Enhanced Glutathione Levels and Oxidoresistance Mediated by
Increased Glucose-6-phosphate Dehydrogenase Expression*
Francesca
Salvemini
,
Annamaria
Franzé
,
Angela
Iervolino
,
Stefania
Filosa
,
Salvatore
Salzano§, and
Matilde
Valeria
Ursini
¶
From the
International Institute of Genetics and
Biophysics, Consiglio Nazionale delle Ricerche, Via Guglielmo Marconi
12, 80125 Naples and the § Center of Endocrinology and
Experimental Oncology, Consiglio Nazionale delle Ricerche, Via Pansini
10, 80131 Naples, Italy
 |
ABSTRACT |
Glucose-6-phosphate dehydrogenase (G6PD) is the
key enzyme of the pentose phosphate pathway that is responsible for the
generation of NADPH, which is required in many detoxifying reactions.
We have recently demonstrated that G6PD expression is induced by a
variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the
functional significance of such G6PD induction after oxidant insult is
still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced
glutathione and a gradual but notable increase in G6PD expression. Both
effects are seen soon after drug addition. Once G6PD activity has
reached the maximum, the GSH pool is restored. We suggest and also
provide the first direct evidence that G6PD induction serves to
maintain and regenerate the intracellular GSH pool. We used HeLa cell
clones stably transfected with the human G6PD gene that display higher
G6PD activity than the parent HeLa cells. Although the activities of
glutathione peroxidase, glutathione reductase, and catalase were
comparable in all strains, the concentrations of GSH were significantly
higher in G6PD-overexpressing clones. A direct consequence of GSH
increase in these cells is a decreased reactive oxygen species
production, which makes these cells less sensitive to the oxidative
burst produced by external stimuli. Indeed, all clones that
constitutively overexpress G6PD exhibited strong protection against
oxidants-mediated cell killing. We also observe that NF-
B
activation, in response to tumor necrosis factor-
treatment, is
strongly reduced in human HeLa cells overexpressing G6PD.
 |
INTRODUCTION |
Reactive oxygen species
(ROSs)1 are produced inside
the cell during oxidative metabolism, and they are involved in human
diseases such as arteriosclerosis, amyotrophic lateral sclerosis,
Down's syndrome, re-perfusion shock syndrome, and cancer (1-3).
Abnormal production of ROSs can damage macromolecules such as nucleic
acids, lipids, and proteins and therefore participate in necrotic cell death and apoptosis (4). Inside the cell, ROSs are scavenged by both
enzymatic and non-enzymatic antioxidant pathways. Reduced glutathione
(GSH), a cysteine-containing tripeptide, is required to maintain the
normal reduced state of the cells and to counteract all the deleterious
effects of oxidative stress. GSH is synthesized inside the cells
through a complex biochemical pathway composed of several well known
enzymes (5). During the reaction of H2O2 scavenging, GSH is oxidized to GSSG by the enzyme GSH peroxidase (6).
The reduction of GSSG to GSH is catalyzed by GSSG reductase, which uses
NADPH as reducing potential. NADPH is also required for the formation
of active catalase tetramers. This latter enzyme catalyzes the
reduction of H2O2 in H2O and
O2 (7, 8). Catalase is mainly peroxisomal, whereas the GSSG
reductase/GSH peroxidase cycle is active in the cytoplasm. The NADPH
required for the production of both GSH and catalase is produced by the
pentose phosphate pathway (9, 10).
Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting
enzyme of the pentose phosphate pathway, has long been regarded as
important in the biosynthesis of the sugar moiety of nucleic acids
(11). Until recently, the role of this housekeeping enzyme in the cell
response to oxidative stress was limited to human erythrocytes that
lack any other NADPH-producing route (12, 13). However, recent results
have demonstrated that this enzyme also plays a protective role against
ROSs in nucleated eucaryotic cells that possess alternative routes for
the production of NADPH. Indeed, in the lower eucaryote
Saccharomyces cerevisiae, mutants in the G6PD gene are
sensitive to oxidants that specifically deplete the intracellular
pool of GSH (14). Furthermore, mouse ES cells containing G6PD
null mutation are uniquely sensitive to oxidants (15).
