Role of Glutathione S-Transferases in Protection against Lipid Peroxidation

OVEREXPRESSION OF hGSTA2-2 IN K562 CELLS PROTECTS AGAINST HYDROGEN PEROXIDE-INDUCED APOPTOSIS AND INHIBITS JNK AND CASPASE 3 ACTIVATION*

Yusong YangDagger , Ji-Zhong ChengDagger , Sharad S. Singhal§, Manjit SainiDagger , Utpal PandyaDagger , Sanjay Awasthi§, and Yogesh C. AwasthiDagger

From the Dagger  Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555 and the § Department of Chemistry and Biochemistry, University of Texas, Arlington, Texas 76019

Received for publication, January 19, 2001, and in revised form, March 5, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H2O2 cytotoxicity. Treatment with 30 µM H2O2 for 48 h or 40 µM PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H2O2 treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H2O2 suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.


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INTRODUCTION
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Treatment with hydrogen peroxide or the agents that lead to oxidative stress due to the generation of reactive oxygen species (ROS)1 is known to cause apoptosis in a variety of cell lines of different origin (1-10). 4-Hydroxy-2-nonenal (4-HNE), a stable end product of lipid peroxidation (LPO), has also been shown to cause apoptosis in a variety of cell lines (11-15). Although the mechanisms of H2O2- and 4-HNE-induced apoptosis are not completely understood, there appear to be some common features associated with the apoptotic signaling pathways during H2O2- and 4-HNE-induced apoptosis. For example, activation of the stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK), increased phosphorylation of c-Jun, activation of caspase 3 (CPP32) and degradation of poly(ADP-ribose) polymerase (PARP) have been shown to be associated with apoptosis induced by both H2O2 (7, 9, 16) and 4-HNE (11, 13). These observations raise obvious questions about the possible involvement of LPO products in the mechanisms of apoptosis and suggest the possibility that LPO products generated during oxidative stress may be involved in the signaling mechanisms of H2O2-induced apoptosis.

In aerobic organisms, ROS are continually generated, and during oxidative stress caused by stimuli such as infection and exposure to xenobiotics, their overproduction causes various deleterious effects including increased LPO. In order to protect against these harmful ROS, aerobic organisms have developed a number of cellular defenses (17). The antioxidant defense system may be considered to be composed of non-enzymatic and enzymatic components. The low molecular weight antioxidants including vitamins A and E, ascorbate, urate, and GSH comprise the non-enzymatic component, although the antioxidant enzymes including superoxide dismutases (SOD), catalase (CAT), and glutathione peroxidases (GPxs) comprise the enzymatic component. Among the enzymatic mechanisms, GPxs (EC 1.11.1.9), which provide defense against ROS-mediated LPO, play an important role in protection mechanisms against oxidative stress. At least four selenium-dependent GPx isoenzymes designated as cellular GPx (GPx-1) (18), gastrointestinal GPx (GPx-2) (19), plasma GPx (GPx-3) (20, 21), and phospholipid hydroperoxide GPx (GPx-4) (22, 23) have been characterized in mammalian tissues. Among these isoenzymes, only GPx-4, which is associated with membranes, can use phospholipid hydroperoxides (PL-OOH) as substrates, and the in situ reduction of PL-OOH in biological membranes by GPx-4 has been demonstrated (24, 25).

In addition to the selenium-dependent GPx activities, mammalian cells also have selenium-independent GPx activity displayed by glutathione S-transferases (GSTs, EC 2.5.1.18). GSTs, which belong to a supergene family of phase II detoxification enzymes, are involved in the conjugation of a wide range of electrophilic xenobiotics, including carcinogens and mutagens, to the endogenous nucleophile GSH (26-28). At least six gene families coding for the Alpha, Mu, Pi, Theta, Kappa, and Zeta class GSTs have been reported in humans (27, 29, 30). The selenium-independent GPx activity of GSTs toward organic hydroperoxides was first characterized in rat liver (31), and in humans, this activity was shown to be predominantly expressed by the cationic GSTs (32), which belong to the Alpha class (33). In human liver, the cationic Alpha class GST isoenzymes2 hGSTA1-1 and hGSTA2-2 account for the bulk of GST proteins (34). Both GSTA1-1 and GSTA2-2 can utilize fatty acid hydroperoxides (FA-OOH) as well as PL-OOH as substrates (35). However, neither the GSH-dependent reduction of PL-OOH by GSTs in biological membranes in situ has been demonstrated nor has the physiological significance of the GPx activities of hGSTA1-1 and hGSTA2-2 been systematically investigated. A subgroup of the Alpha class GSTs having substrate preference for 4-HNE is also present in mammals including humans (36-39). The physiological role of these GST isoenzymes is also not clear. Therefore, the present studies were designed to elucidate the role of the Alpha class GSTs in the protective mechanism against LPO. In the studies presented in this communication, we have first assessed the capability of recombinant hGSTA1-1 and hGSTA2-2 for GSH-dependent reduction of PL-OOH (GPx activity) in biological membranes in situ, and we examined the effect of overexpression of hGSTA2-2 in human erythroleukemia cells (K562) on LPO during oxidative stress. We then examined the role of hGSTA2-2 overexpression in the mechanisms of H2O2-induced apoptosis in K562 cells. Results of these studies demonstrate for the first time that hGSTA1-1 and hGSTA2-2 can reduce PL-OOH in the biological membranes in situ and that the overexpression of hGSTA2-2 protects K562 cells from H2O2-induced LPO and cytotoxicity. More importantly, our results show that the transfection of K562 cells with hGSTA2 attenuates H2O2-induced apoptosis by suppressing SAPK/JNK activation and caspase 3-mediated PARP cleavage.

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Materials-- Epoxy-activated Sepharose 6B, GSH, 1-chloro-2,4-dintrobenzene (CDNB), cumene hydroperoxide (CU-OOH), linoleic acid, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma. 4-HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI). Dilinoleoylphosphatidylcholine hydroperoxide (PC-OOH), Dilinoleoylphosphatidylethanolamine hydroperoxide (PE-OOH), 9-hydroperoxylinoleic acid (9-LOOH), and 13-hydroperoxylinoleic acid (13-LOOH) were synthesized as described previously (40). Lipid hydroperoxides and 4-HNE were stored at -70 °C under nitrogen atmosphere. Ampholines and other supplies for isoelectric focusing were obtained from Amersham Pharmacia Biotech. All reagents for SDS-PAGE and Western transfer were purchased from Bio-Rad. The polyclonal antibodies raised against the Alpha, Mu, and Pi class GSTs were the same as those used in our previous studies (40). Human tissue samples were obtained from the autopsy service at the University of Texas Medical Branch with no diagnosed disorders related to investigated organs, and blood was obtained from the University of Texas Medical Branch blood bank. The use of human tissues in the present studies was approved by the Institutional Review Board of the University of Texas Medical Branch.

Purification of GSTs-- In order to obtain recombinant GSTs, the cDNAs for hGSTA1 and hGSTA2 were cloned into the pET30a(+) (Novagen, Madison, WI) as described previously (35). The pET expression constructs were transformed into Escherichia coli BL21(pLysS) (Stratagene, La Jolla, CA) and cultured in LB medium containing 50 µg/ml kanamycin. When the cultures reached an absorbance of 0.6 at 600 nm, isopropyl-1-thio-beta -D-galactopyranoside was added at a final concentration of 500 µM to induce the expression. The cells were cultured overnight at 30 °C in the presence of isopropyl-1-thio-beta -D-galactopyranoside. The BL21 cells were lysed by sonication in 10 mM potassium phosphate buffer, pH 7.0, containing 1.4 mM beta -mercaptoethanol (buffer A) and centrifuged for 45 min at 28,000 × g at 4 °C. The supernatants were collected and subjected to affinity chromatography using GSH linked to epoxy-activated Sepharose 6B (40). The recombinant GSTs were eluted from the GSH affinity resin with 50 mM Tris-HCl, pH 9.6, containing 10 mM GSH and 1.4 mM beta -mercaptoethanol. Purification of "total GSTs" from K562 cells by GSH affinity chromatography was performed as we described previously (41).

