioxidant PDTC was observed to efficiently inhibit antiCD95 antibody-induced chromatin fragmentation in Ju-

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Figure 2. Concentration-dependent inhibition of thymocyte apoptosis by thiram. Thymocytes were incubated at 37 °C for 5 h with various concentrations of thiram in the presence (9) or absence (0) of 25 µM etoposide. The extent of apoptotic DNA fragmentation was then quantitated using diphenylamine. Data bars represent mean ( SD (n ) 3), while an asterisk indicates a significant difference between a thiram-treated sample and its respective control (t-test, p < 0.01). Cell viability, as determined by trypan blue exclusion, was determined in samples of the etoposide-treated cells as shown.

Figure 1. Dithiocarbamate inhibition of apoptotic chromatin degradation in both thymocytes and Jurkat T lymphocytes. (Top) Thymocytes (5 × 106 cells/mL) were incubated with 25 µM etoposide in the presence of various concentrations of PDTC (O). The copper chelator BCPS (100 µM) was also included in half of the samples (b). Cells were collected after 5 h, and the extent of apoptotic DNA fragmentation was quantitated using diphenylamine. For each PDTC concentration, a significant difference between samples incubated with or without BCPS is indicated by an asterisk (t-test, p < 0.01). (Bottom) Jurkat cells (5 × 106 cells/mL) were preincubated with various concentrations of PDTC or DSF for 30 min as indicated before exposure to 250 ng/mL anti-CD95 (Fas/APO-1) antibody. Cells were collected 3 h later, and chromatin fragmentation was analyzed by agarose gel electrophoresis. Table 1. Inhibition of Etoposide-Induced Thymocyte Chromatin Fragmentation by Both Reduced DCs and DC Disulfidesa treatment

% DNA fragmentation

untreated etoposide etoposide + 10 µM PDTC etoposide + 10 µM DEDC etoposide + 5 µM DSF

16 ( 1 67 ( 2 44 ( 1* 31 ( 10* 21 ( 1*

a Cells (5 × 106 mL-1) were incubated for 6 h in the presence of 25 µM etoposide together with 10 µM PDTC, 10 µM DEDC, or 5 µM DSF as indicated. The cells were then collected, and the degree of apoptotic DNA fragmentation was quantified using diphenylamine. Data values represent mean ( SD for triplicate samples, and an asterisk indicates a significant difference between DC-treated samples relative to the etoposide-only control (t-test, p < 0.05).

rkat T cells (Figure 1, bottom). Using an alternative MTT reduction assay of cell viability, the ED50 for PDTC inhibition of apoptosis was approximately 20 µM (data not shown). The inhibitory action of DCs on apoptosis has previously been attributed to their antioxidant functions (15-17). These are well established in vitro and include both direct radical scavenging and chelation of redox-active metals (21, 22). However, using whole

cells we have recently found that despite these antioxidant properties PDTC can actually induce a prooxidant state in thymocytes (18). We were therefore interested in investigating whether development of a more oxidized intracellular environment is also necessary for DCs to exert an inhibitory effect on T cell apoptosis. Reduced DCs Require Copper To Inhibit Apoptosis. PDTC is known to act as a copper ionophore: it transports extracellular copper across membranes so that the metal becomes concentrated inside the cells (18). Because we have found that copper can also oxidize both PDTC and DEDC to their corresponding disulfides,2 the potential clearly exists for DCs to become oxidized in a copper-dependent fashion during a cell culture experiment. We have previously shown that BCPS, a negatively charged metal chelator with a high affinity for copper, inhibits both copper uptake and subsequent oxidative stress in PDTC-treated thymocytes (18). The same reagent also completely prevented PDTC from inhibiting both etoposide-induced and spontaneous apoptotic DNA fragmentation of thymocytes (Figure 1, top, and data not shown). BCPS was also effective in preventing PDTC and DEDC from inhibiting anti-CD95 antibody-induced apoptosis of Jurkat cells (data not shown). DC Disulfides Are More Potent Inhibitors of Apoptosis than Their Reduced Counterparts. Since DCs can potentially be oxidized by Cu2+ ions naturally present in the FBS component of cell culture medium, we investigated whether DC disulfides themselves could also directly exert an inhibitory effect on lymphocyte apoptosis. Both DSF and thiram were effective inhibitors of etoposide-induced thymocyte apoptosis (Table 1 and Figure 2). On a molar basis, they were more potent inhibitors than either PDTC and DEDC, with an ED50 of less than 5 µM, against internucleosomal DNA fragmentation. DSF and thiram were also more potent inhibitors of CD95-mediated Jurkat T cell apoptosis than either of the reduced DCs (see Figure 1, bottom, data on thiram not shown). Thus, the antiapoptotic activities previously described for reduced DCs are also displayed when the compounds are oxidized to disulfides, with the latter being effective at lower concentrations. In contrast 2 Burkitt, M. J., Bishop, H. S., Milne, L., Tsang, S. Y., Provan, G. J., Nobel, C. S. I., Orrenius, S., and Slater, A. F. G. Manuscript in preparation.