In every cell line so far tested, as well as in lymphocyte primary
cultures, we demonstrated that G6PD expression is enhanced by oxidative
stress induced by agents that either increase the intracellular
concentration of O
2 or decrease the GSH pool. The mechanism
regulating G6PD expression appears to affect the rate of transcription
initiation (16).
Here, we confirm that G6PD expression is induced by drugs that decrease
the intracellular GSH pool; indeed, this increase is blocked by
treatment with antioxidants that specifically replenish the
intracellular GSH. We also observed that, rapidly after drug treatment,
GSH decreases and G6PD is enhanced. When G6PD reaches maximal
induction, GSH pool is restored, suggesting that GSH equilibrium could
be dependent from G6PD expression.
We have isolated and extensively characterized HeLa clones that
overexpress G6PD and thus have increased G6PD activity. We observe that
the activity of other enzymes in these cells, such as GSSG reductase,
GSH peroxidase, catalase, or 6PGD (the second enzyme of pentose
phosphate pathway) remain unchanged. Furthermore, these cells feature
increased intracellular levels of GSH and consequently, lower ROSs
levels with respect to control cells. The G6PD-overexpressing cells
exhibit a markedly decreased NF-
B DNA binding activity, produced by
the TNF-
-dependent ROS burst, and a marked increase in
cellular resistance to apoptosis induced by hydrogen peroxide and
TNF-
.
In conclusion, the results reported here suggest that G6PD is part of
an inducible mechanism of cell response to oxidative stress;
furthermore, they support the hypothesis that G6PD plays a key role in
the control of intracellular reductive potential by increasing the
intracellular content of glutathione, which, in turn, decreases ROS
levels. This overall intracellular reduced environment may facilitate
cellular protection against oxidant injuries.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines and Culture Conditions--
The human hepatoma Hep3B
cell line and HeLa cell line were grown in Dulbecco's modified minimal
essential medium. Media were supplemented with 10% fetal calf serum
and 1% penicillin/streptomycin (Life Technologies, Inc.). The cells
were cultured and stably transfected as described elsewhere (17). The
plasmid pGD15neo has been described (18); it also contains a Neo gene
driven by the SV40 early promoter. Control HeLa cells were transfected with the same plasmid devoid of all the human G6PD coding sequences. Cells were harvested 48 h after transfection, and clones were selected and cultured in 400 µg/ml G418. No significant change in
G6PD levels (mRNA, protein, and activity) was detectable between wild type and vector control-transfected clones (HeLa-neo).
Cell Treatment and Enzyme Assay--
All the experiments
described here were performed on actively growing cells. To summarize
briefly, the medium was removed and cells were incubated in
phosphate-buffered saline containing the indicated concentrations of
H2O2 (Merck), NAC, PDTC (Sigma), and diamide
(Calbiochem); the concentration of each oxidant used was empirically
determined. After a 30-min incubation at 37 °C, cells were rinsed
with phosphate-buffered saline and fresh medium was added.
G6PD activity was determined, as already described (19), by measuring
the rate of production of NADPH. Since 6PGD, the second enzyme of the
pentose phosphate pathway, also produces NADPH, both 6PGD and total
dehydrogenase activity (G6PD+6PGD) were measured separately, as
described elsewhere (20), in order to obtain accurate enzyme activity.
G6PD activity was calculated by subtracting the activity of 6PGD from
total enzyme activity.
Catalase, glutathione reductase, glutathione peroxidase activities, and
total protein concentration were determined according to Aebi (21),
Carlberg (22), Mannervick (23), and Bradford (24), respectively.
G6PD Electrophoresis--
Cellulose acetate gel electrophoresis
and staining for G6PD was carried out according to published methods
(25), with the following modifications. (a) Gel running
buffer was 85 mM Trizma base, pH 9.2, 35 mM
glycine, 58 mM sucrose, 0.2 mM EDTA, 36.6 mM NADP. (b) Length of run was 90 min at 100 V
at room temperature. (c) Gel staining solution was 0.1 mM Tris-Cl, pH 8.0, 0.2 mg/ml glucose 6-phosphate, 0.1 mg/ml NADP, 0.15 mg/ml dimethylthiazol-2-diphenyltetrazolium bromide,
0.1 mg/ml phenazine methosulfate, 20 mM
MgCl2.
