Importance of glucose-6-phosphate
dehydrogenase activity in cell death
WANG-NI TIAN, LEIGH D. BRAUNSTEIN, KIRA APSE, JIONGDONG PANG,
MARK ROSE, XIAONI TIAN, AND ROBERT C. STANTON
Renal Division and Department of Medicine, Joslin Diabetes Center, Beth Israel Deaconess
Medical Center and Harvard Medical School, Boston, Massachusetts 02115
oxidative stress; pentose phosphate pathway; apoptosis
A COMPLEX INTERPLAY OF intracellular signals and metabolic processes is involved in the regulation of cell death
(22). Two principal patterns of cell death have been
described, necrosis and apoptosis. Necrosis is associated with inflammation (22), whereas apoptosis (programmed cell death) is a regulated process that is
usually associated with chromatin condensation and
nuclear fragmentation (22). Recent evidence indicates
that cell death may be due to a predetermined genetic
program, external triggers, or intracellular stimuli
(22). Disturbance of this interplay of extracellular and
intracellular factors may trigger cell death.
Although many signals and metabolic events may be
important in the regulation of cell death, the intracellular redox level, in particular, has been shown to play a
critical role. For example, cell death has been associated with an increase in intracellular levels of reactive
oxygen species (ROS) (9, 27). Administration of oxidants such as H2O2 causes cell death. In particular,
administration of relatively low concentrations of H2O2
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to cells will cause apoptosis, whereas higher concentrations will cause necrosis (3). Antioxidants can prevent
cell death (44). For example, exposure of cells to the
antioxidant N-acetyl-L-cysteine (NAC) prevents cell
death (44). Also, Bcl-2, a critical antiapoptotic gene, has
been suggested to work, at least in part, through an
antioxidant pathway (11). Thus regulation of the intracellular redox potential is critical for the control of cell
death.
The intracellular redox potential is determined by
the concentrations of oxidants and reductants. A critical modulator of the redox potential is NADPH, the
principal intracellular reductant in all cell types. Glucose-6-phosphate dehydrogenase (G6PDH), the ratelimiting enzyme of the pentose phosphate pathway
(PPP; Fig. 1), determines the amount of NADPH by
controlling the metabolism of glucose via the PPP (14).
It has been traditionally thought that G6PDH was a
typical ‘‘housekeeping’’ enzyme that was regulated solely
by the ratio of NADPH to NADP (14). But research from
our lab as well as others suggests that this enzyme is
highly regulated and plays important roles in a variety
of cellular processes (14, 32, 37). Work from our lab and
others has shown that G6PDH is under close transcriptional, translational, and posttranslational control (14,
32, 37). Specifically, our lab has demonstrated that
growth factors can rapidly activate G6PDH and stimulate translocation of G6PDH (32, 37). We have further
shown that specific growth factor receptor-associated
signaling proteins can affect G6PDH (37). In addition,
we recently showed that G6PDH activity plays a critical role in cell growth via its role in intracellular redox
regulation (36). In particular, overexpression of G6PDH
stimulated cell growth (as determined by increased
[3H]thymidine incorporation), whereas inhibition of
G6PDH abrogated growth factor stimulation of [3H]thymidine incorporation (36). We also showed that lack of
NADPH but not ribose 5-phosphate was responsible for
the growth suppression caused by inhibition of G6PDH
(36). Others have also found that inhibition of G6PDH
abrogated mitogen-stimulated cell proliferation (7, 8).
Considering the importance of G6PDH for cellular
antioxidant defenses and the well-documented role of
oxidant stress in many models of cell death, we studied
the role of G6PDH in cell death.
In this paper, we show that inhibition of G6PDH
potentiated H2O2-mediated cell death. Overexpression
of G6PDH rendered the cells more resistant to H2O2mediated cell death. Serum deprivation, another stimulator of cell death, caused a decrease in G6PDH activity
and an increase in ROS. This serum deprivationinduced increase in ROS was almost completely abro-
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
C1121
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Tian, Wang-Ni, Leigh D. Braunstein, Kira Apse,
Jiongdong Pang, Mark Rose, Xiaoni Tian, and Robert
C. Stanton. Importance of glucose-6-phosphate dehydrogenase activity in cell death. Am. J. Physiol. 276 (Cell Physiol.
45): C1121–C1131, 1999.—The intracellular redox potential
plays an important role in cell survival. The principal intracellular reductant NADPH is mainly produced by the pentose
phosphate pathway by glucose-6-phosphate dehydrogenase
(G6PDH), the rate-limiting enzyme, and by 6-phosphogluconate dehydrogenase. Considering the importance of
NADPH, we hypothesized that G6PDH plays a critical role in
cell death. Our results show that 1) G6PDH inhibitors
potentiated H2O2-induced cell death; 2) overexpression of
G6PDH increased resistance to H2O2-induced cell death; 3)
serum deprivation, a stimulator of cell death, was associated
with decreased G6PDH activity and resulted in elevated
reactive oxygen species (ROS); 4) additions of substrates for
G6PDH to serum-deprived cells almost completely abrogated
the serum deprivation-induced rise in ROS; 5) consequences
of G6PDH inhibition included a significant increase in apoptosis, loss of protein thiols, and degradation of G6PDH; and 6)
G6PDH inhibition caused changes in mitogen-activated protein kinase phosphorylation that were similar to the changes
seen with H2O2. We conclude that G6PDH plays a critical role
in cell death by affecting the redox potential.
