, Ji-Zhong Cheng , Sharad S. Singhal , Manjit Saini …Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation (LPO) under normal conditions as well as during the oxidative

hGSTA2-2 overexpression blocks H2O2 induced apoptosis

1

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 Yang∗, Ji-Zhong Cheng∗, Sharad S. Singhal# , Manjit Saini∗, Utpal Pandya∗,

Sanjay Awasthi# and Yogesh C. Awasthi∗1

From the ∗Department of Human Biological Chemistry and Genetics, University of Texas

Medical Branch, Galveston, Texas 77555 and #Department of Chemistry and

Biochemistry, University of Texas at Arlington, Arlington, Texas 76019

Running title: hGSTA2-2 overexpression blocks H2O2 induced apoptosis.

¶ Supported in part by NIH grants EY 04396 (YCA) and CA 77495 (SA)

1To whom correspondence should be addressed:

Yogesh C. Awasthi, Ph. D., Department of Human Biological Chemistry and Genetics,

7.138 Medical Research Building, University of Texas Medical Branch, Galveston, Texas

77555-1067.

Tel.: 409-772-2735; Fax: 409-772-6603; E-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 7, 2001 as Manuscript M100551200 by guest on M

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Summary

The physiological significance of the Se-independent glutathione peroxidase

(GPx) activity of glutathione S-transferases (GSTs), associated with the major alpha-class

isozymes hGSTA1-1 and hGSTA2-2, is not known. In present studies we demonstrate

that these isozymes show high GPx activity towards phospholipid hydroperoxides (PL-

OOH) and they can catalyze glutathione (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 (LPO)

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, while 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 c-Jun N-terminal kinases (JNK), while in hGSTA2-2 overexpressing cells,

only a slight activation of JNK activity is observed at 6 h which declines to basal levels

within 24 hr. 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 towards H2O2 suggesting that the alpha-class

GSTs play an important role in regulation of the intracellular concentrations of the LPO

products which may be involved in the signaling mechanisms of apoptosis.

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Introduction

Treatment with hydrogen peroxide or the agents which lead to oxidative stress

due to the generation of reactive oxygen species (ROS)2 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). While 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

vitamine A and E, ascorbate, urate and glutathione (GSH) comprise the non-enzymatic


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component. While the antioxidant enzymes including superoxide dismutases (SOD),

catalase (CAT) and glutathione peroxidases (GPxs) comprise the enzymatic component.

Among the enzymatic machanisms, 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 Se-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 isozymes, 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 Se-dependent GPx activities, mammalian cells also have Se-

independent GPx activity displayed by Glutathione S-transferases (GSTs; E.C. 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 Se-independent GPx activity of GSTs towards

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 isozymes3

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


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biological membranes in situ has been demonstrated, nor the physiological significance

of the GPx activities of hGSTA1-1 and hGSTA2-2 has 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

isozymes is also not clear. Therefore, 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 examined the effect of overexpression of hGSTA2-2 in

human erythroleukemia cells (K562) on LPO during oxidative stress. Thereupon, we

have 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.

Experimental Procedures

MaterialsEpoxy-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 Chemical Co.(St

Louis, MO). 4-HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI).


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Dilinoleoyl phosphatidylcholine hydroperoxide (PC-OOH), Dilinoleoyl

phosphatidylethanolamine hydroperoxide (PE-OOH), 9-hydroperoxy linoleic acid (9-

LOOH) and 13- hydroperoxy linoleic 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 (Piscataway, NJ). All reagents for SDS-PAGE and

Western transfer were purchased from Bio-Rad (Hercules, CA). 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 autopsy service at

the University of Texas Medical Branch (UTMB) with no diagnosed disorders related to

investigated organs and blood was obtained from UTMB blood bank. The use of human

tissues in the present studies was approved by the Institutional Review Board of UTMB.