Immunoblot--
15 µg of protein lysates were separated by
SDS-polyacrylamide gel electrophoresis and electroblotted to
nitrocellulose. Nitrocellulose filter was incubated with rabbit
anti-human G6PD antibody supplied by U. Benatti (26) in 3% bovine
serum albumin. For detection of rabbit antibodies, the filter was
incubated with goat anti-rabbit IgG conjugated with horseradish
peroxidase (Bio-Rad) and developed with a substrate of 0.5 mg/ml
4-cloronaphthol and 0.33% hydrogen peroxide in 0.1 M
Tris-HCl. Immunoblots using an antibody raised against human
-actin
(Amersham Corp.) were performed according to the manufacturer's
instructions. They were revealed with the ECL kit from Amersham, and
autoradiographs were recorded onto X-Omat AR films (Eastman Kodak
Co.).
Electrophoretic Mobility Shift Assays--
Gel shift assays were
performed using standard methods; a double-stranded
B
oligonucleotide, whose sequence has already been described (27), was
used as probe. The oligonucleotide was labeled using Klenow enzyme. The
radiolabeled probe was incubated with HeLa nuclear extract for 30 min
at room temperature. For oligonucleotide competition analysis, a
100-fold molar excess of cold competitor was also added to the mixture.
In order to carry out DNA binding assays in which antibodies were
included, antibodies were added to the binding assay mixtures and
incubated for 2 h on ice before the addition of radiolabeled
probe. The p65 antibody was purchased from Santa Cruz Biotechnology.
HeLa cell nuclear extracts were made using Lee's method (28). DOC
treatment was performed by incubating the cytosolic fraction from
unstimulated cells for 15 min with 0.8% DOC and 1% Nonidet P-40
before electrophoretic mobility shift assay (29). A Molecular Dynamics
PhosphorImagerTM system was used to analyze the gels.
Intracellular Glutathione Measurement--
Intracellular reduced
glutathione content was estimated using the Bioxitech GSH-400 enzymatic
method (OXIS) in accordance with the manufacturer's
instructions. Briefly, total cellular protein material (from 6 × 106 cells) was precipitated in 5% metaphosphoric acid and
the resulting supernatant was used for the test. The level of GSH
present in each cell type was calculated according to standard
curves of increasing GSH concentrations.
Fluorescent Measurement of Intracellular ROSs--
Formation of
ROSs was measured using DCFH-DA according to Royall and Ischiropoulos
(30). Cells (6 × 106) were pre-loaded with 5 mM DCFH (Molecular Probes) in the culture medium for 30 min. Measurement were carried out in duplicates using a FACScan (Becton
Dickinson) flow cytometer. Dead cells and debris were excluded by
forward/side scatter gating .
Induction of Apoptosis--
Cells were plated 24 h before
the assay at a density of 1 × 106 cells/10-cm plate.
Apoptoic cell death was induced by H2O2 and TNF-
used at the indicated concentration. 24 h after induction of apoptosis, cells were washed in phosphate-buffered saline and then
resuspended in the same buffer and fixed with ice cold 70% ethanol.
Fixed cells were stained with 50 µg/ml propidium iodide and DNA
content was analyzed using a FACScan (Becton Dickinson) flow cytometer
by red fluorescence. Apoptotic cells appear as a broad hypodiploid DNA
peak preceding the peak of diploid DNA from viable cells. Cells
containing a lower amount of DNA and a side scatter higher than that of
G0/G1 cells were considered to be apoptotic
(31, 32).