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G6PDH ACTIVITY AFFECTS CELL DEATH
gated by providing cells with substrates for G6PDH. By
evaluating the pattern of cell death, we show that
G6PDH inhibitors greatly enhanced apoptosis. Last, to
begin to determine the intracellular signaling proteins
that G6PDH activity may affect, we show that H2O2
and G6PDH inhibitors altered mitogen-activated protein kinase (MAP kinase) phosphorylation in a similar
manner. We conclude that G6PDH plays a critical role
in cell death, likely by affecting the intracellular redox
potential.
EXPERIMENTAL PROCEDURES
Materials. Cell culture media, growth factors, and serums
were obtained from Life Technologies. Rabbit anti-rat G6PDH
antibody was generously provided by Dr. Rolf Kletzien
(Upjohn). Fluorescent dyes were obtained from Molecular
Probes. FLAG antibody was purchased from EastmanKodak. Extracellular signal-regulated kinase II was from
New England Biolabs. All other chemicals were obtained from
Sigma.
Cell culture. BALB/c A31 and BALB/c simian virus 40
(SV40) 3T3 fibroblasts, as well as COS-7 cells, were grown in
DMEM containing 10% calf serum. K-562 and RIN5mAF cells
were grown in RPMI 1640 medium supplemented with 10%
inactivated FCS. PC12 cells were grown in DMEM containing
5% calf serum and 5% horse donor serum; 1% penicillinstreptomycin was present in all culture media. In all experiments, separated plates of control cells (without any inhibitor) were simultaneously incubated with cells treated with
inhibitor. Bovine aortic endothelial cells (BAEC) were cultured from freshly obtained bovine aorta and maintained in
low-glucose DMEM plus 10% calf serum.
Assessment of cell viability. Cell death was measured by
trypan blue uptake.
Enzyme activity measurements. Enzyme activities in cell
lysates were measured in a spectrophotometer as previously
described (37).
Measurement of G6PDH and phosphogluconate dehydrogenase activity in intact cells. The method employed by Van
Noorden and colleagues (4, 15) was used. Cells were incu-
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Fig. 1. Pentose phosphate pathway. Rate-limiting enzyme is glucose6-phosphate dehydrogenase (G6PDH). Activation of two dehydrogenase enzymes, G6PDH and phosphogluconate dehydrogenase (PGD),
results in production of NADPH, H⫹, and ribose 5-phosphate.
bated with medium containing 1⁄3 volume of 1 mM tetranitroblue tetrazolium dye, 0.5 mM glucose 6-phosphate (G6P), and
0.5 mM NADP for 8 h. The increase in blue color is dependent
on the production of NADPH, which reduces the tetrazolium
dye and produces a blue color. The intensity of the blue color
was measured spectrophotometrically. The specificity of this
method for G6PDH has been previously determined (4, 15)
and verified by us in control experiments (see Providing
substrates for G6PDH to serum-deprived cells abrogates intracellular ROS accumulation).
Measurement of intracellular ROS. Intracellular accumulation of ROS was measured fluorometrically. The nonfluorescent dye 28,78-dichlorofluorescin diacetate (DCFDA) is freely
permeable to cells. DCFDA is hydrolyzed to 28,78-dichlorofluorescin (DCF) inside the cells, where it converts upon interaction with ROS to a fluorescent DCF (23). DCF fluorescence
reading of the samples was conducted using a microplate
fluorometer (Cambridge Technology) with the excitation filter
set at 485 nm and the emission filter set at 530 nm.
Measurement of protein thiols. Thiols were assayed using
DTNB (18). Briefly, cell lysate was precipitated and washed
twice with TCA. Cellular proteins were suspended in Tris·HCl
(pH 7.6). Twenty minutes after the addition of DTNB, the
absorbance was measured at 412–520 nm.