Purified GSTs were dialyzed against buffer A, and an aliquot of the dialyzed enzyme was subjected to isoelectric focusing (IEF) in an LKB-8100 column using ampholines in the pH ranges of 3.5-10.0 and a 0-50% (w/v) sucrose density gradient. After IEF at 1600 V for 24 h, 0.8-ml fractions were collected and monitored for GST activity with CDNB determined in alternate fraction and pH measured in every 5th fraction.

Peroxidized Erythrocyte Membranes-- The erythrocyte membranes were prepared according to the method described by Awasthi et al. (42) with slight modifications as described below. The blood was washed twice with Hanks' buffer containing 5 mM KCl, 0.3 mM KH2PO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM glucose, 100 µM phenylmethylsulfonyl fluoride (PMSF), 100 µM EDTA, and 1.4 mM beta -mercaptoethanol. The erythrocyte membranes (ghosts) were prepared by repeated washing with 10× volume of lysis buffer containing 10 mM Tris-HCl, pH 7.4, 1.4 mM beta -mercaptoethanol, 100 µM EDTA, and 100 µM PMSF at 22,000 × g until the ghosts were free of the visible color of hemoglobin. The ghosts were stored under nitrogen at -70 °C to minimize auto-oxidation. The membrane lipid peroxidation was induced by incubating ghosts with 1 mM hydrogen peroxide and 1 mM ferrous sulfate at 37 °C for 1 h in the dark, and the reaction was stopped by adding 25 µM desferrioxamine (25).

Determination of PL-OOH-- The peroxidized membranes suspended in 10 mM Tris-HCl, pH 7.4, 1.4 mM beta -mercaptoethanol, and 0.1 mM EDTA were extracted with 2 volumes of chloroform/methanol (2:1, v/v). After centrifugation at 4,000 × g for 5 min at 4 °C, the organic phase was collected, and chloroform was evaporated under a stream of nitrogen. The amounts of membrane PL-OOH recovered were determined by the microiodometric assay (25, 43) as briefly described. Samples were treated with 300 µl of deoxygenated glacial acetic acid/chloroform mixture (3:2, v/v) and 20 µl of deoxygenated potassium iodide solution (1.2 mg/ml) in the dark, and after incubating the reaction mixture for 5 min at 25 °C, 0.9 ml of 20 mM cadmium acetate was added. The reaction mixture was centrifuged at 4,000 × g for 2 min, and the absorbance of supernatant was determined at 353 nm. The readings were converted to PL-OOH content (nmol/mg protein) using an extinction coefficient of 21.9 × 103 M-1 cm-1.

Enzyme Assays-- GST activity toward CDNB was determined spectrophotometrically at 340 nm by the method of Habig et al. (44). One unit of GST activity was defined as the amount of enzyme catalyzing the conjugation of 1 µmol of CDNB with GSH per min at 25 °C. GPx activity toward hydroperoxide substrates was determined using the glutathione reductase (GR)-coupled assay as we described previously (40). Briefly, 1 ml of reaction mixture containing 3.2 mM GSH, 0.32 mM NADPH, 1 unit of GR, and 0.82 mM EDTA in 0.16 M Tris-HCl, pH 7.0, was preincubated with an appropriate amount of GST at 37 °C for 5 min. The reaction was started by addition of appropriate hydroperoxide substrates (prepared in methanol). The final concentration of all the hydroperoxide substrates in the reaction mixture was 100 µM. The consumption of NADPH was monitored at 340 nm for 4 min at 37 °C. One unit of GPx activity was defined as the amount of enzyme necessary to consume 1 µmol of NADPH per min. A non-substrate blank as well as a non-enzyme additional blank was used to correct for non-GR-dependent NADPH oxidation and non-enzymatic peroxidase activity.

For determination of the antioxidant enzymes and the enzymes involved in GSH homeostasis, the cells were homogenized in buffer A containing 1.4 mM beta -mercaptoethanol by sonication, and 28,000 × g supernatant of the homogenate was assayed for enzyme activities. CAT activity was determined by the method described by Beers and Sizer (45). Total SOD activity was determined according to the method of Paoletti and Mocali (46), and beta -mercaptoethanol was excluded from the extracts used for assays. One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of NADPH oxidation of the control by 50%. GR activity was determined by the method of Carlberg and Mannervick (47), and gamma -glutamylcysteine synthetase (gamma -GCS) activity was determined by the method of Seelig and Meister (48). GSH was determined using the whole lysates prepared without beta -mercaptoethanol according to the method of Beutler et al. (49).

Immunoprecipitation of GPx Activity in Human Tissue Extracts-- Human tissues were rinsed with PBS and homogenized in buffer A on ice. The homogenates (10% w/v) were centrifuged at 28,000 × g for 45 min at 4 °C, and the supernatants after dialysis against 200× buffer A with three changes were used for immunoprecipitation studies. The IgG fraction of the polyclonal antibodies against human Alpha class GSTs used for immunoprecipitation of the GPx activity was purified using DEAE-cellulose ion exchange chromatography and protein A immunoaffinity chromatography as we described previously (50). In immunotitration experiments, fixed aliquots (100 µl) of 28,000 × g supernatants containing 50 µg of protein were incubated with increasing amounts of purified anti GST-Alpha antibodies (0.25 to 2.5 µg IgG) at 4 °C. Equal amounts of purified preimmune serum were used in controls, and additional controls containing only buffer were also used. After 2 h of incubation, 20 µl of protein A-Sepharose beads (Sigma) were added to the reaction mixtures and incubated overnight at 4 °C. The reaction mixtures were centrifuged at 10,000 × g for 30 min, and the GPx activities toward CU-OOH and PC-OOH were determined in the supernatants. When required, the proteins from immunoprecipitated pellets and the supernatant fraction were subjected to Western blotting using biotin-labeled antibodies against human Alpha class GSTs followed by detection with streptavidin-horseradish peroxidase according to the manufacturer's (Amersham Pharmacia Biotech) suggested protocol to exclude the detection of IgG.

Cell Cultures-- K562 cells were cultured in RPMI 1640 medium containing L-glutamine and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO2 humidified atmosphere. Cells were passaged twice every week and maintained in log phase growth at 2 × 105 to 5 × 105/ml to avoid spontaneous differentiation.

Stable Transfection with hGSTA2-- Based on the cDNA sequences of hGSTA2, polymerase chain reaction primers were designed to amplify the coding sequence of hGSTA2 from the 5'-Stretch Plus human lung cDNA library in the pTriplEx vector (CLONTECH), and amplified cDNA was subcloned into the pTarget-T mammalian expression vector (Promega). K562 cells were transfected with pTarget-T/hGSTA2 vector or with the vector alone using liposome-based TransfastTM transfection reagent (Promega). In these experiments, 1 × 106 cells were incubated with liposome-encapsulated vectors (2 µg of DNA/6 nmol of cationic lipid) in 1 ml of serum-free media at 37 °C. After 4 h of incubation, 5 ml of complete medium was added, and cells were cultured for 20 h. Thereafter, stable transfectants were isolated by selection on 400 µg/ml G418 for ~2 weeks. Single clones of stably transfected cells were isolated by limiting dilution. Several G418-resistant stable clones were selected for further characterization by Western blotting and enzyme assays and maintained in medium containing 400 µg/ml G418.