 |
RESULTS |
G6PD Expression Is Enhanced by GSH-depleting Drugs: This Effect Is
Specifically Counteracted by NAC--
We have already reported that
several oxidative drugs, whose final effect is to decrease the
intracellular GSH pool, enhanced the expression of G6PD and that this
regulation is achieved at the levels of transcription of this gene
(16). In the first experiment, human Hep3B cells were treated with
diamide, a powerful sulfhydryl group-oxidizing agent that specifically
causes the depletion of glutathione without any other observable effect
(33). During the same experiment, we tested the antioxidant ability of
both NAC and PDTC. NAC is a GSH precursor (34), whereas PDTC is a
metal-chelating agent, which complexes Fe3+ and other metal
ions; these, by means of the Fenton reaction, participate in the
production of hydroxyl radicals from H2O2 (35). Hep3B cells were exposed to 500 µM diamide for 30 min in
phosphate-buffered saline; the buffer was then replaced with fresh
medium. In the cases in which NAC (30 mM) or PDTC (500 µM) were used, each antioxidant was added to the cells
1 h before diamide treatment. Two hour after cell treatments, G6PD
activity was analyzed by both spectrophotometric assay and cellulose
acetate gel electrophoresis. This last method allows a specific
determination of G6PD activity without the interference of other
NADPH-producing enzymes (25). During the same experiments, we also
measured the levels of G6PD protein by immunoblot analysis of total
cellular proteins probed with anti-G6PD antiserum (26). In the same
immunoblot,
-actin protein levels was also measured as reference
(Fig. 1). As reported in Table
I, we found that G6PD activity was
2.1-fold increased in diamide-treated cells; this increase is abolished
by NAC pretreatment (see also Fig. 1B for cellulose acetate
gel electrophoresis analysis of G6PD activity). A similar result was
observed when another GSH precursor, glutathione diethyl ester, was
used instead of NAC (data not shown). PDTC does not counteract diamide
effect but, rather, determines an enhancement of G6PD protein levels
and of G6PD activity comparable to that observed with diamide
(2.1-fold). This is not surprising since this compound can exert either
antioxidant or pro-oxidant effects in different situations (36).
Indeed, it was demonstrated that PDTC and other dithiocarbamates may
cause oxidation of GSH by means of a non-radical mechanism (35). We
further demonstrated that the PDTC-mediated increase of G6PD is also
counteracted by NAC (Table I).

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Fig. 1.
Regulation of G6PD expression by oxidants and
antioxidants. A, Western blot analysis of G6PD protein
levels made with extracts of Hep3B cells treated with oxidants and/or
antioxidants, as described under "Results". Protein extracts from
Hep3B cells left untreated (lane 1) or treated
with 30 mM NAC (lane 2), 500 µM PDTC (lane 3), or 500 µM diamide (lane 4), NAC + diamide
(lane 5), and PDTC + diamide (lane
6) were analyzed. G6PD protein was detected with rabbit
anti-human G6PD polyclonal antibodies. The membrane was then stripped,
and ECL-Western blot analysis was performed with monoclonal antibody
against -actin to verify the loading. A representative experiment is
presented. B, G6PD enzyme activity was estimated using
cellulose acetate gel electrophoresis of cell lysates. 2 µg of
protein extracts from human blood cells (control
lane) and Hep3B cells left untreated (lane
1) or treated with 30 mM NAC (lane
2), 500 µM PDTC (lane
3), or 500 µM diamide (lane
4) were analyzed using a staining method allowing the
specific determination of G6PD activity. A representative experiment is
presented.
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Table I
Effects of oxidants and antioxidants on G6PD activity in Hep3B cells
Hep3B cells were treated for 30 min in phosphate-buffered saline with
or without (No addition) 500 µM diamide, 500 µM BSO, or 500 µM PDTC (listed in the first
column) preceded or not (No addition) by pretreatment with either 30 mM NAC or 500 µM PDTC for 1 h in growth
medium (listed in the first row). The buffer was then replaced with
fresh medium and cells were collected after 2 h. At the end of
incubation period G6PD activity was determined and defined as nanomoles
of reduced NADP/min/mg of protein as described under "Experimental
Procedures." Results are given as mean ± S.D. of three
independent experiments. ND, not determined.
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As shown in Fig. 1A, the results obtained by activity
determination, perfectly parallel to what was observed using protein analysis. Hence, the GSH-mediated regulation of G6PD activity results
from regulated levels of this protein. Identical results were obtained
when BSO, an effective inhibitor of
-glutamylcysteine synthetase
(the enzyme that catalyzes the first step in biosynthesis of GSH) (6),
was used in place of diamide to reduce the intracellular GSH content
(Table I).
Results comparable to those reported in Table I were obtained in HeLa
cells after diamide or BSO treatments (data not shown).