Tagged protein and expression. The FLAG-containing
G6PDH construct was generated by PCR. The FLAG sequence was tagged to the carboxy terminus of G6PDH. cDNA
of G6PDH was a kind gift from Dr. Ye-Shih Ho (Institute of
Chemical Toxicology, Detroit, MI). To prepare the FLAGG6PDH fusion protein, G6PDH-FLAG cDNA was subcloned
into the expression vector pcDNA3 (Invitrogene). PC12 cells
were transfected by gene pauser (Bio-Rad) and selected in
geneticin-containing medium. The expression of epitopetagged G6PDH was confirmed by Western blotting using
monoclonal antibody against FLAG epitope, as well as by
enzyme activity (36).
Determination of DNA fragmentation. After incubation
with or without H2O2 or G6PDH inhibitors for a specified
time, cells were spun down and the pellet was then incubated
at 50°C for 1 h in lysis buffer containing 10 mM EDTA, 50 mM
Tris · HCl (pH 8.0), 0.5% (wt/vol) Triton X-100, and 0.5 mg/ml
proteinase K. Samples were then incubated with additional
0.5 mg/ml RNase A at 50°C for another 3 h. After heating to
70°C, samples were mixed with gel buffer containing 10 mM
EDTA (pH 8.0), 1% (wt/vol) low-gelling-temperature agarose,
0.25% (wt/vol) bromphenol blue, and 40% (wt/vol) sucrose
before loading into dry wells of 2% (wt/vol) agarose gel
containing 0.5 mg/ml ethidium bromide. After electrophoresis, DNA was visualized under ultraviolet light (305 nm).
DNA staining for microscopy. Cells were fixed with 4%
paraformaldehyde for 15 min and then stained with 0.4
mg/100 ml 48,68-diamidine-28-phenylindole dihydrochloride
(DAPI) at 4°C for 2 h. Cells were then washed twice and
mounted onto glass slides. An Olympus microscope was used
for fluorescence detection. For each treatment, 10 fields (100
cells/field) were counted.
Determination of p42 and p44 MAP kinase phosphorylation. Subconfluent PC12 cells were serum starved for 24 h
and then pretreated for 10, 20, or 30 min with one of the
following: DMEM alone, the G6PDH inhibitor dehydroepiandrosterone (DHEA; 200 µM), the oxidant menadione
(200 µM), or H2O2 (1 and 5 mM). In some experiments,
epidermal growth factor (EGF; 10 ng/ml) was added for 5 min
after the pretreatment. Cells were then lysed, and MAP
kinase p42 and p44 phosphorylation was observed using a
phosphotyrosine-specific antibody (New England Biolabs).
G6PDH ACTIVITY AFFECTS CELL DEATH
Statistics. Student’s t-test was used for statistical analysis.
The statistical significance is represented as follows: * P ⬍
0.05 compared with control, ** P ⬍ 0.01 compared with
control, and *** P ⬍ 0.001 compared with control.
RESULTS
that freshly isolated cells have increased responsiveness of the PPP to oxidative stress.
Serum deprivation is correlated with decreased
G6PDH activity. Our above results indicate that serum
protects cells from H2O2-induced death. Previously,
researchers have shown that serum withdrawal leads
to an increase in ROS (2). Thus deprivation of survival
factors might inactivate antioxidant defense systems
and trigger a ROS-dependent cell death (3, 19).
Because the function of G6PDH is to provide NADPH
for antioxidant defense, we hypothesized that decreased G6PDH activity during serum deprivation may
contribute to a decreased antioxidant defense and thus
lead to an increase in ROS. We utilized an assay system
that measures G6PDH activity in intact cells (see
Measurement of G6PDH and phosphogluconate dehydrogenase activity in intact cells). Figure 3A shows that
serum deprivation led to as much as a two- to threefold
decrease in G6PDH activity compared with cells cultured in serum-containing medium.
Serum deprivation caused large increases in intracellular ROS. Because serum deprivation alone decreased
G6PDH activity, we suspected that an accumulation of
ROS may occur due to a decrease in NADPH availability. Intracellular accumulation of ROS was measured
fluorometrically using the nonfluorescent dye DCFDA,
to which cells are freely permeable. DCFDA is hydrolyzed to DCF inside the cells, where it converts upon
interaction with ROS to a fluorescent DCF. Figure 3B
shows that serum deprivation dramatically increased
the accumulation of ROS inside the cells. Figure 3C
shows that the increase in ROS is time dependent
(compare DMEM with serum).