Lipid Peroxidation-- LPO levels were determined by thiobarbituric acid-reactive substances (TBARS) as described by Wagner et al. (3) with slight modifications. For each determination, 1 × 107 cells were collected by centrifugation at 500 × g for 10 min and washed twice with PBS. The pellet was resuspended in 1 ml of 10 mM potassium phosphate buffer, pH 7.0, containing 0.4 mM butylated hydroxytoluene and vortexed vigorously, and samples were immediately used for TBARS assay. The cellular protein was precipitated by mixing homogenates with 120 µl of saturated trichloroacetic acid solution (250 × g of trichloroacetic acid in 100 ml of water). After centrifugation at 4,000 × g for 15 min, supernatants were quantitatively transferred to glass test tubes and mixed with 2-thiobarbituric acid solution in 0.1 N NaOH with a final concentration of 1.6 mg/ml (total volume 1 ml). The samples were incubated for 30 min at 75 °C. After cooling the samples to room temperature, the absorbances of the samples at 535 nm were measured. LPO levels were expressed as picomoles of malonaldehyde/mg of cell protein. Extinction coefficient for malonaldehyde used was 1.53 × 105 M-1 cm-1 .

Cytotoxicity Assay-- The MTT assay as described by Mosmann (51) and Boekhorst et al. (52) was used with slight modifications to determine the cytotoxicity of H2O2. Briefly, 2 × 104 cells in 190 µl of medium were plated to each well in 96-well flat-bottomed microtiter plates. The medium was supplemented with 10 µl of PBS containing various concentrations of H2O2. Eight replicate wells were used for each concentration of H2O2 used in these experiments. After treatment of cells with H2O2 at 37 °C for 72 h, 10 µl of MTT solution (2 mg/ml in PBS) was added to each well, and the plates were incubated for additional 4 h at 37 °C. The plates were centrifuged at 1200 × g for 10 min, and the medium within the wells was aspirated. The intracellular formazan dye crystals were dissolved by addition of 100 µl of Me2SO to each well and were incubated overnight at room temperature in the dark with constant shaking. The absorbance of formazan at 562 nm was measured using a microplate reader (Elx808 Bio-Tek Instruments). The H2O2 concentrations resulting in a 50% decrease in formazan formation (IC50) were obtained by plotting a dose-response curve.

DNA Laddering-- For the detection of DNA laddering, the cells (3 × 106) were pelleted by centrifugation at 750 × g for 5 min and washed twice with PBS. The genomic DNA from the cells was isolated using Wizard Genomic DNA purification kit (Promega) in which the pellets were lysed in 600 µl of nuclei lysis buffer followed by incubation of the nuclear lysate with 3 µl of RNase (4 mg/ml) for 30 min at 37 °C. The cellular proteins were removed by the addition of 200 µl of protein precipitation solution and centrifuged for 4 min at 14,000 × g. The supernatant fractions containing genomic DNA were concentrated by mixing with 600 µl of isopropyl alcohol. The DNA pellets obtained by centrifugation at 14,000 × g for 1 min were washed by 600 µl of 70% ethanol and solubilized in Tris-EDTA buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0) at 65 °C for 1 h. The concentrations of DNA were determined spectrophotometrically at 260/280 nm. For electrophoresis, DNA samples (2 µg) were loaded on 2% agarose gels containing ethidium bromide. After electrophoresis for 2 h at 50 V, gels were visualized under UV.

TUNEL Assay-- The modified terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assays were performed using the DeadEndTM colorimetric apoptosis detection system (Promega) according to the protocol provided by manufacture with slight modifications. The cells (1 × 104) were cytospinned to the poly-L-lysine pre-coated slides at 750 × g for 5 min. The cells were fixed in 4% paraformaldehyde for 30 min. After washing twice with PBS, the slides were immersed in 0.2% Triton X-100 for 5 min to permeabilize cells. The slides were incubated with biotinylated nucleotide and terminal deoxynucleotidyltransferase in 100 µl of equilibration buffer (200 mM potassium cacodylate, pH 6.6, 25 mM Tris-HCl, pH 6.6, 0.2 mM dithiothreitol; 0.25 mg/ml bovine serum albumin; 2.5 mM cobalt chloride) at 37 °C for 1 h inside a humidified chamber to allow the end labeling reactions to occur. The reaction was stopped by immersing slides in 150 mM sodium chloride, 15 mM sodium citrate, pH 7.4, for 15 min followed by immersion in PBS for 15 min (2 times). Thereafter, the endogenous peroxidases were blocked by immersing the slides in 0.3% H2O2 for 5 min. The slides were treated with 100 µl of horseradish peroxidase-labeled streptavidin solution (1:500 dilution in PBS) and incubated for 30 min at room temperature. Finally, the slides were developed using the peroxidase substrate, H2O2, and the stable chromogen, diaminobenzidine, for 15 min. The slides were rinsed with water and examined under light microscope (Zeiss-3, Germany). The photographs were taken at × 80 magnification.

Western Blot Analysis-- For detection of the expression of hGSTA2-2 in transfected cells, aliquots of 28,000 × g supernatant fraction of the K562 cell homogenate containing 50 µg of protein were subjected to Western blot analysis using the polyclonal antibodies against the human Alpha class GSTs. For detection of PARP, 107 cells were suspended in 100 µl of denaturing lysis buffer containing 62.5 mM Tris-HCl, pH 6.8, 6.0 M urea, 2% SDS, 10% glycerol, 1.4 mM beta -mercaptoethanol, 0.00125% bromphenol blue, 0.5% Triton X-100, and 1 mM PMSF. Cells were sonicated for 3 times for 5 s on ice to disrupt protein-DNA interaction and incubated at 65 °C for 15 min. Samples (20 µl) were applied to 10% SDS-PAGE gels, and Western blot analysis was performed using PARP monoclonal antibody (clone C2-10, PharMingen, San Diego, CA). The 17-kDa subunit of active caspase 3 was detected by SDS-PAGE and immunoblotting 20 µg of 28,000 × g supernatants of cell homogenates using the anti-active caspase 3 antibodies (PharMingen) which recognizes 32-kDa pro-caspase 3 as well.

Solid-phase JNK Assay-- 107 K562 cells were washed with PBS and resuspended in 500 µl of extraction buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Cells were sonicated 4 times for 5 s on ice and then microcentrifuged at 14,000 × g for 10 min. The pellets were discarded, and the supernatants, representing cell extracts, were adjusted to 1 mg/ml protein concentration. Cell lysates (250 µl) were mixed with a 20-µl suspension of GSH-agarose beads in extraction buffer, to which 2 µg of GST-c-Jun-(1-89) were freshly bound. Mixtures were incubated overnight at 4 °C with continuous shaking, and the beads were pelleted by centrifugation at 14,000 × g for 1 min. The beads were washed twice successively with 0.5 ml of extraction buffer followed by 0.5 ml of kinase buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM beta -glycerophosphate, 0.1 mM Na3VO4, 10 mM MgCl2, and 2 mM dithiothreitol (two washes) to remove kinases that have weaker affinity to bind c-Jun-(1-89) than JNK. The pelleted beads were resuspended in 50 µl of kinase buffer supplemented with 100 µM cold ATP and incubated for 30 min at 30 °C. The reaction was terminated by boiling the beads with 25 µl of SDS-polyacrylamide gel sample buffer (3 times) for 5 min. The eluted phosphorylated proteins were subsequently resolved in 12% SDS-PAGE gels and detected using the phospho-c-Jun (Ser-63) antibodies (New England Biolabs).

Statistical Analysis-- The results are expressed as mean ± S.D. Significant differences were evaluated with the unpaired Student's t test or one-way analysis of variance. All statistical tests were carried out at the 5% level of significance.