Rapid G6PD Induction after Diamide Treatment Precedes GSH
Replenishment--
We then compared the kinetics of increase of G6PD
activity and the levels of intracellular GSH after diamide treatment.
Hep3B cells were treated with diamide as reported in Fig. 1; cells were collected at the indicated time points. As reported in Fig.
2, we observed a slight but reproducible
enhancement of G6PD activity (1.3-fold) as soon as 10 min after drug
treatment; simultaneously, GSH was dramatically reduced (80%
reduction). As G6PD activity gradually increases, the levels of
intracellular GSH had restored and, when G6PD reaches the maximum
levels, GSH levels are 30% higher than in untreated control cells.
8 h after treatment, both G6PD activity and GSH levels returned to
normal level.

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Fig. 2.
Kinetic analysis of G6PD activity and GSH
levels. Hep3B cells were treated with 500 µM diamide
as reported in Fig. 1 and collected at the indicated time points. G6PD
activity and GSH levels were determined as described under
"Experimental Procedures." The time point values reported in the
graph represent the fold induction normalized to time 0, for each
cellular type. Results are given as mean ± S.D. of three
independent experiments. Note the GSH replenishment soon after G6PD
induction.
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These observations indicate that G6PD expression is inversely
correlated to GSH intracellular levels and may also suggest that GSH
replenishment may depend on G6PD expression.
Regulation of GSH Levels by G6PD Overexpression--
In order to
assess how G6PD expression may influence GSH levels, cell lines
producing increased levels of G6PD were generated. Human HeLa cells
were stably transfected with an eucaryotic expression vector containing
the entire human G6PD gene (18). Western blot analysis revealed several
cell clones carrying pGd15neo that had increased levels of G6PD
expression. As reported in Fig. 3, two of
these clones were analyzed in more detail. Clone HeLa-Gd3 and clone
HeLa-Gd4 show 3-fold increase in G6PD protein and G6PD activity compared with vector-control transfected clones (HeLa-neo). The increased levels of G6PD protein were also confirmed by the increase in
G6PD mRNA as determined by Northern blot analysis (data not shown).

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Fig. 3.
Characterization of G6PD-overexpressing
clones. A, Western blot analysis of G6PD protein levels
in two HeLa cell clones overexpressing G6PD (Gd3 and Gd4) in comparison
to control HeLa-neo cells. G6PD protein was detected with rabbit
anti-human G6PD serum. B, G6PD activity measured in HeLa-neo
control cells and HeLa-Gd3 and HeLa-Gd4 clones. Note the 3-fold
increase of both G6PD activity and protein respect to HeLa-neo
cells.
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Subsequently, we evaluated the effect of G6PD expression on the
activities of the enzyme of GSSG recycling (GSSG reductase and GSH
peroxidase), on the activity of
H2O2-detoxifying enzyme catalase and on 6PGD,
the second enzyme of the pentose phosphate pathway. We also analyzed
the levels of intracellular GSH. Overexpression and increased activity
of G6PD have no significant effect on GSSG reductase, GSH
peroxidase, catalase, and 6PGD activities; on the other hand, a
2.3-fold increase of GSH levels were detectable in both HeLa-Gd3 and
HeLa-Gd4 clones with respect to control HeLa-neo cells (Table
II).
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Table II
Activity of antioxidant enzymes in HeLa cells overexpressing or not
overexpressing human G6PD
The enzyme activities are defined as following: G6PD and 6PGD,
nanomoles of reduced NADP/min/mg of protein; catalase, micromoles of
H2O2/min/mg of protein assayed at 30 mM
H2O2; GSHPx, nanomoles of NADPH/min/mg of protein
assayed at 2 mM GSH; GR, nanomoles of NADPH/min/mg of
protein; GSH, nmol/106 cells. Numbers are means ± S.D. of
three independent experiments. GR, glutathione reductase; GSHPx,
glutathione peroxidase.
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Overexpression of G6PD Decreases the Intracellular Levels of
ROSs--
We then investigated whether the increased GSH levels
associated with G6PD overexpression might have resulted from decreased levels of ROSs production in those cells. Therefore, we analyzed the
ROS levels in control HeLa-neo, Gd3 (Fig. 3), and Gd4 (not shown)
clones with DCFH-DA, which is converted to DCF by ROS-mediated oxidation (30). Cells stained with DCF were analyzed by
fluorescent-activated cell sorting. The results reported in Fig.