Providing substrates for G6PDH to serum-deprived
cells abrogates intracellular ROS accumulation. It has
been known that G6P and NADP (the substrates for
G6PDH) added to the medium can enter the cells and
be converted to 6-phosphogluconate (6PG) and NADPH
by G6PDH. A number of researchers have used this
method to measure activity of G6PDH in tissues and
cells (4, 15). To be sure that the substrates NADP and
G6P were taken up by the cells, a number of control
experiments were done: 1) there was no change in dye
color when either NADP alone or G6P alone was added
to the cells; 2) only when both substrates for G6PDH
were added was there a change in color; 3) for cells that
are almost completely deficient in G6PDH activity (25),
addition of either substrate alone or both G6PDH
substrates together caused no change in dye color; and
4) using [14C]G6P, we determined that the 14C was
converted into CO2 by the PPP using a previously
described technique (30, 32, 37). This uptake of G6P
was completely blocked by excess cold G6P. These
control experiments do not show whether G6P and
NADP or their metabolites are taken up. Nevertheless,
taken together, these data do suggest that G6P and
NADP or their metabolites can be taken up into the
cells and utilized by G6PDH.
We therefore tested whether increased activity of
G6PDH driven by the addition of G6PDH substrates
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Inhibition of G6PDH potentiates H2O2-induced cell
death in established cells. DHEA and 6-aminonicotinamide (6-ANAD) are known inhibitors of G6PDH (26,
39). To examine the role of G6PDH in the regulation of
cell death, PC12 cells (a neural cell line) and BALB/c
cells (a fibroblast cell line) were incubated with either
50 or 100 µM H2O2 in the absence or presence of 100 µM
DHEA or 5 mM 6-ANAD. Our previous studies indicated that, when cells were incubated with these
concentrations of inhibitors, G6PDH activity was decreased by 40–70% (36). Importantly, we have also
previously shown that inhibition of G6PDH by DHEA
at concentrations used in this study caused a 30–40%
decrease in NADPH levels (36). As shown in Fig. 2A in
the absence of serum, 100 µM H2O2 alone decreased cell
viability. DHEA and 6-ANAD, two structurally different inhibitors of G6PDH, significantly potentiated the
loss of viable cells in the presence of H2O2, suggesting
that inhibition of G6PDH enhanced the detrimental
effects of H2O2 on cell survival. In the presence of serum
but the absence of any G6PDH inhibitor, a modest
concentration of H2O2 (100 µM) did not cause cell death
(data not shown), suggesting that serum can protect
cells from the deleterious effects of modest concentrations of H2O2.
Overexpression of G6PDH increased cell resistance to
H2O2-induced cell death. Because the inhibition of
G6PDH potentiated H2O2-induced cell death, we tested
whether overexpression of G6PDH would provide protection against H2O2-induced cell death. PC12 cells
were stably transfected either with vector alone or with
a construct of G6PDH tagged with an epitope (FLAG
peptide). A clone stably expressing FLAG-G6PDH was
isolated. As shown in Fig. 2B, PC12 cells overexpressing G6PDH were more resistant to H2O2-induced cell
death than control cells that expressed endogenous
levels of G6PDH. This result suggests that increased
G6PDH activity enhanced cellular protection against
H2O2-induced cell death.
Inhibition of G6PDH potentiates H2O2-induced cell
death in nontransformed, freshly isolated cultured cells.
Because BALB/c 3T3 cells are a nontumorigenic but
established cell line and PC12 cells are transformed
cells, freshly isolated cells were studied to determine
whether freshly isolated, nontransformed cells also are
affected by G6PDH inhibition. Figure 2C shows that
DHEA and H2O2 alone as well as the combination of
DHEA and H2O2 significantly enhanced cell death,
although to a lesser degree than in the transformed
cells. The freshly isolated cells were more resistant to
the deleterious effects of both DHEA and H2O2. This
reduced effect of G6PDH inhibition is likely due to the
fact that freshly isolated cells have lower basal activity
of the PPP than transformed cells (30, 32, 37, 41) and
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G6PDH ACTIVITY AFFECTS CELL DEATH
stress-induced cell death. To determine whether G6PDH
inhibition increased apoptosis, DNA fragmentation and
chromatin condensation (42) were evaluated in a variety of cell types.
Figure 4A shows DNA fragmentation in RIN5mAF
cells (a pancreatic ␤-cell line) caused by G6PDH inhibition and by H2O2. DHEA and 6-ANAD caused DNA
fragmentation, characteristic of apoptosis, in several
cell lines tested.
Next we searched for evidence of chromatin condensation, another hallmark of apoptosis. Using immunofluorescence microscopy and DAPI staining of cell nuclei,
we observed a significant increase in chromatin condensation in cells treated with G6PDH inhibitors. Figure 4B shows representative photomicrographs depicting the effect of DHEA on chromatin condensation.
Both G6PDH inhibitors increased chromatin condensation (6-ANAD photo not shown). Also, the detection of
DHEA-enhanced apoptosis was time dependent and
varied in different cell lines, being detectable from 18 to
96 h (data not shown). This chromatin condensation
enhanced by G6PDH inhibition was seen in all cell
lines tested, including PC12, RIN5mAF, COS-7, K-562,
and BALB/c 3T3 cells. Figure 4C shows quantitation of
the DHEA- and 6-ANAD-induced apoptosis. Figure 4C
also shows that the nontransformed BAEC had increased apoptosis after being exposed to DHEA and
H2O2. The dose dependency of the DHEA effect is seen
in Fig. 5 in PC12 cells (EC50 of ⬃60 µM in the absence of
serum). Note that serum rendered the cells more
resistant to the apoptotic effects of DHEA.