Purification of GSTsIn 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 E. coli

BL21( pLysS) (Stratagene, La Jolla, CA) and cultured in LB medium containing 50

µg/ml kanamycin. When the cultures reached an optical density of 0.6 at 600 nm, IPTG

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 IPTG. The BL21 cells were lysed by

sonication in 10 mM potassium phosphate buffer, pH 7.0, containing 1.4 mM β-

mercaptoethanol (buffer A) and centrifuged for 45 min at 28,000g at 4°C. The

supernatants were collected and subjected to affinity chromatography using GSH linked

to epoxy-activated Sepharose 6B (40). The recombinant GSTs was eluted from the GSH-


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affinity resin with 50 mM Tris-HCl, pH 9.6, containing 10 mM GSH and 1.4 mM β-

mercaptoethanol. Purification of “total GSTs” from K562 cells by GSH-affinity

chromatography was performed as described by us 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 to 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 membranesThe 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 β-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 β-mercaptoethanol, 100 µM EDTA and 100 µM PMSF at 22,000g

until the ghosts were free of the visible color of hemoglobin. The ghosts were stored

under nitrogen at –70ºC to minimize auto-oxidation. The membrane lipid peroxidation

was induced by incubating ghosts with 1 mM hydrogen peroxide and 1 mM ferrous

sulfate at 37ºC for 1 hour at dark and the reaction was stopped by adding 25 µM

desferrioxamine (25) .


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Determination of PL-OOH The peroxidized membranes suspended in 10 mM

Tris-HCl, pH 7.4, 1.4 mM β-mercaptoethanol and 0.1 mM EDTA were extracted with 2

volumes of chloroform/methanol (2:1, V/V). After centrifugation at 4000g 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) briefly described below: 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 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 4000g 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-1cm-1.

Enzyme assaysGST 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 minute at 25°C. GPx activity toward hydroperoxide substrates was

determined using the glutathione reductase (GR) coupled assay as described by us

previously (40). Briefly, 1 ml reaction mixture containing 3.2 mM GSH, 0.32 mM

NADPH, 1 unit 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 minutes at 37°C. One unit


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of GPx activity was defined as the amount of enzyme necessary to consume 1 µmol

NADPH per minute. 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 β-

mercaptoethanol by sonication and 28,000g supernatant of the homogenate was assayed

for enzyme activities. Catalase (CAT) activity was determined by the method described

by Beers and Sizer (45). Total superoxide dismutase (SOD) activity was determined

according to the method of Paoletti and Mocali (46) and β-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 γ-

glutamylcysteine Synthetase (γ-GCS) activity was determined by the method of Seelig

and Meister (48). GSH was determined using the whole lysates prepared without β-

mercaptoethanol according to the method of Beutler et al. (49).

Immunoprecipitation of GPx activity in human tissue extractsHuman tissues

were rinsed with PBS and homogenized in buffer A on ice. The homogenates (10% w/v)

were centrifuged at 28,000g for 45 minutes 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 described


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by us before (50). In immunotitration experiments, Fixed aliquots (100 µl) of 28,000g

supernatants containing 50 µg protein were incubated with increasing amount of purified

anti GST-alpha antibodies (0.25 µg to 2.5 µg IgG) at 4°C. Equal amounts of purified

pre-immune 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 10,000g for 30 minutes and the GPx activities towards 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-HRP according to the manufacturer (Amersham Pharmacia

Biotech) suggested protocol to exclude the detection of IgG.

Cell culturesK562 cells were cultured in RPMI 1640 medium containing L-

glutamine, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at

37ºC 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 hGSTA2Based on the cDNA sequences of hGSTA2,

PCR 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 Transfast transfection reagent (Promega). In these experiments, 1 × 106


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cells were incubated with liposome-encapsulated vectors (2 µg DNA/6 nmol of cationic

lipid) in 1 ml of serum-free media at 37ºC. 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 approximately 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 peroxidationLPO 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 500g for 10 min and

washed twice with PBS. The pellet was resuspended in 1ml 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 trichloacetic acid in 100 ml of water). After centrifugation at 4000g 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 pico moles malonaldehyde/mg of cell protein. Extinction

coefficient for malonaldehyde used was 1.53 × 105 M-1cm-1 .