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ABSTRACT
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GPx Activities of GSTA1-1 and GSTA2-2 toward Hydroperoxides-- In order to determine substrate specificities of hGSTA1-1 and hGSTA2-2, recombinant enzymes were prepared by expressing in E. coli. BL21 cells used pET30a(+) expression vector and their subsequent purification was by GSH affinity chromatography (35). Purified hGSTA1-1 and hGSTA2-2 showed a single band at about 25 kDa in denaturing SDS-PAGE gels (data not presented) and had pI values of 9.3 and 8.9, respectively. These isoenzymes were recognized by the antibodies against the cationic Alpha class GSTs of human liver that predominantly consists of hGSTA1-1 and hGSTA2-2. Antibodies against hGSTP1-1, hGSTM1-1, or hGSTA4-4 did not recognize these enzymes (data not presented). These results were consistent with the previously reported properties of hGSTA1-1 and hGSTA2-2 (35). Both enzymes displayed GPx activities and catalyzed GSH-dependent reduction of FA-OOH, PL-OOH, and CU-OOH. However, no detectable activity was observed when H2O2 was used as the substrate (Table I).

                              
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Table I
Specific activities of recombinant (rec) hGSTA1-1 and hGSTA2-2 toward different substrates
Values are means ± S.D., with the number of determinations in parentheses.

GST-mediated Reduction of PL-OOH in Erythrocyte Membranes in Situ-- In order to address the question whether or not GSTA1-1 and GSTA2-2 can catalyze the reduction of intact PL-OOH of biological membranes in situ, we have measured GSH-dependent reduction of PL-OOH present in plasma membranes. These experiments were focused on recombinant hGSTA2-2 because of its relatively higher activity toward isolated PL-OOH as compared with hGSTA1-1. Erythrocyte membranes were chosen as the model because of an easy accessibility of relatively pure membranes (ghosts) in sufficient amounts. Erythrocyte membranes, free of hemoglobin, were prepared as described previously (42) from fresh human blood procured from University of Texas Medical Branch blood bank after Institutional Review Board approval. The membranes were subjected to peroxidation by incubating with H2O2 and trace of Fe2+ as detailed under "Experimental Procedures." Under these conditions, ~670 ± 27 nmol of PL-OOH/mg of membrane protein were generated as measured by the microiodometric assay (25, 43).

These peroxidized membranes were used as the substrate for determining GPx activity of hGSTA2-2 using GR-coupled assay that measures the consumption of NADPH. Membrane preparations (10 µl) containing 3 nmol of PL-OOH were incubated with the reaction mixture containing 0.32 mM NADPH, 3.2 mM GSH, 0.82 mM EDTA, and 1 unit of GR in 0.16 M Tris-HCl, pH 7.0, at 37 °C with a final volume of 1 ml, and NADPH consumption was determined spectrophotometrically. As shown in Fig. 1, a linear rate of NADPH consumption in the presence of GSH alone or GSH with heat-inactivated hGSTA2-2 was observed indicating that GSH alone caused reduction of the peroxidized lipid components of the membrane. We believe that this reduction may be ascribed to the non-enzymatic reduction of membrane PL-OOH by GSH. These results, however, do not exclude the possibility that the traces of GPx-4 like activity reported earlier (53) catalyzed this reduction of PL-OOH. As shown in Fig. 1, addition of varying amounts of hGSTA2-2 to the reaction mixture resulted in a dose-dependent accelerated rate of NADPH consumption indicating that hGSTA2-2 displayed GPx activity toward these substrates in membranes. The specific activity of hGSTA2-2 toward membrane PL-OOH estimated from the curves in Fig. 1 corresponding to 0.1, 0.2, and 0.3 µg of enzyme in the reaction mixture was closely similar (1.28 ± 0.15, 1.27 ± 0.12, and 1.25 ± 0.14 µmol/min/mg protein, respectively). This was considerably lower than activity of hGSTA2-2 toward the purified PL-OOH in the isolated system (Table I). The lower activity of hGSTA2-2 toward membrane PL-OOH in situ may perhaps be attributed to steric factors limiting the availability of the substrates to the active site of the enzyme. Similar results were obtained when hGSTA1-1 was used instead of hGSTA2-2 (data not presented). These results indicated that hGSTA1-1 and hGSTA2-2 catalyzed GSH-dependent reduction of membrane PL-OOH in situ.


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Fig. 1.   In situ reduction of PL-OOH in peroxidized erythrocyte membranes. Erythrocyte membranes were peroxidized by the Fenton reaction as described in the text, and the peroxidized membrane was then used as the substrate to determine GPx activity of hGSTA2-2. Varying amounts of recombinant hGSTA2-2 (0.1-0.3 µg) were preincubated with GPx assay buffer containing 3.2 mM GSH, 0.32 mM NADPH, 1 unit of GSH reductase, and 0.82 mM EDTA in 0.16 mM Tris-HCl, pH 7.0, with a final volume of 1 ml at 37 °C for 5 min. The reaction mixtures containing GSH with and without heat-inactivated hGSTA2-2 were used as controls. The reaction was started by addition of 10 µl of peroxidized membrane preparations (0.43 mg of protein/ml) containing 3.0 nmol of lipid hydroperoxides as determined by the microiodometric assay. The reaction was monitored spectrophotometrically at 37 °C by the rate of NADPH consumption measured as the decrease in the absorbance at 340 nm for 4 min. Means ± S.D. of values from four determinations are shown.

Quantitation of hGSTA2-2-mediated Reduction of Membrane PL-OOH-- The catalytic activity of hGSTA2-2 toward membrane PL-OOH was further confirmed through quantitation of PL-OOH by iodometric titrations. In these experiments, GSH-dependent reduction of PL-OOH by hGSTA2-2 and GPx-1, the major selenium-dependent GPx isoenzyme, was separately quantitated. Peroxidized membrane preparations containing 295 µg of protein and ~205 nmol of PL-OOH were incubated separately at 37 °C with an excess of GSH (4 mM in a total volume of 2 ml in 0.16 M Tris-HCl, pH 7.0) in the presence or absence of enzymes, hGSTA2-2, or GPx-1. The results presented in Table II showed that there was no significant change in the hydroperoxide content in the membrane during the incubation with buffer only. Incubation with GSH alone caused reduction of PL-OOH content from 205 to 116 nmol (about 44% reduction) in 4 min. Addition of 20 µg of hGSTA2-2 in the presence of GSH led to the reduction of about 90% PL-OOH. On the other hand, addition of 20 µg of selenium-dependent GPx-1 did not cause any significant increase in the reduction of PL-OOH over that observed in the presence of GSH only. It is noteworthy that the specific activity of hGSTA2-2 calculated from the data presented in Table II (1.13 ± 0.09 µmol/min/mg protein) was closely similar to that calculated from the data in Fig. 1. These results further confirmed that hGSTA2-2 and hGSTA1-1 catalyzed the reduction of PL-OOH in situ.

                              
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Table II
GSH-dependent reduction of membrane PL-OOH by GSTA2-2 and GPx
Equal amounts of peroxidized membrane preparations containing 295 µg of protein and 205 ± 24.3 nmol of PL-OOH as determined by microiodometric assay were incubated with 4 mM GSH in 0.16 mM Tris-HCl, pH 7.0 with or without 20 µg of recombinant hGSTA2-2 or GPx-1 (Sigma) for 4 min at 37 °C (in a total volume of 2 ml). After the incubation, 2 ml of methanol/chloroform (1:2, v/v) was added to stop the reaction. Residual PL-OOHs were extracted and determined as described under "Experimental Procedures." Means ± S.D. of values from three separate experiments are shown. The final concentration of hGSTA2-2 added to the reaction mixture corresponds to about 0.2 µM (based on molecular mass of 50 kDa for hGSTA2-2).