Although in all cell lines tested apoptosis was universally enhanced by the inhibition of G6PDH, we did find
heterogeneity among different cell types. In some cell
lines, such as PC12, RIN5mAF, COS-7, and K-562 cells,
DHEA induced programmed cell death even without
serum deprivation or the addition of an external oxidant. Notably, the induction of apoptosis in these cell
Fig. 2. A: inhibition of G6PDH by dehydroepiandrosterone (DHEA)
or 6-aminonicotinamide (6-ANAD) enhanced cell death triggered by
oxidative stress. BALB/c 3T3 fibroblasts and PC12 cells were incubated with 50 or 100 µM H2O2 in absence and presence 100 µM DHEA
or 5 mM 6-ANAD for 24 and 48 h. Cell viability was determined by
trypan blue exclusion. Data are expressed as means ⫾ SE of 3
separate experiments, each run in triplicate. * P ⬍ 0.05, *** P ⬍ 0.001
compared with control. B: overexpression of G6PDH rendered PC12
cells more resistant to H2O2-triggered cell death. Overexpression of
G6PDH improved survivability of PC12 cells. Cells were treated with
or without various concentrations of H2O2 for 24 h. Cell viability was
determined by trypan blue exclusion. Data are expressed as means ⫾
SE of 4 separate experiments, each run in triplicate. PC12 cells were
stably transfected with either vector alone or with a construct of
G6PDH tagged with an epitope (FLAG peptide). A clone with a 60%
higher G6PDH expression was chosen as detected by Western blots
using either monoclonal antibody against FLAG epitope or a polyclonal antibody against G6PDH (data not shown). PC12 cells overexpressing G6PDH displayed a higher enzyme activity (1.5 times
control; data not shown). C: inhibition of G6PDH by DHEA enhanced
cell death in nontransformed cells. Bovine aortic endothelial cells
(BAEC) were maintained in 2% serum and incubated with 500 µM
H2O2 in absence and presence of 100 µM DHEA for 3 h. Cell viability
was determined by trypan blue exclusion. Data are expressed as
means ⫾ SE of 3 separate experiments, each run in triplicate. *** P ⬍
0.001 compared with control.
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would ameliorate the rise in ROS due to serum deprivation. Figure 3B shows that the addition of NADP and
G6P to the medium abrogated ROS accumulation to a
level almost as low as in the presence of serum. Note
that only when all substrates for G6PDH were present
was the ROS level greatly decreased (Fig. 3B). Providing the substrates (NADP and 6PG) for phosphogluconate dehydrogenase (PGD), the next enzyme in the
PPP pathway, had only a modest effect on ROS levels.
Figure 3C shows the effects of the antioxidants catalase
and NAC. Catalase and NAC had only modest effects on
reduction of the serum-deprived increase in ROS.
Clearly, provision of the substrates for G6PDH (NADP
and G6P) was the most effective in reducing ROS levels
close to the level seen with serum. These results
suggest that G6PDH activity plays an important role in
cellular defense against the accumulation of ROS.
DHEA and 6-ANAD enhanced apoptosis. Both necrosis and apoptosis have been described in oxidative
G6PDH ACTIVITY AFFECTS CELL DEATH
lines was associated with a relatively high basal apoptosis in the presence of serum. In contrast, BALB/c 3T3
cells had a very low basal level of apoptosis in the
presence of serum, and serum deprivation was required
for DHEA to induce apoptosis in this cell line. Thus it
seems possible that DHEA potentiates an existing
apoptotic potential in susceptible cells.
Because all tested cell lines displaying higher basal
apoptotic level were transformed cells, we suspected
that there might be an association between DHEAtriggered apoptosis and cell transformation. To see
whether cell transformation per se affected the ability
C1125
Fig. 3. A: serum increased G6PDH activity. Enzyme activity was
measured from intact BALB/c 3T3 fibroblasts, which were incubated
in absence or presence of various concentrations of calf serum.
