Selenite negatively regulates caspase-3 through a redox mechanism.

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.

Selenium, an essential trace element in the biological system, has been shown to modulate the functions of a variety of intracellular proteins (1)(2)(3)(4). Selenium exerts many of its functions by being incorporated into the proteins as selenocysteine or selenomethionine (5)(6)(7). The selenoproteins include glutathione peroxidases, formate dehydrogenases, and glycine reductases (5). Selenium can also regulate the functions of many proteins through the oxidation of reactive cysteine residues in the proteins (1)(2)(3)8). Those proteins that can be regulated by selenium through a redox mechanism include Na,K-ATPase, a glucocorticoid receptor, prostaglandin synthase, nuclear factor B and the transcription factor adaptor protein-1 (1-3, 9 -11).
Thus, selenium appears to regulate functions of various proteins, many of which are associated with intracellular signal transmission in diverse cellular events (3,12,13). There are many lines of evidence pointing to selenite and other selenium compounds as playing critical roles in the protection of cells against cell death initiated by various stresses such as ultraviolet (UV) 1 and ionizing irradiation (14 -17). A regulatory mechanism for the anti-death effect of selenite and other selenium compounds, however, remains to be found.
Apoptosis is a cell suicide process with characteristic morphological features that include nuclear membrane breakdown, chromatin condensation and fragmentation, cell membrane blebbing, and the formation of apoptotic bodies (18,19). Many apoptotic stimuli such as tumor necrosis factor-␣, the Fas ligand, UV light, and chemotherapeutic agents have been shown to induce cell death by activating caspases (18). Caspases are a family of cysteine proteases that are major components of the cellular apoptotic machinery. Caspases cleave a number of intracellular proteins after aspartic acid residues. These proteins include lamins, poly(ADP-ribose) polymerase (PARP), fodrin, gelsolin, p21-activated kinase 2, protein kinase C, MEKK1, amyloid ␤-precursor protein, and presenilins (20 -30). It has recently been shown that caspase-3, in particular, is a key player in the DNA fragmentation process and other morphological changes associated with apoptosis (31)(32)(33).
In the present study, we investigated the effect of selenite on UVB-induced cell death, and we focused on caspase-3 activation to understand the mechanism for the anti-death function of selenite. We demonstrate in this report that selenite inhibits both cell death and caspase-3 activation induced by UVB. Selenite also suppresses the catalytic activity of caspase-3 and caspase-3-induced DNA fragmentation in vitro. Furthermore, we provide evidence that the inhibitory action of selenite on caspase-3 occurs through a redox mechanism.

EXPERIMENTAL PROCEDURES
Cell Culture and Viability Assay-Human embryonic kidney 293 (HEK293) cells were routinely maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. When indicated, cells in culture were exposed to chemicals or to ultraviolet B * This work was supported by the Creative Research Initiatives Program of the Korean Ministry of Science and Technology (to E.-J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Preparation of S-100 and Nuclei Fractions from HEK293 Cells-To prepare the S-100 fraction, HEK293 cells were resuspended in a ice-cold buffer containing 20 mM HEPES, pH 7.4, 10 mM KCl, 2 mM MgCl 2 , 1 mM EDTA, 4 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, and 2 g/ml aprotinin. They were then homogenized on ice using a Dounce homogenizer. The cell lysates were subjected to centrifugation at 1000 ϫ g for 10 min at 4°C to remove nuclei and unbroken cells. The soluble fraction was further centrifuged at 100,000 ϫ g for 1 h at 4°C. The resultant soluble fraction (S-100 fraction) was immediately frozen in liquid nitrogen and stored at Ϫ70°C until used for in vitro DNA fragmentation experiments. To prepare the nuclei fraction, HEK293 cells were resuspended in buffer A containing 15 mM HEPES, pH 7.4, 80 mM KCl, 15 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 25 mM sucrose and allowed to swell on ice for 20 min. They were then homogenized with a Dounce homogenizer, and the homogenates were subjected to centrifugation at 1000 ϫ g for 10 min at 4°C. The resultant soluble fraction was layered over buffer A containing 2.3 mM sucrose in a Beckman SW28 centrifuge tube and centrifuged at 22,000 rpm for 90 min at 4°C. The pellet containing nuclei was resuspended in buffer A and immediately used in in vitro DNA fragmentation experiments.