Immunotitration of GPx Activity of Human Tissues toward CU-OOH and PL-OOH-- In order to quantitate the contribution of the Alpha class GSTs in the GSH-dependent reduction of PL-OOH in different human tissues, immunoprecipitation studies using the polyclonal antibodies against human cationic Alpha class GSTs were performed. hGSTA1-1 and hGSTA2-2 which constitute the bulk of the cationic Alpha class GSTs are immunologically similar and can be immunoprecipitated by the polyclonal antibodies raised against the cationic Alpha class GSTs of human liver. Conditions for immunoprecipitation protocols were first standardized to ensure complete immunoprecipitation of the Alpha class GSTs of different human tissues by the anti-GST-Alpha antibodies. It was established that 50 µl of purified anti-GST-Alpha antibodies containing 2.5 µg of IgG were sufficient to immunoprecipitate completely the Alpha class GSTs present in 100 µl of 10% (w/v) extracts of human tissues including testis, liver, lung, heart, and pancreas containing about 50 µg of cytosolic protein. Representative results of Western blot analysis with extracts of human testis are presented in Fig. 2. The pre-immune serum (50 µl) used as the control did not precipitate any Alpha class GSTs because these were exclusively present in the supernatant fraction (Fig. 2A, lane 4) and were not detected in the pellet fraction (Fig. 2A, lane 5). Anti-Alpha class GST antibodies, on the other hand, immunoprecipitated all the Alpha class GSTs because these were absent in the supernatant fraction (Fig. 2A, lane 6) and present only in the pellet (Fig. 2A, lane 7). The results of immunoprecipitation of GPx activities of different human tissues toward CU-OOH and PC-OOH are presented in Fig. 2, B and C, respectively. These results showed that about 80% of GPx activity of human liver toward CU-OOH was immunoprecipitated by anti-GST-Alpha antibodies. Likewise, about 60% of the GPx activity of testis toward CU-OOH was immunoprecipitated by these antibodies. From the extracts of lung, heart, and pancreas where the Alpha class GSTs constitute only a minor portion of total GSTs (28), a relatively smaller fraction of GPx activity toward CU-OOH was immunoprecipitated. In human erythrocytes where complete lack of the Alpha class GSTs is reported (28), the amount of GPx activity toward CU-OOH immunoprecipitated by these antibodies was insignificant. The results of immunoprecipitation of GPx activity toward PC-OOH from human tissues by anti-GST Alpha antibodies also revealed that a major portion of GPx activity of liver and testis extracts was immunoprecipitated by these antibodies. These results suggest that the Alpha class GSTs may play an important role in protection mechanisms against LPO in liver and testis by reducing PL-OOH and interrupting the autocatalytic chain of LPO.


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Fig. 2.   Immunoprecipitation of GPx activity using antibodies against the Alpha class GSTs in different human tissues. A, standardization of the conditions to immunoprecipitate completely the Alpha class GSTs. 100 µl of human testis extracts (0.5 mg/ml) were incubated with 50 µl of protein A-purified anti-Alpha class GST antibodies or pre-immune serum containing 2.5 µg of IgG at 4 °C. After 2 h, 20 µl of protein A-Sepharose beads (sigma) were added to the reaction mixture and incubated overnight at 4 °C. The reaction mixture was centrifuged at 10,000 × g for 30 min, and the proteins recovered in the supernatant and pellet fractions were subjected to Western blot analysis using the biotinylated polyclonal antibodies raised against human cationic Alpha class GSTs followed by streptavidin-horseradish peroxidase. Lane 1, prestained broad range SDS-PAGE standards. Lane 2, 1 µg of Alpha class GSTs purified from human liver as positive control. Lanes 4 and 5, proteins from the immunoprecipitated supernatant and pellet fraction using pre-immune serum, respectively. Lanes 6 and 7, proteins from the immunoprecipitated supernatant and pellet fraction using Alpha class GST antibodies, respectively. B and C, immunoprecipitation of GPx activity toward CU-OOH and PC-OOH in human tissues. 100 µl of different human extracts (0.5 mg/ml) were immunoprecipitated with 50 µl of purified anti-Alpha class GST antibodies () or pre-immune serum (black-square) containing 2.5 µg of IgG as described in A. In controls, serum was replaced by 50 µl of buffer. The proteins recovered in the supernatant were used for determining GPx activity toward CU-OOH (B) and PC-OOH (C). The activities were normalized to the controls. Results are the means ± S.D. of four determinations.

Overexpression of hGSTA2-2 in K562 Cells Protects against LPO-- For investigating the physiological role of Alpha class GSTs against oxidative stress through transfection studies, the human erythroleukemia cell line (K562) was selected because it is known that it does not express any detectable hGSTA1-1 or hGSTA2-2 (41). Transfection of hGSTA2 cDNA in K562 cells and subsequent selection of stably transfected clones resulted in high expression of hGSTA2-2 in K562 as indicated by the results of Western blot analysis. As shown in Fig. 3, only the transfected cells showed expression of hGSTA2-2 which was not detected in either the wild type or vector-alone-transfected cells. Total GST isoenzymes were purified separately from the wild type, vector-transfected, and hGSTA2-transfected cells, and their GST activities toward CDNB and GPx activities toward hydroperoxide substrates are presented in Table III. hGSTA2-transfected cells had only about 1.5-fold higher GST activity toward CDNB. However, about 10-fold higher GPx activities toward various hydroperoxides were observed in the hGSTA2-transfected cells as compared with the wild type or vector-alone-transfected cells. Isoelectric focusing profiles of the GST isoenzymes of vector and hGSTA2-transfected cells presented in Fig. 4 showed that only the cells overexpressing hGSTA2-2 exhibited a peak of GST activity toward CDNB at pH 8.9. Both vector and hGSTA2-transfected cells, however, showed a peak of GST activity at pH 4.8 that was due to hGSTP1-1 which is constitutively expressed in K562 cells (41). These results indicated that hGSTA2-2 transfection resulted in the functional expression of the enzyme.


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Fig. 3.   Expression of hGSTA2-2 in transfected cells. An aliquot of 28,000 × g supernatant fraction of homogenates of the control and transfected K562 cells containing 50 µg of protein was subjected to SDS-PAGE in 12% gel. Western blot analysis was performed using rabbit polyclonal antibodies against human Alpha class GSTs as primary antibodies and peroxidase-conjugated goat anti-rabbit antibodies as secondary antibodies. The blot was developed using horseradish peroxidase color-developing reagent. Lane 1, prestained low range SDS-PAGE standards; lane 2, 0.3 µg of recombinant hGSTA2-2 as the positive control; lanes 4-6, lysates from hGSTA2-transfected, wild type, and vector-transfected K562 cells, respectively.

                              
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Table III
Specific activities of total GSTs from the wild-type, vector-transfected, and hGSTA2-transfected K562 cells
Total GSTs were purified in parallel experiments from equal amounts of cells (1 × 108) using GSH affinity chromatography (41). Values are means ± S.D., with the numbers of determinations in parentheses.


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Fig. 4.   IEF profile of GSTs purified from K562 cells. Total GSTs were purified from 1 × 108 K562 cells by GSH affinity chromatography and were subjected to IEF for 24 h at 1600 V at 4 °C on LKB 8100 liquid column IEF unit as described in the text. The column was eluted in 0.8-ml fractions. GST activity toward CDNB () from hGSTA2-transfected cells (A) and vector-alone-transfected cells (B) was determined in alternate fractions, and pH (black-triangle) was measured in every 5th fraction.