Enzyme activity is expressed as increase in conversion of dye to a
dark blue. Data are from a representative experiment that was
repeated 4 times with similar results. B: addition of substrates for
G6PDH abrogates accumulation of reactive oxygen species (ROS) in
serum-deprived cells. Intracellular accumulation of ROS was measured fluorometrically. Conversion of nonfluorescent dye 2878dichlorofluorescin diacetate (DCFDA) to 28,78-dichlorofluorescin (DCF)
is indication of accumulation of ROS. Cells were either incubated
with combinations of substrates for G6PDH [100 µM NADP and
200 µM glucose 6-phosphate (G6P)] and PGD [100 µM NADP and
200 µM 6-phosphogluconate (6PG)]. Data are means of 4 repeated
DCF fluorescence readings, each run in at least triplicate. *** P ⬍
0.001 compared with control. C: addition of substrates for G6PDH is
more effective than catalase and N-acetyl-L-cysteine (NAC) in reducing accumulation of ROS in serum-deprived cells. Intracellular
accumulation of ROS was measured fluorometrically (see EXPERIMENTAL PROCEDURES ). Cells were either incubated with DMEM alone or
with one of following: substrates for G6PDH (NADP and G6P),
substrates for PGD (NADP and 6PG), catalase (1,000 U/ml), NAC (2
mM), or 1% calf serum. Data are means of 4 repeated DCF fluorescence readings, each run in at least triplicate.
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of DHEA to induce apoptosis, chromatin condensation
was assessed in SV40-transformed BALB/c cells and in
their nontransformed counterpart, BALB/c A31 fibroblasts. The results showed that DHEA caused a similar
extent of apoptosis in the transformed BALB/c cells and
in the nontransformed cells (data not shown). This
result suggests that the degree to which DHEA enhances apoptosis is dependent on cell type rather than
cell transformation.
Inhibition of G6PDH caused a decrease in protein
thiols. Oxidative stress may cause the decrease of
protein thiols, which may consequently impair many
enzymes (5). Because the inhibition of G6PDH may
decrease cellular reducing equivalents, thus limiting
antioxidative defense mechanisms, we tested whether
cell death enhanced by the inhibition of G6PDH is
associated with loss of protein thiols. Protein-bound
sulfhydryl groups were measured using DTNB in control and G6PDH inhibitor-treated PC12 cells. As shown
in Fig. 6, upon the inhibition of G6PDH, protein thiols
significantly decreased, suggesting that the cells had
increased intracellular oxidant levels.
Proteolytic degradation of G6PDH occurs in association with increased cell death. During apoptosis, some
important proteins are ‘‘executed’’ by proteases. In
cultured cells, apoptosis enhanced by DHEA and
6-ANAD was accompanied by the detection of a 46-kDa
fragment of G6PDH (Fig. 7). We have previously shown
the specificity of the G6PDH antibody for G6PDH (32,
36, 37). The amount of the 46-kDa fragment of G6PDH
correlated closely with apoptotic susceptibility in several cell lines tested. For example, BALB/c 3T3 cells,
which have a very low basal apoptotic rate and a
moderate increase in cell death after exposure to
DHEA, showed little to no evidence of the 46-kDa
fragment of G6PDH (data not shown). In contrast, the
RIN5mAF cells, which readily undergo apoptosis after
exposure to DHEA, had significant cleavage of G6PDH
after 48 h of incubation. PC12 cells, which are highly
susceptible to DHEA-induced apoptosis, showed degra-
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Fig. 4. A: G6PDH inhibitors and H2O2 caused DNA fragmentation. Cellular DNA was isolated after cells were
treated in absence and presence of 100 µM DHEA, 5 mM 6-ANAD, or 100 µM H2O2 for 48 h. B: inhibition of G6PDH
enhanced chromatin condensation. Representative micrograph of RIN5mAF cells incubated with one of following
for 48 h and then fixed and stained with 48,68-diamidine-28-phenylindole dihydrochloride (DAPI). Top: DMEM
alone. Bottom: DMEM ⫹ 100 µM DHEA. C: DHEA and 6-ANAD enhanced apoptosis. PC12 and RIN5mAF cells were
incubated with H2O2 (100 µM), DHEA (100 µM), or 6-ANAD (5 mM) for 48 h. BAEC were maintained in 2% serum
and incubated with H2O2 (500 µM) and/or DHEA (100 µM) for 3 h. Apoptotic cells were identified by DAPI staining of
condensed chromatin. * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.001 compared with control.
G6PDH ACTIVITY AFFECTS CELL DEATH
C1127
Fig. 7. G6PDH underwent degradation during apoptosis. Western
analysis of G6PDH in cell lysate using a specific polyclonal antibody
to mammalian G6PDH. PC12 cells were treated for 48 h with or
without 100 µM DHEA, 5 mM 6-ANAD, or 100 µM H2O2.
Fig. 6. Inhibition of G6PDH resulted in decreased protein thiols.
PC12 cells were treated with or without 100 µM DHEA, 5 mM
6-ANAD, 100 µM menadione, or 100 µM H2O2 for either 4 or 24 h in
presence (A) or absence (B) of serum. Cell lysates of control and
inhibitor-treated PC12 cells were then assayed for protein-bound
sulfhydryl groups using DTNB. Data are expressed as means ⫾ SE of
4 separate experiments. * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.001
compared with control.
dation as early as 12 h (data not shown). This correlation between apoptotic tendency and G6PDH degradation suggests that G6PDH may be a target for
proteolysis in apoptosis.