Measurement of Caspase-3 Activity-Cultured cells were lysed with a lysis buffer containing 50 mM Tris, pH 7.4, 1 mM EDTA, 10 mM EGTA, and 10 M digitonin. The soluble fraction of the cell lysate was then assayed for caspase-3 activity using Ac-DEVD-pNA (CLONTECH), a colorimetric substrate for caspase-3, as described in the manufacturer's protocol.
DNA Fragmentation-Cells were exposed to indicated agents, harvested, and lysed in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM ␤-glycerophosphate, 10 mM sodium fluoride, and 5 mM EGTA. Nuclear DNA was extracted from the cell lysates as described previously (34). Fragmented DNA samples were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. In the in vitro DNA fragmentation assay, recombinant human caspase-3 (100 ng, Pharmingen) was added to 80 l of reaction buffer (10 mM HEPES, pH 7.4, 40 mM ␤-glycerophosphate, 50 mM NaCl, 2 mM MgCl 2 , 4 mM EGTA, 2 mM ATP, 10 mM creatine phosphate, 50 g/ml creatine kinase, and 0.2 mg/ml bovine serum albumin) and then incubated with 200 l of the S-100 fraction (ϳ1 mg/ml) and 10 l of the nuclei fraction (ϳ2 ϫ 10 8 nuclei) at 37°C for 140 min. The DNA was extracted and analyzed for fragmentation as described above.
In Vitro Cleavage of GST-ICAD L by Caspase-3-Recombinant GST-ICAD L fusion protein was incubated at 37°C for 1 h with 100 ng of active caspase-3 (Pharmingen) in a reaction buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 20% glycerol. The reaction mixture was then subjected to SDS-polyacrylamide gel electrophoresis through a 12% polyacrylamide gel, and cleaved GST-ICAD L was visualized by Coomassie Brilliant Blue staining. The GST-ICAD L was bacterially expressed using pGEX-4T (Amersham Pharmacia Biotech) and purified on glutathione-agarose.
Immunoblot Analysis for PARP Cleavage-HEK293 cells were harvested and broken using a 26-gauge syringe in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, and 2 g/ml aprotinin. The cell lysate was subjected to centrifugation at 12,000 ϫ g for 10 min at 4°C. The soluble fraction was subjected to SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gel. Twenty micrograms of the soluble fraction was loaded/lane. The separated proteins were then electroblotted onto a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech). Immunoanalysis was performed with a mouse anti-PARP monoclonal antibody (C-2-10, Roche Molecular Biochemicals) and a horseradish peroxidase-conjugated goat anti-mouse IgG antibody. The cleaved PARP was visualized using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).

Selenite Can Suppress the Cell Death Induced by UVB-
Initially we investigated a possible role for selenite during cell death induced by UVB radiation. Exposure of HEK293 cells to 160 J/m 2 UVB resulted in 80% cell death within 24 h (Fig. 1A). The UVB-induced cell death was markedly prevented by pretreatment of the cells with 100 nM sodium selenite. We also found that exposure of HEK293 cells to UVB resulted in apop-totic DNA fragmentation and that selenite prevented this UVB-induced DNA fragmentation (Fig. 1B). We then looked for possible mechanism by which selenite might suppress cell death induced by UVB irradiation.
UVB-induced Cell Death Is Dependent upon Caspase-3-like Protease Activity-Exposure of cells to UVB has been shown to induce activation of the proapoptotic proteins including caspases (35)(36)(37). Indeed, the enzymatic activity of a caspase-3-like protease was markedly enhanced after HEK293 cells had been exposed to UVB (Fig. 2A). We, therefore, tested whether caspase-3 was involved in the UV-induced cell death. HEK293 cells were pretreated with either a relatively general caspase inhibitor, z-VAD, or a caspase-3 inhibitor, z-DEVD, and then exposed to UVB. We then determined cell viability by trypan blue staining (Fig. 2B). UV-induced cell death was prevented efficiently by z-DEVD but not by z-VAD. These data suggest that a caspase-3-like protease is associated with a mechanism that leads to UVB-induced cell death in HEK293 cells.
Selenite Inhibits Caspase-3-like Protease Activity-To further investigate the suppressive effect of selenite on UVBinduced cell death, we examined whether selenite would suppress the UVB-stimulated caspase-3 activity. As expected, exposure of cells to UVB led to enhanced caspase-3-like protease activity in the cell lysate (Fig. 3). Interestingly, treatment of the lysate of UVB-exposed cells with selenite resulted in a decrease in the caspase-3-like protease activity in these cell lysates (Fig. 3). The in vitro inhibitory action of selenite on the caspase-3-like protease activity thus suggests that selenite may modify caspase-3 activity by directly acting on the enzyme. In fact, it has been shown previously that selenite can modulate the functions of various proteins by directly acting on reactive cysteine residues of these proteins (26, 27, 29, 36 -38).