Inhibition of Pro-caspase-3 Processing by DCs

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 639 Table 2. Effect of Diethyl Maleate and Anti-CD95 (Fas/ APO-1) Antibody on the GSH Content of Jurkat T Lymphocytesa

Figure 3. Intracellular ATP in short-term incubations is not depleted by dithiocarbamates. Jurkat T cells (5 × 106 cells/mL) were incubated at 37 °C in the presence of 50 µM PDTC (O), 50 µM DSF (0), or medium alone (b). Aliquots of cells were removed at various times, and their ATP content was determined using a luciferin/luciferase assay. Data points show mean ( SD (n ) 3).

to the reduced molecules, only a minor interference by BCPS on DSF inhibition of thymocyte apoptosis could be seen (data not shown). This latter result was most pronounced at very low DSF concentrations (less than 500 nM) and probably reflects some degree of extracellular (or intracellular) reduction of DSF, presumably by serum proteins like albumin. Taken together, these findings are compatible with DC disulfides being the active antiapoptotic molecules. They also indicate that copper is not the inhibitory agent but rather the mediator of DC oxidation. DCs Do Not Decrease Cellular ATP in Short Incubations. DCs are toxic to lymphocytes in long-term incubations, causing apoptosis at low concentrations (less than 50 µM) and necrosis when applied at higher doses (18). One important parameter determining whether a cell will undergo apoptosis or necrosis following a toxic insult is its energy status and, in particular, the availability of ATP (34, 35). Apoptosis is considered to be a high-energy-demanding phenomenon (35). For example, it has been shown that apoptotic chromatin condensation induced by CD95-activated caspases requires ATP (36). Given previous reports that DSF inhibits oxidative phosphorylation (37), we monitored ATP levels in Jurkat cells for 5 h after the addition of either 50 µM PDTC or 50 µM DSF. As seen in Figure 3, both compounds actually caused the intracellular ATP levels to increase slightly over the incubation period as compared to untreated cells. This finding shows that DCs and their respective disulfides do not deplete intracellular ATP, indicating that their inhibition of apoptotic processes is not indirectly mediated through a short-term depletion of cellular ATP. GSH Depletion Does Not Affect DC Disulfide Inhibition of Apoptosis. Another possible explanation for the antiapoptotic effects of DSF and thiram is that their disulfide is nonenzymatically reduced inside the cells. The resulting reduced DC would then be available to exert an antioxidant effect that inhibits apoptosis. The increased potency of the DC disulfides may simply be a reflection of their increased membrane permeability compared to the reduced molecule. To investigate this issue, we decreased the reductive power of cells by depleting GSH, a major cellular reductant, with diethyl maleate (DEM). Treating Jurkat T cells with 200 µM

DSF (µM)

Fas Ab

0 1 5 10

-

0 1 5 10

-

GSH

DSF (µM)