H2O2, menadione, and DHEA stimulated phosphorylation of p42 and p44 MAP kinase in serum-deprived
cells. DHEA attenuated the EGF-stimulated phosphorylation of p42 and p44 MAP kinase. Many signaling
pathways have been implicated in causing apoptosis.
We hypothesized that, if G6PDH inhibition is causing
cells to be more susceptible to oxidative stress, then
signaling proteins associated with apoptosis should be
affected by G6PDH inhibitors in a manner similar to
the effects of other oxidants. Initially, we evaluated the
MAP kinase pathway that has been shown to be
implicated in multiple physiological processes including cell death (43). The effects of the oxidants (H2O2 and
menadione) as well as the G6PDH inhibitor DHEA on
the phosphorylation of the critical MAP kinase proteins, p42 and p44, were examined. Figure 8, top, shows
that H2O2 and menadione stimulated phosphorylation
of p42 and p44 in the absence of growth stimuli. DHEA
also had a modest although lower stimulation of p42
and p44 phosphorylation, suggesting that DHEA inhibited phosphorylation of p42 and p44 in a manner
similar to known oxidants.
It has been shown that EGF prevents apoptosis in a
variety of cell types and that EGF activates the MAP
kinase pathway. We therefore hypothesized that DHEA
might decrease EGF-induced phosphorylation of p42
and p44. Figure 8, bottom, shows that the stimulation
of p42 and p44 phosphorylation by EGF was significantly attenuated by DHEA and to a lesser extent by
oxidant exposure (Fig. 8, bottom). These results indicate that both DHEA and H2O2 similarly alter phosphorylation patterns of the p42 and p44 MAP kinases. Thus
the attenuation of EGF-induced phosphorylation by
DHEA may be one of the mechanisms underlying
DHEA-induced cell death.
Last, we tested the effects of PD-98059, the MAP
kinase inhibitor, on cell survival. It has been reported
that activation of p42 and p44 MAP kinase is part of a
cell survival cascade (29, 44). Thus stimulation of p42
and p44 phosphorylation in response to H2O2, menadione, and DHEA likely reflects a cellular protective
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Fig. 5. DHEA enhanced apoptosis in a dose-dependent manner.
PC12 cells were exposed to DHEA for 48 h in presence or absence of
serum. Data are means of 3 experiments.
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G6PDH ACTIVITY AFFECTS CELL DEATH
response to increased oxidative stress. PD-98059 alone
increased cell death (data not shown). In combination
with DHEA, PD-98059 significantly enhanced cell death
over DHEA or PD-98059 alone (by Student’s t-test, P ⬍
0.01; data not shown).
DISCUSSION
G6PDH plays an important role in cell death by
regulating intracellular redox levels. A principal finding
of our study was that alterations in G6PDH activity can
significantly alter oxidative stress-induced cell death.
As noted in the introduction, there is ample evidence
showing that an increase in ROS induces cell death (9,
27). This increase in ROS may be due to overproduction
of ROS from extracellular or intracellular processes, or
there may be a decrease in endogenous antioxidant
defense. Much work in recent years has been centered
on such intracellular antioxidants as superoxide dismutase, GSH, and catalase. Alterations in the activities of
these enzyme systems have been implicated as causes
of diseases (e.g., amyotrophic lateral sclerosis) as well
as cell death (17). Yet the antioxidant defense mechanisms ultimately rely on the adequate production of
NADPH for reducing equivalents during oxidative stress
(19). The principal source of NADPH is the PPP, and
many studies have shown that under oxidative stress
G6PDH and the PPP are elevated (28, 38). Although
there are other metabolic pathways that produce
NADPH, research has shown that the PPP is the
predominant source of NADPH required to defend
against oxidative stress. For example, the work by
Pandolfi et al. (24) using G6PDH-deficient cell lines
shows that other sources of NADPH do not adequately
replace the lack of NADPH production by G6PDH. That
is, the G6PDH-deficient cells had decreased growth
rates and cloning efficiencies and were highly sensitive
to oxidative stress compared with cells expressing
endogenous levels of G6PDH. Thus G6PDH is critical
for NADPH production and is the principal source of
NADPH in a large number of cell types. However, in
liver, adipose tissue, pancreatic ␤-cells, and macrophages, NADP⫹-dependent malate dehydrogenase may
play a significant role in NADPH production.