Dithiothreitol and ␤-Mercaptoethanol Reverse the Effect of Selenite on Caspase-3 Activity-If selenite suppresses the catalytic activity of caspase-3 through modifying critical cysteine residues on the enzyme, the action of selenite on caspase-3 could be reversible by sulfhydryl-reducing agents. We tested this possibility using reducing agents such as dithiothreitol (DTT), ␤-mercaptoethanol, and glutathione (Fig. 4). In vitro treatment of purified recombinant caspase-3 with 100 nM selenite resulted in dramatic inhibition of the enzymatic activity (Fig. 4A). Furthermore, DTT and ␤-mercaptoethanol reversed the inhibitory action of selenite on caspase-3. Glutathione did not affect the inhibitory action of selenite.
We also examined the effect of selenite on the in vitro cleavage of ICAD L by caspase-3. ICAD L is an inhibitor of caspase-activated DNase, an enzyme responsible for the induction of DNA fragmentation during apoptotic cell death. Caspase-3 cleaved ICAD L in vitro, and this cleavage was blocked by selenite (Fig. 4B). Inhibition of the ICAD L cleavage by selenite was reversed by DTT and ␤-mercaptoethanol. Taken together, these data strongly suggest that selenite suppresses enzymatic activity of caspase-3 through oxidizing critical sulfhydryl groups on the enzyme and that the reducing agents DTT and ␤-mercaptoethanol can reverse the selenite-induced oxidation of the redox-sensitive cysteine residues of caspase-3. We also examined the effect of selenite on in vitro DNA fragmentation stimulated by caspase-3 (Fig. 4C). For the in vitro DNA fragmentation experiment, intact nuclei and a cytosolic S-100 fraction were prepared from HEK293 cells. Incubation of the S-100 fraction and the isolated nuclei with caspase-3 resulted in DNA fragmentation. The caspase-3-dependent DNA fragmentation was suppressed in the presence of selenite, and the inhibitory effect of selenite was reversible by either DTT or ␤-mercaptoethanol. Other thiol reactive agents such as N-ethylmaleimide, azodicarboxylic acid bisdimethylamide, and o-iodosobenzoate also repressed the caspase-3-dependent DNA fragmentation (Fig. 4C).
Selenite Represses Intracellular Caspase-3-like Protease Activity in Intact Cells-We examined whether selenite could inhibit intracellular caspase-3-like protease activity in intact cells. Exposure of HEK293 cells to UVB-stimulated caspase-3like protease activity and pretreatment of the cells with 100 nM selenite resulted in suppression of the UVB-stimulated caspase-3-like protease activity (Fig. 5A). The inhibitory effect of selenite on the intracellular caspase-3-like protease activity was attenuated by DTT and ␤-mercaptoethanol but not by glutathione.
One physiological substrate for caspase-3 is PARP, a 116-kDa protein, which can be cleaved into 85-and 31-kDa fragments during apoptotic cell death (38). We examined whether selenite could suppress PARP cleavage by caspase-3-like protease in vivo. PARP cleavage was detected after HEK293 cells were exposed to UVB radiation (Fig. 5B). Pretreatment of cells

FIG. 2. UVB-induced cell death depends on caspase-3-like protease activity.
A, HEK293 cells were exposed to 160 J/m 2 UVB radiation and then incubated for the indicated times at 37°C. Cells were harvested, lysed, and assayed for caspase-3-like protease activity using a colorimetric substrate, Ac-DEVD-pNA. B, HEK293 cells were pretreated with 200 M z-VAD-fmk or z-DEVD-fmk for 12 h and then exposed to 160 J/m 2 UVB radiation. After the UVB treatment, cells were incubated further for 24 h at 37°C, harvested, and examined for cell viability by trypan blue exclusion. Asterisks indicate statistically significant difference between the two values (p Ͻ 0.01).