Fas Ab

GSH

0 µM Diethyl Maleate 36 ( 2 0 34 ( 2 1 40 ( 1* 5 39 ( 2 10

+ + + +

28 ( 3 25 ( 2 41 ( 3* 41 ( 12

200 µM Diethyl Maleate 15 ( 1 0 14 ( 1 1 17 ( 1* 5 18 ( 2 10

+ + + +

8(1 8(1 14 ( 1* 17 ( 2

a Cells (5 × 106 mL-1) were incubated in the presence or absence of 200 µM DEM for 30 min to deplete GSH and then washed into fresh cystine-free medium to prevent resynthesis of GSH. AntiCD95 (Fas/APO-1) antibody (250 ng mL-1) was then added as indicated, and the cells were cultured for another 2 h. Intracellular GSH was determined after monobromobimane derivatization (25) and is expressed as nmol mg of protein-1. Data values are mean ( SD (n ) 3), and significant differences relative to each 0 µM DSF control are indicated by an asterisk (t-test, p < 0.05).

Figure 4. DSF inhibition of anti-CD95 (Fas/APO-1) antibodyinduced chromatin fragmentation is not affected by depletion of intracellular GSH. Jurkat cells (5 × 106 cells/mL) were preincubated for 30 min with 0 µM (A) or 200 µM (B) DEM, washed into fresh cystine-free medium, and then cultured with various combinations of anti-CD95 (Fas/APO-1) antibody and DSF as indicated. The cells were collected after 3 h, and the extent of apoptotic DNA fragmentation was visualized after agarose gel electrophoresis.

DEM for 30 min, washing the cells to remove the oxidant, and then resuspending them in cystine-free RPMI to prevent resynthesis of GSH depleted intracellular GSH to approximately 40% of control levels (see Table 2). The extent of anti-CD95 antibody-induced cell death was similar in both normal and GSH-depleted cells (Figure 4). DSF also inhibited apoptosis in both cases, with, if anything, a somewhat better inhibition of apoptosis being seen in the GSH-depleted cells (Figure 4B, MTT data not shown). Examination of Table 2 also reveals that the GSH content of Jurkat T cells undergoing apoptosis decreased, which is consistent with our previous report (38). Significantly, apoptotic GSH depletion was reversed in the presence of DSF, independent of whether the background thiol content of the cells was normal (untreated) or low (DEM treated). Similar results were obtained by studying DSF inhibition of CD95-mediated apoptosis in Jurkat cells depleted of GSH by a 6 h incubation in cystine-free media (GSH depleted to 20% of controls, data not shown), excluding the possibility that

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Figure 5. DSF inhibition of processing of the caspase-3 proenzyme in Jurkat cells exposed to anti-CD95 (Fas/APO-1) antibody. Cells were preincubated for 1 h with the concentrations of DSF indicated, before initiating apoptosis by the addition of anti-CD95 (Fas/APO-1) antibody (500 ng/mL). Cells were collected after another 75 min, lyzed, and then subjected to SDS-PAGE. Proteins were blotted to nitrocellulose, and caspase-3 was detected using an antibody raised against the p17 subunit of the mature protease. Migration positions of the proform (32 kDa) and mature p17 subunit (17 kDa) of caspase-3 are indicated on the left side of the figure. Table 3. Induction of Caspase-3-like Proteolytic Activity in Response to Anti-CD95 (Fas/APO-1) Antibody Is Reduced when Jurkat T Lymphocytes Are Exposed to DSF 15 or 30 min after the Antibodya treatment untreated Fas Ab only Fas Ab + 10 µM DSF Fas Ab + 50 µM DSF Fas Ab + 10 µM DSF Fas Ab + 50 µM DSF

time of DSF addition (min)

caspase-3-like activity

15 15 30 30

1.15 18.03 6.48 1.70 9.23 2.13

a Jurkat cells (5 × 106 mL-1) were incubated with 500 ng mL-1 anti-CD95 (Fas/APO-1) antibody. DSF (10 or 50 µM) was added 15 or 30 min later where indicated. Caspase-3 activity was assayed 1 h after addition of the antibody, using DEVD-AMC as a synthetic substrate. Enzyme activity is expressed as pmol of AMC cleaved min-1 106 cells (average rates from triplicate assays are shown).