Cytotoxicity assayThe MTT assay as described by Mosmann (51) and

Boekhorst et al (52) was used with slight modifications to determine the cytotoxicity of


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H2O2. Briefly, 2 ×104 cells in 190 µl 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 1200g for 10 minutes

and the medium within the wells was aspirated. The intracellular formazan dye crystals

were dissolved by addition of 100 µl of DMSO to each well and incubating 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 750g 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 RNAase (4 mg/ml) for 30 min at 37°C. The cellular

proteins were removed by addition of 200 µl of protein precipitation solution and

centrifuged for 4 min at 14,000g, The supernatant fractions containing genomic DNA

were concentrated by mixing with 600 µl of isopropanol. The DNA pellet obtained by

centrifugation at 14,000g for 1 min were washed by 600 µl of 70% ethanol and

solublized 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


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nm. For electrophoresis, DNA samples (2 µg) were loaded on 2% agarose gels containing

ethidium bromide. After electrophoresis for 2 hours at 50 V, gels were visualized under

UV.

TUNEL assayThe modified TUNEL (terminal deoxynucletidyltransferase

dUTP nick end labeling) assays were performed using the DeadEnd 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 750g 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

deoxynucleotidyl transferase (TdT) in 100 µl of equilibration buffer (200 mM potassium

cacodylate, pH 6.6; 25 mM Tris-HCl, pH 6.6; 0.2 mM DTT; 0.25 mg/ml BSA; 2.5 mM

cobalt chloride) at 37ºC 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 ×). Thereafter, the endogenous peroxidases were blocked by immersing the slides

in 0.3% H2O2 for 5 min. The slides were treated with 100µl 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 (DAB) 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 analysisFor detection of the expression of hGSTA2-2 in


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transfected cells, aliquots of 28,000g supernatant fraction of the K562 cell homogenate

containing 50 µg protein were subjected to Western blot analysis using the polyclonal

antibodies against the human alpha-class GSTs. For detection of PARP, 107 cells were

suspendend in 100 µl of denaturing lysis buffer containing 62.5 mM Tris-HCl (pH6.8),

6.0 M urea, 2% SDS, 10% glycerol, 1.4 mM β-mercaptoethanol, 0.00125% bromophenol

blue, 0.5% Triton X-100 and 1 mM PMSF. Cells were sonicated for 3 × 5 sec 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 of 20 µg of 28,000g

supernatants of cell homogenates using the anti-active caspase 3 antibodies (Pharmingen)

which recognizes 32 kDa pro-caspase 3 as well.

Solid-phase JNK assay107 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 β-glycerophosphate, 1 mM Na3VO4, 2.5 mM

sodium pyrophophate, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Cells were

sonicated 4 times for 5 sec on ice and then microfuged at 14,000g for 10 min. The pellets

were discarded, and the supernatants, representing cell extracts, were adjusted to 1mg/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 14,000g for 1 min. The beads were washed twice

successively with 0.5 ml of extraction buffer followed by 0.5 ml of kinase buffer


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containing 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 0.1 mM Na3VO4, 10

mM MgCl2 and 2 mM DTT (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×) 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 AnalysisThe 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.

Results

GPx activities of GSTA1-1 and GSTA2-2 towards hydroperoxides In order to

determine substrate specificities of hGSTA1-1 and hGSTA2-2, recombinant enzymes

were prepared by expressing in E. Coli. BL21 cells using pET30a(+) expression vector

and their subsequent purification 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 value of 9.3 and 8.9, respectively. These

isoenzymes were recognized by the antibodies against the cationic alpha-class GSTs of

human liver which predominantly comprise 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


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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).

GST mediated reduction of PL-OOH in erythrocyte membranes in situIn 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 towards isolated PL-

OOH as compared to 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 UTMB blood bank after

Institutional Review Board approval. The membranes were subjected to peroxidation by

incubating with H2O2 and trace of Fe2+ as detailed in the Experimental Procedure Section.