Measurement of LPO, using TBARS as the index, showed that hGSTA2-transfected cells had significantly reduced levels of LPO (Fig. 5). The attenuation of lipid peroxidation by hGSTA2-2 overexpression was much more pronounced in cells when LPO was induced by the addition of H2O2 and traces of Fe2+. To assess the possibility whether hGSTA2-2 overexpression would affect the activities of antioxidant enzymes, such as SOD, CAT, and GPx as well as gamma -glutamylcysteine synthetase (gamma -GCS) and GR involved in maintaining GSH homeostasis, the activities of these enzymes were determined in vector-transfected and hGSTA2-transfected K562 cells. In vector-transfected and hGSTA2-transfected cells, the activities of SOD (13.46 ± 0.31 and 14.32 ± 0.79 unit/mg protein, respectively) and CAT (21.68 ± 0.66 and 23.69 ± 1.19 µmol/min/mg protein, respectively) were similar. GPx activity toward H2O2 in the vector-transfected cells (2.70 ± 0.23 nmol/min/mg protein) and hGSTA2-transfected cells (2.72 ± 0.18 nmol/min/mg protein) was also similar. Likewise, the GSH levels and the activities of gamma -GCS and GR of the control and hGSTA2-transfected cells were similar (data not presented). These results indicated that hGSTA2 transfection did not affect the levels of antioxidant enzymes and GSH homeostasis of K562 cells. Taken together, these results further confirm that the Alpha class GSTs protect membranes from LPO during oxidative stress by reducing PL-OOH.


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Fig. 5.   Effect of hGSTA2-2 overexpression on LPO in K562 cells. 1 × 107 K562 cells were incubated with RPMI complete medium or RPMI complete medium containing 100 µM H2O2 and 50 µM FeSO4 for 30 min. The cells were pelleted by centrifugation, washed with PBS, and homogenized in 10 mM potassium phosphate buffer, pH 7.0, containing 0.4 mM butylated hydroxytoluene. The whole homogenate was immediately taken for TBARS assay as described in the text. The values (means ± S.D., n = 3) are presented in the bar graph. The asterisk indicates a significant difference from the controls (p < 0.01).

hGSTA2-2 Overexpression Protects against H2O2 Cytotoxicity-- To investigate whether hGSTA2-2 overexpression confers resistance against H2O2 cytotoxicity, the effect of H2O2 was compared in the wild type, vector-transfected, and hGSTA2-transfected cells. The IC50 values of H2O2 as determined in three independent experiments were found to be 20.33 ± 2.46, 22.67 ± 1.44, and 32 ± 2.64 µM for the wild type, vector-transfected, and hGSTA2-transfected cells, respectively. Results of a representative experiment are presented in Fig. 6. These results indicated that hGSTA2-2 overexpression confers about 1.5-fold resistance to oxidative stress in K562 cells and show that hGSTA2-2, or the Alpha class GSTs in general, play an important role in the protection mechanisms against the low levels of H2O2, which may be constantly generated in aerobic organisms.


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Fig. 6.   Effect of hGSTA2 transfection on cytotoxicity of H2O2 to K562 cells. Aliquots of cells in log-phase growth from wild type (), vector-transfected (open circle ), and hGSTA2-transfected (black-down-triangle ) K562 cells were washed twice, resuspended in PBS, and inoculated at a density of 2 × 105 cells/ml (100 µl/well) into 8 replicate wells with various H2O2 concentrations (0-50 µM) in a 96-well plate. The MTT assays were performed as described in the text. Blank (no cells) subtracted A590 values were normalized to control (cells without H2O2 treatment). Representative results from one of the three independent experiments on H2O2 cytotoxicity are presented.

hGSTA2-2 Overexpression Protects against H2O2-induced Apoptosis-- Exposure to H2O2 and a variety of agents causing oxidative stress are known to induce apoptosis (1-10). Therefore, we compared the extent of H2O2-induced apoptosis in the control and hGSTA2-transfected K562 cells. Assessment of apoptosis by DNA fragmentation through TUNEL assay and DNA laddering studies clearly indicated that the cells transfected with hGSTA2 were relatively more resistant to H2O2-induced apoptosis. As shown in Fig. 7 by the appearance of dark brown color in the nuclei (TUNEL assay), the wild type and vector-alone-transfected cells showed remarkable apoptosis when exposed to 30 µM H2O2 for 48 h. However, in the cells transfected with hGSTA2 only a minimal DNA fragmentation was observed under these conditions. These results were consistent with those of DNA laddering experiments (Fig. 8A) which showed characteristic internucleosomal degradation of the DNA only in the control cells but not in the hGSTA2-transfected cells. Taken together, these results established that hGSTA2-2 overexpression attenuates H2O2-induced apoptosis.


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Fig. 7.   Detection of DNA fragmentation in situ by TUNEL assay in K562 cells. Wild type (A), vector-transfected (B), and hGSTA2-transfectd K562 cells (C) were treated with 30 µM H2O2. After 48 h, cells were cytospinned and fixed in 4% paraformaldehyde. DNA fragmentation was detected by TUNEL assay as described in the text. The nuclei of apoptotic cells were stained dark brown. The photographs were taken at × 80 magnification.


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Fig. 8.   The effect of overexpression of hGSTA2-2 in K562 cells on H2O2-, PC-OOH-, and 4-HNE-induced apoptosis. The wild type, vector-transfected, and hGSTA2-transfected K562 cells were treated with 30 µM H2O2 for 48 h (A), 40 µM PC-OOH for 8 h (B), or 40 µM 4-HNE for 8 h (C) in RPMI complete medium. After the incubations, genomic DNA was extracted and electrophoresed on 2% agarose gel. Lanes 1-3 in all panels represent the wild type, vector-transfected, and hGSTA2-transfected K562 cells, respectively. Apoptosis was examined by the appearance of characteristic DNA laddering.

Effect of hGSTA2-2 Overexpression on 4-HNE and PL-OOH-induced Apoptosis-- 4-HNE, a highly reactive but relatively stable end product of LPO, is known to induce apoptosis in K562 cells (15) and various other cells (11-14). Therefore, we investigated if hGSTA2-2 overexpression could protect the cells against 4-HNE-induced apoptosis. Results of these experiments as presented in Fig. 8C showed that hGSTA2-2 overexpression did not provide any noticeable protection against apoptosis caused by 4-HNE. These results are consistent with our earlier studies (50) showing that 4-HNE is not the preferred substrate for hGSTA2-2. Attenuation of 4-HNE-induced apoptosis by overexpression of mGSTA4-4 which utilizes 4-HNE as the preferred substrate has been demonstrated previously (15). Since hGSTA2 transfection did not affect CAT, SOD, or GPx activity toward H2O2 in K562 cells, our results demonstrate that hGSTA2-2 provides protection to K562 cells against H2O2-induced apoptosis by reducing PL-OOH and thereby breaking the autocatalytic chain of LPO. This idea is supported by the results showing that apoptosis in K562 cells induced by PC-OOH is attenuated by hGSTA2-2 overexpression (Fig. 8B) which failed to protect against the apoptotic effect of 4-HNE, a product downstream to PL-OOH in the LPO chain. These results suggest a role of LPO products in the initiation of oxidative stress-induced apoptosis and suggest that the apoptosis caused by H2O2 may be, at least in part, initiated by PL-OOHs which are the main substrates for the cationic Alpha class GSTs.