our finding was influenced by the method chosen to deplete GSH. These experiments indicate that DSF is not dependent on intracellular GSH to inhibit apoptosis transduced through the CD95 receptor and therefore probably does not need to be reduced inside cells to exert its antiapoptotic action. Consistent with our previous report (18), DSF was also observed to elevate the intracellular GSSG content of Jurkat T cells (2-fold by 30 min, data not shown). GSSG is known to be a physiological regulator of protein function through its ability to form mixed disulfides with protein thiols (39). This therefore represents another potential mechanism by which DSF could alter cellular function. We observed that exposure to DEM also depleted intracellular GSSG, so that the absolute GSSG concentration in cells treated with DEM plus DSF was lower than in untreated cells undergoing normal apoptosis (data not shown). However, as already described, no decrease in the ability of DSF to inhibit CD95 apoptosis could be detected following DEM treatment (see Figure 4). This permits the additional conclusion that DSF does not require an increase in intracellular GSSG levels to inhibit T cell apoptosis. DC Disulfides Inhibit Caspase Activation. One of the proteases known to be centrally involved in CD95transduced apoptosis is pro-caspase-3 (8). This protease is proteolytically processed from two 32 kDa proforms to an active tetramer consisting of two p17 and two p12

Figure 6. DSF inhibition of processing of pro-caspase-3 in Jurkat cytosols induced with cytochrome c and dATP. S-100 cytosol was prepared from 4.5 × 108 Jurkat cells as described (22). Processing of the caspase-3 proenzyme was initiated by the addition of cytochrome c (400 nM) and dATP (0.9 mM), and reactions were terminated after 30 min. DSF (1, 10, 50, or 100 µM) and DTT (1 mM) were included in the incubations where indicated. At the end of the reaction, the samples were divided into two aliquots: one for direct analysis of pro-caspase-3 processing by Western blot and the other for measurement of DEVD-AMC cleaving activity (see the legend to Figure 5 and Experimental Procedures for details). Enzyme activity in each sample is presented beneath its respective lane on the gel and is expressed as pmol of AMC min-1 mg of protein-1 (average from triplicate samples).

subunits (2). Processing of the 32 kDa proform is undertaken in vitro by other caspases (4, 5), as well as it involves some steps of autocatalysis (4). Since this processing event is thiol-dependent, we decided to investigate whether it was sensitive to oxidative inhibition by DC disulfides. DSF was found to exert a pronounced concentrationdependent inhibition of p17 subunit accumulation after Jurkat cells were exposed to anti-CD95 antibody (Figure 5). This inhibition occurred at DSF concentrations very similar to those needed to affect other apoptotic parameters, such as chromatin fragmentation. Inhibition of pro-caspase-3 processing was also associated with a decrease in the total amount of DEVD-AMC cleaving activity recoverable in the cells (Table 3). Pro-caspase-3 processing and eventual activity were both significantly inhibited even when DSF was added 15 or 30 min after the antibody (Table 3 and data not shown), eliminating the possibility that the DC disulfide somehow directly interfered with the antibody-receptor interaction. Because CD95-stimulated apoptosis of Jurkat cells requires the active participation of mature caspase-3 (8), these results suggest that DC disulfides exert some of their inhibitory effect by blocking the proteolytic activation of this enzyme. To investigate this issue further, we used a recently developed in vitro model of pro-caspase-3 activation consisting of organelle-free S-100 cytosolic extracts from untreated Jurkat cells activated by the addition of cytochrome c and dATP (27). When pro-caspase-3 processing was initiated in this way, a clear dose-dependent inhibitory effect of DSF was observed. This was evidenced by a reduced accumulation of processed p17 polypeptide and by the presence of less caspase-3-like proteolytic activity at the end of the experiment (Figure 6). DSF was again active in a concentration range resembling that required to inhibit whole cell apoptosis (1-50 µM). Significantly, the inhibitory effect of DSF was abolished if an excess of DTT was included in the incubation (Figure 6), while in additional experiments 100 µM PDTC was found to exert no inhibitory effect on