Under these conditions, approximately 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 which 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


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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

amount of hGSTA2-2 to the reaction mixture resulted in a dose-dependent accelerated

rate of NADPH consumption indicating that hGSTA2-2 displayed GPx activity towards

these substrates in membranes. The specific activity of hGSTA2-2 towards membrane

PL-OOH estimated from the curves in Fig. 1 corresponding to 0.1, 0.2, and 0.3 µg

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 towards the purified PL-OOH in isolated system (Table I). The lower activity

of hGSTA2-2 towards 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.

Quantitation of hGSTA2-2 mediated reduction of membrane PL-OOHThe

catalytic activity of hGSTA2-2 towards 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 Se-dependent GPx

isoenzyme was separately quantitated. Peroxidized membrane preparations containing

295 µg of protein and approximately 205 nmol of PL-OOH were incubated separately at


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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 205nmol to 116 nmol (about 44% reduction) in 4

min. Addition of 20 µg 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 Se-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 catalyze the reduction of PL-OOH in situ.

Immunotitration of GPx activity of human tissues towards CU-OOH and PL-

OOHIn 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 immunoprecipiated by the polyclonal

antibodies raised against the cationic alpha-class GSTs of human liver. Conditions for

immunoprecipitation protocols were first standardized to ensure complete

immunoprecipitaion 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 completely immunoprecipitate the alpha-class


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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 as

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 as 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 towards CU-

OOH and PC-OOH are presented in Fig. 2B and Fig. 2C, respectively. These results

showed that about 80% of GPx activity of human liver towards CU-OOH was

immunoprecipiated by anti-GST alpha antibodies. Likewise, about 60% of the GPx

activity of testis towards CU-OOH was immunoprecipiated 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), relatively smaller fraction of GPx activity towards CU-OOH

was immunoprecipitated. Human erythrocytes where complete lack of the alpha-class

GSTs is reported (28), the amount of GPx activity towards CU-OOH immunoprecipiated

by these antibodies was insignificant. Results of immunoprecipitation of GPx activity

towards 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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Overexpression of hGSTA2-2 in K562 cells protects against LPOFor

investigating the physiological role of alpha-class GSTs against oxidative stress through

transfection studies, 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 isozymes 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 towards CDNB. However,

about 10 fold higher GPx activities towards various hydroperoxides were observed in the

hGSTA2-transfected cells as compared to the wild type or vector alone transfected.

Isoelectric focusing profiles of the GST isozymes of vector and hGSTA2-transfected cells

presented in Fig. 4 showed that only the cells overexpressing hGSTA2-2 exhibited a peak

of GST activity towards CDNB at pH 8.9. Both vector and hGSTA2-transfected cells

however, showed a peak of GST activity at pH 4.8 which 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.

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


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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 γ-Glutamylcysteine synthetase (γ-GCS) and GR

involving 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 towards 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) were also similar. Likewise, GSH levels and the

activities of γ-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.

hGSTA2-2 overexpression protects against H2O2 cytotoxicityTo 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


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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.

hGSTA2-2 overexpression protects against H2O2 induced apoptosisExposure

to H2O2 and a variety of agents causing oxidative stress are known to induce apoptosis

(1-10). We therefore compared the extent of H2O2 induced apoptosis in the controls 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.

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


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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 towards 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 that the apoptosis

caused by H2O2 may be, at least in part, initiated by PL-OOH 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

poly(ADP ribose) polymerase (PARP) into a 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


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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).

Effect of hGSTA2-2 overexpression on SAPK/JNK activitySAPK/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. 10A and Fig. 10B 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 after 2 h of 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

baseline level 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


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cleavage. Furthermore, attenuation of SAPK/JNK activation by hGSTA2-2

overexpression suggests a role of PL-OOH in their activation.