4 clearly show a significant decrease in
the intensity of DCF fluorescence resulting from the overexpression of
G6PD (44% of the control cells).

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Fig. 4.
G6PD overexpression in HeLa cells decreases
the level of reactive oxygen species by acting on the glutathione
metabolism. In vivo estimation of intracellular ROSs
level by fluorescent-activated cell sorting analysis using DCFH-DA
probe. DCF was measured as described under "Experimental
Procedures." In A the results are presented as
fluorescence histograms determined in a representative experiment. In
B data are presented as DCF fluorescence index that was
calculated as the ratio between the DCF fluorescence index of each
sample and that measured in control HeLa-neo cells. Results are given
as mean ± S.D. of three independent experiments. a,
control HeLa-neo cells; b, HeLa-Gd3 clones; c and
d, control HeLa-neo cells and HeLa-Gd3 clone treated for
24 h with 1 mM BSO before intracellular ROSs
determination. Similar results were obtained using HeLa-Gd4 clone (not
shown).
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The question remained as to whether the G6PD overexpression-mediated
lower ROS intracellular levels resulted from a higher detoxification of
ROSs due to elevated concentrations of reduced glutathione;
alternatively, G6PD may have directly reduced the basic ROS formation
and, as a consequence, increased the cellular concentration of
glutathione. The production of ROSs was therefore analyzed in
glutathione-depleted control HeLa-neo cells as well as Gd3 and Gd4
clones by using the in vivo conversion of DCFH-DA to
fluorescent DCF. Glutathione depletion was induced by 24-h incubation
of the cells with 1 mM BSO, a glutathione synthesis inhibitor (6). This treatment depleted glutathione by >99% in all
types of cells (data not shown) and increased the rate of ROS formation
(Fig. 4). It is of interest that the G6PD-mediated lower rate of ROS
formation, which was clearly detectable in non-BSO-treated cells, was
almost completely abolished by the glutathione depletion. This supports
the hypothesis that G6PD acts at the level of glutathione metabolism
rather than the level of ROS formation.
TNF-
-mediated Activation of NF-
B Is Attenuated by G6PD
Overexpression--
The ability of G6PD expression to decrease ROS
levels was investigated using targets that are particularly sensitive
to oxidative burst. We therefore analyzed the TNF-
-mediated
activation of the transcription factor NF-
B in HeLa cells together
with Gd3 and Gd7 clones. Most inducers of NF-
B seem to rely on the
production of ROSs, as evidenced by the inhibitory effect of several
antioxidants, including NAC (33). A causal link between ROS production
and TNF-
-mediated NF-
B induction was clearly demonstrated (27, 37). The activation of NF-kB by TNF-
was analyzed by in
vitro DNA binding and electrophoretic mobility shift assays.
Nuclear extracts were prepared from control HeLa cells as well as Gd3 and Gd4 clones that were either left untreated or exposed to TNF-
. DNA binding assays were performed using a DNA probe encompassing the
B motif (see "Experimental Procedures"). As seen in Fig. 4, 2-h
treatment with 25-200 units/ml TNF-
induced, in control HeLa cells,
the binding of a protein factor to
B oligonucleotide. Competition
experiments revealed that the binding to the radioactive
B DNA was
no more detectable when increasing concentration of nonradioactive
B
DNA were added to the binding mixture. A supershift band was also
observed when the reaction mixture was incubated with an antibody that
recognizes the p65/RelA subunit of NF-
B. Hence, TNF-
induced the
binding of NF-
B to the
B oligonucleotide in HeLa cells. The
binding was greatly reduced in Gd3 cells (Fig. 5) and Gd4 cells (not shown), which
overexpress G6PD with respect to control HeLa-neo cells. In order to
determine the amount of inducible NF-
B present in the cytoplasm of
HeLa-neo and HeLa-Gd3 cells, we treated cytoplasmic fraction obtained
from both cells with 0.8% DOC and 1% Nonidet P-40. This treatment
allows the dissociation of I
B-
from NF-
B and thus restores the
DNA binding ability of this factor. As shown in Fig. 5
(lanes 13 and 15), DOC treatment promotes similar levels of NF-
B binding to
B DNA in HeLa-neo and
HeLa-Gd3. This indicates that the increase in G6PD activity did not
alter the intrinsic ability of NF-
B to bind DNA, but rather at least
partially impairs the process that leads to the activation of this
factor due to oxidative stress.