Inhibition of Pro-caspase-3 Processing by DCs

Figure 7. Cytochrome c/dATP-induced in vitro activation of pro-caspase-3 influenced by the redox state of GSH. Processing of the caspase-3 proenzyme was initiated in S-100 Jurkat cytosols by the addition of cytochrome c and dATP, and samples were taken and assayed as in Figure 6. The reaction buffer also included various concentrations of GSH and GSSG as indicated. Concentrations are expressed as GSH:GSSG ratios with the added total GSH + GSSG concentration always being 5 mM. The S-100 cytosols contained around 200 µM GSH and 2 µM GSSG on their own as determined by HPLC. For comparison 10 mM DTT was included in one sample. Enzyme activity in each sample is presented beneath its respective lane on the gel and is expressed as pmol of AMC min-1 mg of protein-1 (average from triplicate samples).

pro-caspase-3 processing (data not shown). Both observations are consistent with the oxidized DC disulfide being the inhibitory entity. Glutathione Regulation of Caspase-3 Processing. The potent effects of DSF on pro-caspase-3 activation raised the possibility that other disulfides may induce similar inhibitory effects. We therefore investigated whether the physiological disulfide GSSG would also inhibit caspase-3 activation. When a 5 mM glutathione redox buffer was included in the cytochrome c/dATP activation system, normal pro-caspase-3 activation occurred at 5 mM GSH. As the redox buffer shifted to a more oxidized state, however, protease activation was progressively supressed (Figure 7). Thus, an important physiological disulfide exerts inhibitory effects on caspase-3 proenzyme processing similar to the disulfide toxicant DSF, illustrating the potential for this event to be endogenously regulated by thiol redox.

Discussion Given the potent antioxidant actions possessed by DCs in vitro (21, 22), until now it has been assumed that one or more of these activities explain the observed inhibitory effect of DCs on apoptotic cell death (15-17). However, the corresponding disulfides of these compounds are known to induce two-electron oxidation of reduced thiols. In the presence of hydrogen peroxide and GSH, for example, DCs redox cycle such that GSSG formation is promoted (i.e., they mimic the action of GSH peroxidase, see ref 23), while due to their copper-chelating activity they can also act as ionophores to drive the accumulation of the metal into cells (18, 40, 41). We have recently found that unlike copper-GSH complexes (42), the corresponding copper-DC complex can slowly redox cycle.2 The DC disulfides formed during this cycle are potent thiol-oxidizing agents of both GSH and protein thiols (23, 48). Thus although DCs are one-electron radical-scavenging antioxidants, they can also promote thiol oxidation. Both actions must be considered when trying to understand the biological effects of these compounds. Three lines of evidence lead us to conclude that the prooxidant effects of DCs are responsible for their anti-