Effect of hGSTA2-2 Overexpression on H2O2-induced Activation of Caspase 3-- Activation of caspase 3 resulting in the proteolytic cleavage of the 116-kDa native PARP into an 89-kDa peptide is reported to be associated with H2O2-induced apoptosis (7, 16, 54). The results presented in Fig. 9 showed that caspase 3-mediated PARP cleavage was not observed in the wild type or vector-alone-transfected K562 cells after 24 h of H2O2 exposure. However, after 48 h of exposure to 30 µM H2O2, PARP cleavage was observed in these cells. In contrast, no detectable PARP cleavage was observed in hGSTA2-transfected cells even after 48 h of H2O2 exposure. These results further confirm that hGSTA2-2 overexpression provides protection against H2O2-induced apoptosis in K562 cells and are consistent with the suggested role of caspase 3 in H2O2-induced apoptosis (5, 7, 8, 55). The activation of caspase 3 only in the wild type and vector-transfected K562 cells was further confirmed by Western blot analysis of the cell extracts using antibodies recognizing both the 32-kDa unprocessed pro-caspase 3 and the 17-kDa subunit of the active caspase 3. Results of these experiments showed that pro-caspase 3 (CPP32) was cleaved into active caspase 3 (17 kDa) only in the control cells and not in the hGSTA2-transfected K562 cells (data not presented).


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Fig. 9.   Effect of hGSTA2-2 overexpression on H2O2-induced PARP cleavage. Cells were incubated with 30 µM H2O2 in the medium for the indicated times. Cell lysates were subjected to Western blot analysis using the monoclonal antibody against PARP (Clone C2-10) which recognizes the full-length PARP (116 kDa) as well as its 89-kDa fragment. Lanes 1-3, lysates from the wild type, vector-transfected, and hGSTA2-transfected cells, respectively, treated with H2O2 for 24 h. Lanes 4-6, lysates from the wild type, vector-transfected, and hGSTA2-transfected cells, respectively, treated with H2O2 for 48 h.

Effect of hGSTA2-2 Overexpression on SAPK/JNK Activity-- SAPK/JNKs, members of the mitogen-activated protein kinases (MAPKs)-related family, are activated in response to oxidative stress and other kinds of cellular stress (56-62), and their activation may be required for apoptosis (60-62). We thereafter investigated whether hGSTA2-2 overexpression affected SAPK/JNK activity in K562 cells stressed with H2O2. Results presented in Fig. 10, A and B, showed that exposure to 30 µM H2O2 markedly stimulated SAPK/JNK activities in the wild type and vector-alone-transfected K562 cells as indicated by the increased phosphorylation of c-Jun. The SAPK/JNK activity was increased as early as 2 h after H2O2 exposure, peaked after 6 h, and thereafter was maintained at peak levels for at least 24 h. As shown in Fig. 10C, overexpression of hGSTA2-2 significantly inhibited the activation of SAPK/JNK upon H2O2 exposure. Only a slight increase in SAPK/JNK activity was observed in these cells after 2 and 6 h of exposure, but in contrast to the control cells, this activity returned to base-line levels after 24 h of exposure (Fig. 10C). Consistent with the earlier suggestions, our results suggest that SAPK/JNK activation is an early event during H2O2-induced apoptosis and perhaps precedes DNA fragmentation, caspase 3 activation, and PARP cleavage. Furthermore, attenuation of SAPK/JNK activation by hGSTA2-2 overexpression suggests a role of PL-OOH in their activation.


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Fig. 10.   Effect of hGSTA2-2 overexpression on H2O2-induced SAPK/JNK activation in K562 cells. Cells were incubated with 30 µM H2O2 for the indicated times. Cell extracts containing 250 µg of proteins from the wild type (A), vector-transfected (B), and hGSTA2-transfected (C) cells were incubated overnight with 2 µg of GST-c-Jun-(1-89) fusion protein. After extensive washing, the kinase reaction was performed in the presence of 100 µM cold ATP as described in the text. Phosphorylation of c-Jun at Ser-63 was detected by Western blot analysis using Phospho-c-Jun (Ser-63) antibody. beta -Actin expression was shown to confirm the same amount of protein incubated with c-Jun.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of GSTs in detoxification of electophilic xenobiotics including carcinogens through the conjugation of these electrophiles to GSH is well established (26-28, 63). However, the physiological significance of selenium-independent GPx activity of GSTs, primarily associated with the Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not clear, and systematic studies in this area are lacking. In this communication, we provide strong evidence for an important role of the Alpha class GST isoenzymes, hGSTA1-1 and hGSTA2-2, in the protection mechanisms against LPO. Our results demonstrate that (i) these isoenzymes show high GPx activities toward the physiological substrates, PL-OOH generated during LPO; (ii) GSH-dependent reduction of PL-OOH by these isoenzymes occurs in biological membranes in situ; (iii) overexpression of hGSTA2-2 attenuates LPO in K562 cells under normal conditions as well as during oxidative stress; and (iv) overexpression of hGSTA2-2 in K562 cells attenuates the cytotoxic effects of H2O2 and other oxidants and protects against H2O2-induced apoptosis by blocking SAPK/JNK and caspase 3 activation.

Previous studies have suggested that GST-catalyzed GSH-dependent reduction of PL-OOH requires the prior release of FA-OOH by phospholipase A2 (64). Later studies in our laboratory have suggested, however, that intact PL-OOH can be used as substrates by the Alpha class GSTs (40). Our present results show that both recombinant hGSTA1-1 and hGSTA2-2 have relatively high activity toward PL-OOH (Table I). Consistent with the results of our previous studies (35), these results show that a prior release of FA-OOH from PL-OOH is not a prerequisite for the GSH-dependent reduction of PL-OOH by the Alpha class GSTs. GSTs are cytosolic enzymes, which raises the question whether these enzymes can catalyze the reduction of PL-OOH in situ in the biological membranes and, if so, how? By using two independent approaches to quantitate the reduction of PL-OOH in erythrocyte membranes, we demonstrate for the first time that the Alpha class GSTs can indeed catalyze the GSH-dependent reduction of PL-OOH present in biological membranes (Fig. 1 and Table II). The specific activities of hGSTA2-2 toward membrane PL-OOH derived from both approaches are similar, indicating the validity of these methods. However, the specific activity of hGSTA2-2 toward the membrane PL-OOH is remarkably lower than that toward the isolated PL-OOH. This may not be surprising because the steric factors may limit the access of the enzyme to membrane PL-OOH. The kcat (0.94 s-1) of hGSTA2-2 for membrane PL-OOH calculated from data in Table II is low. However, GSTs are known for their low catalytic efficiency which is compensated by their unusually high cellular abundance (65). The mechanisms through which presumably cytosolic GSTs catalyze the reduction of membrane PL-OOH using GSH, a hydrophilic co-substrate, are not known and should be elucidated.

We have shown previously that the cationic Alpha class GSTs including hGSTA1-1 and hGSTA2-2 are not expressed in K562 human erythroleukemia cells (41). These cells therefore provide a suitable model for studying the protective role of GSTs against oxidative stress. Our results demonstrate the attenuation of LPO in K562 cells overexpressing hGSTA2-2. Similar protection was observed in cells overexpressing hGSTA1-1 (data not presented). Under normal physiological conditions, cells overexpressing hGSTA2-2 showed a lesser extent of LPO as compared with the wild type and vector-transfected cells. This protection was much more pronounced in cells subjected to oxidative stress by including 100 µM H2O2 and traces of Fe2+ in the medium (Fig. 5). These results strongly support the role of the Alpha class GSTs against LPO caused by oxidative stress because the levels of the antioxidant enzymes and those regulating GSH homeostasis remain unaltered in the transfected cells. Furthermore, hGSTA2-2 shows no detectable GPx activity toward H2O2 (Table I) suggesting that the protective role of GSTs against LPO is attributed to their ability to reduce PL-OOHs which propagate the autocatalytic chain of LPO through continuous generation of free radicals. The functional relevance of the GPx activity of GSTs toward PL-OOH appears to be relatively more important to liver as opposed to other tissues because of the relatively higher oxidative stress in this organ due to ROS generated during the metabolism and the biotransformation of xenobiotics by the cytochrome P-450 system. The Alpha class GSTs constitute the bulk of GST protein of liver which has been estimated to be about 3-5% of the total soluble proteins of this organ (28, 34). Our results show that more than half of the total GPx activity of liver toward PL-OOH is contributed by the Alpha class GSTs. In the majority of the extrahepatic tissues such as brain, lung, heart, and pancreas where hGSTP1-1 which does not show that GPx activity is the predominant GST isoenzyme (28), only a minimal GPx activity due to GSTs is observed. However, in testis, a major portion of GPx activity toward PL-OOH is contributed by GSTs. Testes are rich in the Alpha class GSTs (28, 34), and it may be necessary to protect this tissue from ROS-induced damage. The importance of GSTs in the protection against oxidative stress in testes is underscored by recent studies showing that GST activity of germ cells is increased upon H2O2 exposure, and the inhibition of GSTs leads to enhanced LPO (66).