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Fig. 5.
G6PD overexpression attenuates
TNF- -mediated NF- B binding activity. Nuclear extracts were
prepared from HeLa-neo cells (lanes 1-5) and
HeLa-Gd3 clones (lanes 6-10) treated with
increased quantity (25-200 units/ml) of TNF- . NF- B binding
activity was analyzed by electrophoretic mobility shift assay as
described under "Experimental Procedures." In A, an
autoradiograph of a typical experiment is presented. Unlabeled
competitor (lane 11) or antiserum that recognize
the p65 subunit of NF- B (lane 12) were added
to cell extracts to control the specificity of the binding. In
lanes 12-15, equal amounts of cytoplasmic
extracts of either HeLa-neo and HeLa-Gd3 were incubated with B
probe. The mixtures were either left untreated (lanes
12 and 14) or treated for 15 min with 0.8% DOC
in the presence of 1% Nonidet P-40 before electrophoretic mobility
shift assay (lanes 13 and 15). In
B, results are reported as fold induction over the control
(untreated cells). Results are given as mean ± S.D. of three
independent experiments. Note the parallel but attenuated
TNF- -mediated induction of NF- B binding activity in HeLa-Gd3
clone respect to control cells.
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Sensitivity to Apoptotic Signals Is Influenced by Levels of G6PD
Expression--
Since G6PD overexpression raises the intracellular
glutathione content and, as consequence, decrease the intracellular ROS production, we investigated whether this phenomenon can influence the
survival of these cells exposed to oxidants burst. Apoptosis was
produced in adherent cells treated with both hydrogen peroxide and
TNF-
at the indicated concentrations. In this experiment, we also
used parent HeLa cells as control. 24 h after treatment, the rate
of apoptotic death was measured on cells fixed in 70% ethanol, stained
with propidium iodide, and subsequently analyzed for DNA content by
flow cytometry. The data shown in Fig. 6
clearly demonstrate that cell death was strongly decreased in
G6PD-overexpressing cells (Gd3 and Gd4) in comparison to control cells
(HeLa and HeLa-neo). Thus, the levels of G6PD have a dramatic effect on
the susceptibility to apoptotic cell death induced by both agents.

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Fig. 6.
Flow cytometry analysis of
H2O2 and TNF- induced apoptosis in HeLa,
HeLa-neo, HeLa-Gd3, and HeLa-Gd4 clones. After incubation with
either H2O2 and TNF- for 24 h, control
and treated cells were fixed in 70% ethanol, stained with propidium
iodide, and subsequently analyzed for DNA content by flow cytometry.
A, representative flow cytometry determinations of apoptotic
cell death. DNA content frequency histograms are reported.
a, e, and i, HeLa cells; b,
f, and j, HeLa-neo clone; c,
g, and k, Gd3-neo clone; d,
h, and l, Gd4-neo clone. a-d,
untreated cells; e-f, cells treated with 500 µM H2O2; i-l, cells
treated with 100 units/ml TNF- . B, rate of apoptotic cell
death induced by hydrogen peroxide and TNF- in HeLa, HeLa-neo,
HeLa-Gd3, and HeLa-Gd4 cells. Results are given as mean ± S.D. of
three independent experiments.
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DISCUSSION |
We have already shown that G6PD expression is up-regulated
by oxidants through a mechanism acting mainly on the rate of
transcription of this gene (16). Here we show that this effect is
specifically counteracted by NAC, an antioxidant that replenishes the
GSH pool (35). Furthermore, we found that G6PD increase precedes full GSH restoration, through kinetic analysis of G6PD expression after drug
treatment. G6PD overexpression in HeLa cells therefore determines an
increase in GSH and a consequent decrease of intracellular ROSs. We
show that G6PD expression controls intracellular GSH without
interfering with the activity of the other enzymes involved in the
peroxide/hydroperoxide-detoxifying pathway. As a result, cells
overexpressing G6PD have an overall reduced state. Consequently, downstream effects induced by ROSs burst are buffered in these cells.