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apoptotic action. First, and most significant, we observed that although chelation of external copper removed the antiapoptotic effect of reduced DCs (Figure 1, top), it had little effect on the inhibitory activity of DC disulfides. This implies that a copper-dependent oxidation was essential if the reduced DCs were to be inhibitory. Second, the oxidized DC disulfides were more potent inhibitors of apoptosis than their reduced counterparts (ED50 values 5-10-fold lower). Third, depleting intracellular GSH (the major cellular thiol reductant) had no effect on the ability of DC disulfides to inhibit CD95mediated apoptosis (Figure 4). Although GSH depletion was incomplete, this finding is inconsistent with an intracellular reduction of DC disulfides being required for their antiapoptotic effects. T cell apoptosis is known to require the proteolytic activation of caspase proenzymes like pro-caspase-3 (1, 2, 8). Since activation of these apoptosis-specific proteases themselves requires caspase-like activity (4, 5), this event is vulnerable to interference by thiol oxidants. In Jurkat cells treated with anti-CD95 antibody, we observed that DSF inhibited processing of the procaspase-3 at the same concentrations as it blocked apoptosis (Figure 5), suggesting action of the DC disulfide at this step. Results from a cell-free model system of procaspase-3 activation provided further support for this conclusion (Figure 6). DSF was observed to exert a concentration-dependent inhibition on proenzyme activation, which was reversed when DTT was included in the reaction buffer as a thiol reductant. PDTC, however, was without activity under these conditions (all buffers were nominally copper-free). Interestingly, in the presence of DTT, generation of the active p17 fragment of caspase-3 actually appeared to be enhanced (see Figure 6, lanes with DTT), hinting at the possiblity of some endogenous disulfide regulation of the process. The most likely explanation for the inhibition of procaspase-3 processing by DC disulfides is that by forming a mixed disulfide with one or more cysteine residues on the putative caspase that cleaves pro-caspase-3, processing is inhibited. This residue may be an active site cysteine nucleophile of the protease (which should be very redox active), or it could be another redox-sensitive thiol on the protein. However, the nature of this putative caspase is so far unknown. Clues about an upstream caspase in the different systems used in this study come from CD95 apoptosis, where caspase-8 has been proposed to be upstream of caspase-3 (6, 7). Interestingly, caspase-8 has recently, along with two other caspases (caspase-6 and caspase-10), been shown to cleave pro-caspase-3 in vitro (4, 5). However, the physiological relevance of this observation remains uncertain. Neither of the abovementioned caspases has so far been shown to be involved in the cytochrome c/dATP in vitro activation model. On the contrary, both caspase-8 and caspase-10 are unlikely to be involved, since they require ligation to receptors like CD95 to be activated (6, 7). Except for the requirement for cytochrome c, dATP, and an unidentified cytosolic factor, nothing is so far known about the activator(s) of pro-caspase-3 in this system (27). Whether any of the three putative pro-caspase-3 activators, like caspase-6, caspase-8, and caspase-10, are targets for DSF, or/and if it is another yet uncharacterized caspase(s), awaits further studies. The possibility of an endogenous disulfide, such as GSSG, to exert an inhibitory effect on pro-caspase-3 processing is illustrated by our results in Figure 7. This

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would mean that a cellular stress which increases intracellular levels of GSSG would have the potential to effect the induction of the apoptotic program via modulation of the caspase cascade. However, in the case of DSF, this seems very unlikely judging from the higher inhibitory power of DSF (DSF being 100-1000-fold more active than GSSG on a molar basis in blocking pro-caspase-3 activation) and the fact that depleting GSSG did not affect DSF inhibition. It thus appears that activation of this cascade requires the intracellular thiol redox state to be predominantly reducing. This may explain the frequent observation in toxicology that necrosis and apoptosis are mutually exclusive. A correct redox state is therefore probably crucial for successful apoptosis. Consistent with this scheme, the superoxide anion has recently been proposed to be inhibitory for CD95-mediated apoptosis in a human melanoma cell line (25). It is also noteworthy that although oxidants, such as hydrogen peroxide, are known inducers of apoptosis, cell death typically occurs many hours after the oxidant has decayed (43-46). We argue from this that although oxidative stress is a potent inducer of apoptosis, the event itself requires that the cell exists in a reduced state during the time in which the caspases cleave their substrates. The eventual accumulation of peroxidized products presumably reflects a secondary loss of antioxidant capacity in the terminal apoptotic cell remnant (38, 47). Finally, the paradoxical observation that DCs inhibit apoptosis in the short term (15-17, this paper) yet induce the same event after longer incubation (18) also requires comment. DCs are extensively metabolized via several different pathways in cells, generating CS2 and various isothiocyanates, sulfoxides, and sulfones (see refs 4851). We propose that although the DC disulfides themselves are antiapoptotic by virtue of their interaction with caspases, with time this effect is lost as the inhibitor is metabolized. The resulting release of copper exacerbates the oxidative stress already being experienced by the cells, and engagement of the apoptotic pathway (now free from DC disulfide inhibition) results in cell death.

Nobel et al.

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Acknowledgment. We are grateful to Dr. Donald Nicholson (Merck Frost Centre for Therapeutic Research, Canada) for his gift of the anti-p17 antibody and to Dr. Arne Lundin (Biotema AB, Sweden) for his help with the ATP measurements. Anna Carin Hellerqvist is greatly appreciated for her help with the manuscript. This work was supported by grants from the Karolinska Institutet and the Swedish Medical Research Council (Project No. 03X-2471).

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