Discussion

The role of GSTs in detoxification of electophilic xenobiotics including

carcinogens through the conjugation of these electrophiles to GSH is well estabilished

(26-28, 63). However, the physiological significance of Se-independent GPx activity of

GSTs, primarily associated with the alpha-class isozymes 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 isozymes, hGSTA1-

1 and hGSTA2-2 in the protection mechanisms against LPO. Our results demonstrate that

(i) these isozymes show high GPx activities towards the physiological substrates, PL-

OOH generated during LPO; (ii) GSH–dependent reduction of PL-OOH by these

isozymes 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 phopholipase A2 (64). Later

studies in our laboratory have, however, suggested 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 towards PL-OOH (Table I).

Consistent with the results of our previous studies (35), these results show that a prior


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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 that whether these enzymes can catalyze the reduction of PL-OOH in situ in the

biological membranes and if so how? 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 memebranes (Fig.1, Table II). The specific activities of hGSTA2-2

towards membrane PL-OOH derived from both approaches are similar indicating validity

of these methods. However, the specific activitity of hGSTA2-2 towards the membrane

PL-OOH is remarkably lower than that towards 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 previously shown 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 lesser extent of LPO as compared to the wild-type


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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 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 towards H2O2 (Table I) suggesting that the protective

role of GSTs against LPO is attributed to their ability to reduce PL-OOH which

propagate the autocatalytic chain of LPO through continous generation of free radicals.

The functional relevance of the GPx activity of GSTs towards PL-OOH appears to be

relatively more important to liver as opposed to other tissues because of 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 to 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 towards PL-OOH is contributed by the alpha-class GSTs.

In majority of the extrahepatic tissues such as brain, lung, heart and pancreas where

hGSTP1-1 which does not show 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 towards PL-OOH is contributed by GSTs. Testis are rich in the alpha-

class GSTs (28, 34) and it may be required to protect this tissue from ROS induced

damage. The importance of GSTs in the protection against oxidative stress in testis 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).


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The proxidants, including H2O2 are known to induce apoptosis in a variety of

cell lines (1-10). 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 of K562 cells from

H2O2 induced apoptosis. These results also show that caspase 3 mediated PARP cleavage

is also compromised in hGSTA2-2 overexpressing cells which 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 can not 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 to PLOOH in LPO cascade of reactions may be involved in H2O2 induced

apoptosis in K562 cells. This contention finds the support in the results of experiment

showing that while 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


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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 previously demonstrated 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 at least in part, be attributed 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 studies 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.

MAPK family which includes ERK-1/2, SAPK/JNK and p38 MAPK is involed

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,


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ceramide and oxidative stress is reported to be accompanied with the activation of

SAPK/JNK (9, 56, 59-61). Blockage of SAPK/JNK activation using a dominant-negative

SEK1, which can not 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 which lasts for at least 24 hr in the control cells

which are prone to H2O2 induced apoptosis (Fig. 10A and 10B). 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 which 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 have suggested that transient activation of SAPK/JNK may lead to cell

proliferation/differentiation while sustained activation of SAPK/JNK may lead to

apoptosis (58, 62). It may be speculated that the levels of LPO products which are

regulated by hGSTA2-2 may be one of the determinants of the differential (transient or

sustained) activation of JNK. These speculation, however, need to be substantiated by

further studies.


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FOOTNOTES:

2The abbreviations used are: ROS, reactive oxygen species; GSH, glutathione; GSTs,

glutathione S-transferases; GPx, glutathione peroxidase; LPO, lipid peroxidation; MAPK,

mitogen activated kinases; SAPK/JNK, stress-activated protein kinases/c-Jun N-terminal

kinases; PARP, poly(ADP-ribose) polymerase; GR, glutathione reductase; γ-GCS, γ-

glutamylcysteine Synthetase; SOD, superoxide dismutase; CAT, catalase; PL-OOH,

phospholipid hydroperoxides; PC-OOH, dilinoleoyl phosphatidylcholine hydroperoxide;

PE-OOH, dilinoleoyl phosphatidylethanolamine hydroperoxide; 9-LOOH, 9-

hydroperoxy linoleic acid; 13-LOOH, 13-hydroperoxy linoleic acid; CU-OOH, cumene

hydroperoxide; CDNB, 1-chloro-2,4-dinitrobenzene; 4-HNE, 4-hydroxy-2-nonenal.