The pro-oxidants, including H2O2, are known to induce apoptosis in a variety of cell lines (1-10). The role of H2O2 and ROS in general in the mechanism of apoptosis is suggested by a number of studies showing that the stimuli promoting apoptosis also lead to increased formation of H2O2 and ROS (4-6, 67-69). However, the mechanisms of H2O2-induced apoptosis and the involved signaling pathway are not completely understood. Results of our TUNEL assay (Fig. 7) and DNA laddering (Fig. 8A) experiments clearly show that overexpression of hGSTA2-2 protects K562 cells from H2O2-induced apoptosis. These results also show that caspase 3-mediated PARP cleavage is also compromised in hGSTA2-2-overexpressing cells that are resistant to H2O2-induced apoptosis (Fig. 9). The observed attenuation of apoptosis in hGSTA2-2-overexpressing cells may be attributed to their ability to accelerate the reduction of PL-OOH. hGSTA2-2 cannot directly detoxify H2O2 because it has no activity toward this substrate. The overexpression of hGSTA2-2 also does not affect the enzyme activities involved in antioxidant functions and regulation of GSH homeostasis which may otherwise lead to the detoxification of H2O2 or other ROS generated during H2O2 exposure. Taken together, our results suggest that PL-OOH or the LPO products downstream of PL-OOH in the LPO cascade of reactions may be involved in H2O2-induced apoptosis in K562 cells. This contention finds support in the results of experiments showing that although the treatment of the control K562 cells with PC-OOH causes apoptosis, the cells overexpressing hGSTA2-2 are resistant to the apoptotic effect of PC-OOH under these conditions (Fig. 8B).

Another physiological hydroperoxide, 5-hydroperoxyeicosatetraenoic acid (5-HPETE), has also been implicated in the mechanisms of H2O2-induced apoptosis in K562 cells. Treatment of K562 cells with H2O2 causes activation of 5-lipoxygenase activity leading to the generation of 5-HPETE which is suggested to be involved in the mechanisms of apoptosis (70). We have demonstrated previously that hGSTA2-2 can effectively catalyze the reduction of 5-HPETE through its GPx activity (35). Therefore, it may be speculated that the observed inhibition of H2O2-induced apoptosis in hGSTA2-2-overexpressing cells may be attributed, at least in part, to enhanced GSH-dependent reduction of 5-HPETE by hGSTA2-2. Further studies are needed to explore this possibility.

4-HNE, a stable end product of LPO, has been shown to cause apoptosis in a variety of cell lines (11-15). Overexpression of hGSTA2-2 failed to protect K562 cells from apoptosis caused by 4-HNE which is generated later than PL-OOH in the cascade of LPO chain reactions. This may be expected because hGSTA2-2 does not show high activity for catalyzing the conjugation of 4-HNE to GSH (50). It has been shown that hGSTA4-4 (38, 39) and its rat and mouse orthologs rGSTA4-4 (37) and mGSTA4-4 (36) use 4-HNE as the preferred substrate. Unpublished studies3 in our laboratory show that mGSTA4 transfection in HL-60 cells provides protection against 4-HNE-induced apoptosis further suggesting the role of GSTs in regulation of the intracellular concentrations of the products of LPO, particularly PL-OOH and their downstream products.

The MAPK family, which includes ERK-1/2, SAPK/JNK, and p38 MAPK, is involved in the regulation of cellular proliferation, differentiation, and apoptosis (71-73). A number of studies indicate that the activation of SAPK/JNK may play a crucial role in the control of apoptotic cell death. Apoptosis caused by stimuli such as TNF Alpha, UV radiation, ceramide, and oxidative stress is reported to be accompanied by the activation of SAPK/JNK (9, 56, 59-61). Blockage of SAPK/JNK activation using a dominant-negative SEK1, which cannot phosphorylate and activate SAPK/JNK, protects against the apoptosis induced by various agents (60, 61) suggesting the involvement of SAPK/JNK in the initiation of apoptosis. Our studies on the effect of hGSTA2-2 overexpression on SAPK/JNK activation in K562 cells show that H2O2 treatment causes an early (within 2 h) and persistent activation of SAPK/JNK that lasts for at least 24 h in the control cells that are prone to H2O2-induced apoptosis (Fig. 10, A and B). The cells overexpressing hGSTA2-2, which are resistant to H2O2-induced apoptosis, show only a slight and transient activation of SAPK/JNK upon treatment with H2O2. Only a slight activation of SAPK/JNK in these cells was observed after 2 h of H2O2 treatment that peaked after 6 h, declined thereafter, and became comparable to the untreated cells within 24 h (Fig. 10C). The significance of this transient activation of SAPK/JNK in cells overexpressing hGSTA2-2 is not understood and must be investigated further. It may be pointed out that previous studies (58, 62) have suggested that transient activation of SAPK/JNK may lead to cell proliferation/differentiation whereas sustained activation of SAPK/JNK may lead to apoptosis. It may be speculated that the levels of LPO products that are regulated by hGSTA2-2 may be one of the determinants of the differential (transient or sustained) activation of JNK. These speculations, however, need to be substantiated by further studies.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants EY 04396 (to Y. C. A.) and CA 77495 (to S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Human Biological Chemistry and Genetics, 7.138 Medical Research Bldg., University of Texas Medical Branch, Galveston, TX 77555-1067. Tel.: 409-772-2735; Fax: 409-772-6603; E-mail: ycawasth@utmb.edu.

Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M100551200

2 The nomenclature of GSTs is based on Ref. 74.

3 J.-Z. Cheng, Y. Yang, S. S. Singhal, M. Saini, U. Pandya, P. Zimniak, S. Awasthi, and Y. C. Awasthi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; GSTs, glutathione S-transferases; GPx, glutathione peroxidase; LPO, lipid peroxidation; MAPK, mitogen-activated protein kinases; SAPK/JNK, stress-activated protein kinases/c-Jun N-terminal kinases; PARP, poly(ADP-ribose) polymerase; GR, glutathione reductase; gamma -GCS, gamma -glutamylcysteine synthetase; SOD, superoxide dismutase; CAT, catalase; PL-OOH, phospholipid hydroperoxides; PC-OOH, dilinoleoylphosphatidylcholine hydroperoxide; PE-OOH, dilinoleoylphosphatidylethanolamine hydroperoxide; 9-LOOH, 9-hydroperoxylinoleic acid; 13-LOOH, 13-hydroperoxy linoleic acid; CU-OOH, cumene hydroperoxide; CDNB, 1-chloro-2,4-dinitrobenzene; 4-HNE, 4-hydroxy-2-nonenal; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; IEF, isoelectric focusing; TBARS, thiobarbituric acid-reactive substances; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase dUTP nick end labeling; FA-OOH, fatty acid hydroperoxides; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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