FIG. 3. Selenite inhibits caspase-3-like protease activity in cell
lysates of UVB-exposed HEK293 cells. HEK293 cells were exposed to 160 J/m 2 UVB radiation and then incubated for 8 h at 37°C. Cell lysates prepared from the cultured cells were then incubated with indicated concentrations of sodium selenite for 2 h on ice and analyzed for caspase-3-like protease activity using Ac-DEVD-pNA. Asterisks denote statistically significant changes from the value of control (ϩUVB alone) (p Ͻ 0.01).
with 100 nM selenite completely blocked the UVB-induced PARP cleavage. Our data thus suggest that selenite inhibited caspase-3-like protease activity in the intact cells. DISCUSSION In the present study, we show that exposure of cells to UVB induces the activation of a caspase-3-like protease activity as well as cell death, whereas z-DEVD, a caspase-3 inhibitor, can effectively block the UVB-induced cell death. These observations suggest that a caspase-3-like protease is a major component of the cellular machinery leading to UVB-induced cell death. Participation of the caspase-3-like protease in UVBinduced cell death had been demonstrated previously (36,37). Our data indicate that selenite treatment suppresses both cell death and DNA fragmentation induced by UVB radiation. Fur-thermore, selenite also prevented the stimulation of the caspase-3-like protease activity and PARP cleavage that occurs in UVB-exposed cells. PARP is one of the well studied physiological substrates of caspase-3 (38). Our findings suggest that inhibition of the caspase-3-like protease activity may be a major mechanism by which selenite treatment protects HEK293 cells from UVB-induced cell death.
Caspases are cysteine proteases that contain cysteine in their catalytic domain (39). Site-directed mutagenesis studies have shown that replacement of cysteine in the active site with other amino acid abolishes the catalytic activity of caspases (40). Furthermore, the enzymatic activity of caspase-3 was lost when reactive thiol groups on the enzyme were modified chemically (41). It is interesting in this context that selenite is capable of oxidizing thiol groups on proteins (1)(2)(3)8). We proposed, therefore, that at least one mechanism by which selenite treatment can prevent the caspase-3-like protease activation is the oxidation of critical cysteine residues on the caspase-3-like protease. This hypothesis is consistent with our data using thiol reducing agents, DTT and ␤-mercaptoethanol. Both DTT and ␤-mercaptoethanol prevented the inhibitory effect of selenite on caspase-3 activity in cleaving the substrate, Ac-DEVD-pNA. The reducing agents also reversed the suppression by selenite of caspase-3-induced in vitro DNA fragmentation.
Internucleosomal DNA fragmentation, which has been long considered a biochemical hallmark of apoptosis, appears to occur at a late stage in many of the apoptotic events. There are accumulating lines of evidence that caspases play a pivotal role in the activation of nuclear DNA fragmentation (19,42). Caspases, especially caspase-3, have been shown to activate a  5. Selenite represses caspase-3-like protease activity and PARP cleavage in UVB-exposed HEK293 cells. HEK293 cells were pretreated with 100 nM sodium selenite for 48 h, exposed to 160 J/m 2 UVB light, and then incubated in complete medium for 8 h at 37°C. A, cell lysates (100 g) were treated with 10 mM DTT, 10 mM ␤-mercaptoethanol (␤-ME), or 10 mM glutathione (GSH), respectively and then assayed for caspase-3-like protease activity using Ac-DEVD-pNA. Asterisks denote statistically significant changes from the value of control (ϩUVB alone) (p Ͻ 0.01). B, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis on a 12% polyacrylamide gel and analyzed for PARP cleavage by immunoblot probed with a mouse anti-PARP monoclonal antibody. caspase-activated DNase (CAD/DFF40) through cleaving its inhibitor (ICAD/DFF45) (24,(43)(44)(45)(46). In our study, exposure of cells to UVB resulted in the stimulation of internucleosomal DNA fragmentation, and the UVB-induced DNA fragmentation could be prevented by pretreatment of cells with z-DEVD (data not shown). This suggests that a caspase-3-like protease is associated with the mechanism that operates in the UVBinduced DNA fragmentation. Recombinant active caspase-3 protein also enhanced the in vitro DNA fragmentation in isolated nuclei of HEK293 cells. Pretreatment of caspase-3 with selenite resulted in suppression of the in vitro DNA fragmentation activated by caspase-3. These data suggest that inhibition of the caspase-3-like protease activity may be an important mechanism by which selenite represses the UVB-induced nuclear DNA fragmentation.
Selenite and other selenium compounds modulate a variety of cellular activities including cell survival and death (47)(48)(49). Our findings in this study that selenite suppresses caspase-3like protease activity through a redox mechanism may contribute significantly to a better understanding of the mechanism by which selenite participates in functions of cell survival and cell death. However, whether selenite also modulates other types of caspases besides caspase-3 with this kind of mechanism still needs to be investigated.