3The nomenclature of GSTs is based on ref. 74. by guest on March 18, 2020

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Figure Legends

FIG. 1. In situ reduction of PL-OOH in peroxidized erythrocyte membranes.

Erythrocyte membranes were peroxidized by Fenton reaction as described in 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 µg to 0.3 µg) were

preincubated with GPx assay buffer containing 3.2 mM GSH, 0.32 mM NADPH, 1 unit

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.

FIG. 2. Immunoprecipitation of GPx activity using antibodies against the alpha-

class GSTs in different human tissues. A, Standardization of the conditions to

completely immunoprecipitate 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 10,000g for 30 min and the proteins

recovered in the supernatant and pellet fractions were subjected to Western blot analysis


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using the biotinylated polyclonal antibodies raised against human cationic alpha-class

GSTs followed by streptavidin-HRP. 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 α-class GST antibodies,

respectively. B and C, Immunoprecipitation of GPx activity towards 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 (■) 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 towards CU-OOH (B) and PC-OOH (C). The activities were

normalized to the controls. Results are the means ± S.D. of four determinations.

FIG. 3. Expression of hGSTA2-2 in transfected cells. An aliquot of 28,000g

supernatant fraction of homogenates of the control and transfected K562 cells containing

50 µg 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 HRP 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, 5 and 6, lysates from hGSTA2-transfected, wild-type and vector-

transfected K562 cells, respectively.


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FIG. 4. Isoelectric focusing (IEF) profile of GSTs purified from K562 cells. Total

GSTs were purified from 1× 108 K562 cells by GSH-affinity chromatography and

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 fraction, and pH (▲) was measured in every 5th fraction

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 thiobarbituric acid reactive substances (TBARS) assay as described in the text.

The values (means ± S.D., n=3) are presented in the bar graph. Asterisk indicates

significantly difference from the controls (P < 0.01).

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 (○), and hGSTA2-

transfected (▼) K562 cells were washed twice, resuspended in PBS, and inoculated at a

density of 2 ×105 cells/ml (50 µl/well) into 8 replicate wells with various H2O2

concentration (0 µM-50 µM) in a 96-well plate. The MTT assays were performed as

described in the text. Blank (no cells) subtracted OD590 values were normalized to control

(cells without H2O2 treatment). Representative results from one of the three independent

experiments on H2O2 cytotoxicity are presented.


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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.

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, 2, 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.

FIG. 9. Effect of hGSTA2-2 overexpression on H2O2 induced Poly(ADP-ribose)

Polymerase (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, 2 and 3, lysates from the wild-

type, vector-transfected, and hGSTA2-transfected cells, repectively, treated with H2O2 for

24 h. Lane 4, 5 and 6, lysates from the wild-type, vector-transfected, and hGSTA2-

transfected cells, respectively, treated with H2O2 for 48 h.


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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 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 of cold ATP as described in the text. Phosphorylation of c-Jun at Ser

63 was detected by Western blot analysis using Phospho-c-Jun (Ser63) antibody. β-actin

expression was shown to confirm same amount of protein incubated with c-Jun.

.


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Table I

Specific activities of recombinant hGSTA1-1 and hGSTA2-2 toward different substrates

Specific activity (µmol/min/mg)

Substrate rec-hGSTA1-1 rec-hGSTA2-2

CU-OOHa 12.60 ± 0.57 (3) 14.54 ± 0.72∗ (3)

L-OOHa, b 6.50 ± 0.31 (3) 7.80 ± 0.67∗ (3)

PC-OOHa 7.20 ± 0.14 (3) 10.21± 0.28∗∗ (3)

H2O2a N.D. (4) N.D. (4)

CDNBc 19.87 ± 0.21 (4) 10.94 ± 0.36∗∗ (4)

Values are means ± S.D., with the number of determinations in parentheses. a GPx

activities of the GSH-affinity purified recombinant enzymes were determined using 100

µM hydroperoxide and 3.2 mM GSH in assay buffer, pH 7.0 at 37°C as described in the

text; b Mixture of 9- and 13-LOOH; c GST activity was determined using 1.0 mM CDNB

and 1.0 mM GSH in assay buffer, pH 6.5 at 25°C as described in the text. Statistically

significant differences were evaluated with the unpaired Student’s t test and indicated by

∗ (P< 0.05) and ∗∗ (P<0.01).