We found that G6PD overexpression interferes negatively with the
activation of the transcription factor NF-
B by TNF-
and decreases
susceptibility to apoptotic signals induced by hydrogen peroxide and
TNF-
.
It has been recently suggested that the primary physiological role of
G6PD in mammalian cells is the defense against oxidative injuries (15,
38). The question remains as to how G6PD exerts this protective effect.
Several pieces of evidence indicated that the formation of GSH from its
oxidized form, GSSG, is dependent on NADPH produced by the pentose
phosphate pathway and that this pathway can be activated in response to
GSH depletion (39). In agreement with these observations, we suggest
that levels of G6PD may have a dominant role in the control of output
of GSH and thus in the maintaining of an intracellular redox potential. An alternative explanation could be that G6PD is involved in a mechanism that promotes glutathione storage. Therefore, the exact biochemical mechanism underlying the antioxidant properties of G6PD
enzyme require a more thorough investigation.
Besides the enzymes directly involved in the production and
detoxification of ROSs, several proteins have been shown to decrease the net intracellular generation of ROSs and consequently interfere with the downstream effects of oxidative stress. Some proteins, such as
bcl-2, have been proposed to control intracellular ROSs levels in a
GSH-independent way (40). Other, such as the small heat shock protein
hsp27, lead to a decreased production of ROSs as derived from the
increased intracellular GSH content (41). In this last case, the
hsp27-dependent G6PD enhanced expression was proposed as an
explanation of this
phenomenon.2
Furthermore, decreased ROSs production has been reported in cells with
constitutive decreased expression of transaldolase, the key enzyme of
nonoxidative branch of pentose phosphate pathway (42), or in cells
overexpressing GSH peroxidase (43). In both cases lower ROSs were
derived from an increased GSH/GSSG ratio. In this paper we show that
G6PD shares the same properties, but, more interestingly, it is subject
to rapid up-regulation in response to oxidative stress. This could be a
general rule, since we have already shown that G6PD expression is
increased in several human cell lanes by drugs that ultimately lead to
GSH depletion (16, 17). Furthermore, we have recently demonstrated
that, in primary cultures of human and bovine lymphocytes,
pesticide-mediated genotoxic effects are coupled to oxidative stress
and G6PD up-regulation (44, 45). However, as G6PD enzyme levels were
found to be positively regulated by a number of stimuli, such as
platelet-derived growth factor (20) and ischemia reperfusion (46, 47),
it would be interesting to investigate whether G6PD-enhanced expression can always be related to change in the cellular redox conditions.
It is well known that G6PD is expressed in all cell types, although at
varying levels (19). We suggest that G6PD serves as a critical
determinant of tissue- and cell type-specific sensitivity to oxidative
stress signals. However, we cannot exclude the possibility that other
pentose phosphate pathway enzymes, or enzymes involved in the control
of peroxide/hydroperoxide scavenging pathway, may have an alternative
role to G6PD in different cell types.
 |
ACKNOWLEDGEMENTS |
We thank André Patrick Arrigo and Guido
Rossi for the critical review of the manuscript and helpful advice. We
also thank Maria Terracciano for skillful technical assistance.
 |
FOOTNOTES |
*
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: International
Inst. of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Via Guglielmo Marconi 12, 80125 Naples, Italy. Tel.: 39-81-7257-248; Fax: 39-81-5936-123; E-mail: ursini{at}iigbna.iigb.na.cnr.it.
The abbreviations used are:
ROS, reactive oxygen
species; G6PD, glucose-6-phosphate dehydrogenase; BSO, L-buthionine-(S,R)-sulfoximine; 6PGD, 6-phosphogluconate dehydrogenase; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetylcysteine; DOC, sodium deoxycholate; DCFH-DA, dichlorofluorescein diacetate; DCF, dichlorofluorescein; TNF-
, tumor
necrosis factor-
.
2
A. P. Arrigo, personal communication.
 |
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