From the 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
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ABSTRACT |
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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.
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.
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
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-
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 Determination of PL-OOH--
The peroxidized membranes suspended
in 10 mM Tris-HCl, pH 7.4, 1.4 mM
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 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 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
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 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.
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).
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.
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.
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.
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.
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
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.
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.
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).
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.
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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-
-D-galactopyranoside. The BL21 cells
were lysed by sonication in 10 mM potassium phosphate
buffer, pH 7.0, containing 1.4 mM
-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
-mercaptoethanol. Purification of "total
GSTs" from K562 cells by GSH affinity chromatography was performed as we described previously (41).
-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
-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).
-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.
-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
-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
-glutamylcysteine synthetase (
-GCS) activity was determined by the method of Seelig and Meister (48). GSH was determined using the whole lysates prepared
without
-mercaptoethanol according to the method of Beutler et
al. (49).
1 cm
1
.
-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.
-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
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Specific activities of recombinant (rec) hGSTA1-1 and hGSTA2-2 toward
different substrates
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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.
GSH-dependent reduction of membrane PL-OOH by GSTA2-2
and GPx
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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 (
) 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.
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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.
Specific activities of total GSTs from the wild-type,
vector-transfected, and hGSTA2-transfected K562 cells
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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 (
) was measured in every
5th fraction.
-glutamylcysteine synthetase (
-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
-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).
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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 (
), and
hGSTA2-transfected (
) 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.
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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.
View larger version (56K):
[in a new window]
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.
View larger version (55K):
[in a new window]
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.
View larger version (83K):
[in a new window]
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. -Actin expression was shown to confirm the same amount of
protein incubated with c-Jun.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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FOOTNOTES |
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* 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.
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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;
-GCS,
-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.
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REFERENCES |
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