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Table II

GSH-dependent reduction of membrane PL-OOH by GSTA2-2 and GPx

Incubation Conditions Residual PL-OOH content (nmol)

No incubation 205.2 ± 24.3

Peroxidized membranes + buffer only 210.0 ± 32.0

Peroxidized membranes + GSH 115.7 ± 8.2

Peroxidized membranes + GSH+ hGSTA2-2

25.2 ± 1.9

Peroxidized membranes + GSH + GPx-1 107.5 ± 11.3Equal 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-OOH were extracted and determined as described under Experimetal

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 weight of 50 kDa for hGSTA2-2).


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Table III

Specific activities of Total GSTs from the wild-type, vector-transfected, and hGSTA2-

transfected K562 cells

Specific activity (µmol/min/mg protein)

SubstrateWild-type K562

Vector-transfected K562

hGSTA2-transfectedK562

CDNB a 33.88 ± 2.14 (4) 35.08 ± 6.20 (4) 53.37 ± 7.42 (4)

CU-OOH b 0.38 ± 0.06 (4) 0.43 ± 0.04 (4) 4.2 ± 0.6 (3)

PC-OOH b 0.38 ± 0.06 (4) 0.36 ± 0.07 (4) 3.5 ± 0.4 (3)

PE-OOH b 0.34 ± 0.06 (4) 0.26 ± 0.04 (4) 3.0 ± 0.4 (3)

9-LOOH b 0.36 ± 0.07 (4) 0.20 ± 0.03 (4) 3.5 ± 0.3 (3)

13-LOOH b 0.37 ± 0.06 (4) 0.26 ± 0.04 (4) 4.1 ± 0.5 (3)

Total GSTs were purified in parallel experiments from equal amount of cells (1× 108)

using GSH-affinity chromatography (41). Values are means ± S. D., with the numbers of

determinations in parentheses. a GST activity determined at 25°C. b GPx activities

determined at 37°C.


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Time (min)

0 1 2 3 4

Ab

sorb

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340

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-0.015

-0.010

-0.005

0.000

GSH onlyGSH + heat inactivated hGSTA2-2GSH + 0.1 µg hGSTA2-2GSH + 0.2 µg hGSTA2-2GSH + 0.3 µg hGSTA2-2

Yang et al. Fig. 1


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liver testis pancreas lung heart erythrocytes

G

Px

act

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CU

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liver testis pancreas lung heart

GP

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kDa 1 2 3 4 5 6 7

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28

Yang et al. Fig. 2


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108 −−

50 −−

27 −−

Yang et al. Fig. 3


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Fraction Number

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GS

T a

cti

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WIld typeVector-transfectedhGSTA2-transfected

without H2O2 100 µµM H2O2 + 50 µµM Fe2+

* *

Yang et al. Fig. 5


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0 5 10 15 20 25 30 35 40 45 50

surv

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Yang et al. Fig. 6


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H2O2 PC-OOH 4-HNE

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52.5 –

36.2 –

118 – 81 –

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Awasthi and Yogesh C. AwasthiYusong Yang, Ji-Zhong Cheng, Sharad S. Singhal, Manjit Saini, Utpal Pandya, Sanjay

induced apoptosis and inhibits JNK and caspase 3 activationOverexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide

Role of glutathione S-transferases in protection against lipid peroxidation.

published online March 7, 2001J. Biol. Chem.

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, Ji-Zhong Cheng , Sharad S. Singhal , Manjit Saini …Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation (LPO) under normal conditions as well as during the oxidative

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