The PPP via the nonoxidative branch may also affect
cell survival directly and/or by providing substrates for
glycolysis. First, Bank et al. (1) showed that levels of
transaldolase, an enzyme in the nonoxidative branch of
the PPP, can regulate via regulation of the levels of the
two dehydrogenases, G6PDH and PGD. Increases in
transaldolase led to decreased levels of G6PDH and
PGD as well as increased ROS levels and increased
apoptosis, whereas decreased transaldolase had the
opposite effects. These results, however, also support
our hypothesis that G6PDH activity by producing
NADPH is important for cell survival. Second, the
nonoxidative branch of the PPP also provides substrates for glycolysis. Thus altered flux through the
PPP may alter substrate availability for glycolysis,
leading to changes in ATP and NADH levels. These
metabolic changes could affect cell survival. For example, reduced levels of NAD and NADH have been
shown to be associated with decreased growth. Mazurek
et al. (20) showed that AMP-induced inhibition of a
breast cancer cell line is likely caused by reduced
glycolytic carbon flux leading to reductions in NAD and
NADH levels. Thus effects of the inhibition of PPP on
glycolysis could potentially inhibit cell growth via
effects on NAD/NADH levels in addition to changes in
NADPH. Also, Street et al. (34) showed that 6-ANAD
enhanced irradiation-induced killing of tumor cells.
This increased killing was associated with decreased
activity of both the PPP and glycolysis. Thus the
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Fig. 8. Top: H2O2, menadione, and DHEA
stimulate phosphorylation of p42 and p44
mitogen-activated protein kinase (MAP kinase). Western blot from cell lysates using
a specific antibody to phosphorylated p42
and p44 MAP kinase. PC12 cells were
incubated with H2O2 (1 and 5 mM), DHEA
(200 µM), or menadione (200 µM) for 10,
20, or 30 min. Bottom: H2O2 and DHEA
attenuate epidermal growth factor (EGF)induced phosphorylation of p42 and p44
MAP kinase. After preincubation of serumstarved PC12 cells with H2O2 (1 and 5 mM)
or DHEA (200 µM) for 10, 20, or 30 min,
cells were then exposed to EGF for 5 min.
G6PDH ACTIVITY AFFECTS CELL DEATH
colleagues (5, 40) further demonstrated the significance
of thiol/disulfide in cell biology. They found that cell
death was preceded by the loss of protein thiols.
Phosphofructokinase activity was greatly decreased
following exposure to GSSG, likely due to changes in
subunit associations. Glutathione oxidation state is
dependent on G6PDH as NADPH is the reductant for
GSSG (19). Gilbert and colleagues (5, 40) also showed
similar effects on thiolase I, fatty acid synthase, and
other enzymes. Thus decreased protein thiol content
caused by G6PDH inhibition is consistent with our
hypothesis that G6PDH plays a critical role in cell
death by regulating intracellular redox levels.
Decreased G6PDH activity leads to enhancement of
apoptosis. The effect of oxidants on cell function appears to be related to its intracellular concentration (3,
9, 35). For example, low levels of oxidants (1–5 µM)
appear to be stimulatory to cell growth and have been
implicated as downstream signals for the growth factors, such as platelet-derived growth factor (35).
Midrange concentrations of oxidants (50–100 µM) have
been suggested to cause apoptosis, whereas high concentrations of oxidants (500–1,000 µM) have been shown
to cause necrosis. Because we and others had previously shown that DHEA can inhibit growth factorstimulated cell growth (10, 36), we were interested in
determining whether G6PDH inhibition led to apoptosis and/or necrosis. Our data show that G6PDH inhibition clearly led to an increase in the numbers of
apoptotic cells.
Notably, our data showed that serum was relatively
protective against cell death and that cells exposed to
serum have a relatively increased antioxidant defense.
Importantly, serum deprivation led to a decrease in
G6PDH activity (Fig. 3A). Because serum deprivation
enhanced DHEA-induced cell death (Fig. 5), we believe
that serum deprivation-induced cell death is due, at
least in part, to inhibition of G6PDH.
Also of interest, our data showed that there is
considerable cell specificity with respect to the susceptibility of a cell to undergo apoptosis. For example, in
BALB/c 3T3 fibroblasts, only in the absence of serum
did DHEA and 6-ANAD enhance apoptosis, whereas, in
PC12, RIN5mAF, COS-7, and K-562 cells, even in the
presence of serum, DHEA and 6-ANAD enhanced apoptosis. All of the cells that have increased susceptibility
to apoptosis following exposure to G6PDH inhibition
are transformed cells. Previous work by a number of
researchers showed that cancer cells in vivo and transformed cells in culture have significantly increased
activities of G6PDH to levels as high as 20-fold greater
than nontransformed cells (41). Thus it is intriguing to
speculate that specific inhibition of G6PDH could differentially induce more apoptosis in stressed cells or cells
with higher apoptotic tendency and thus may offer
therapeutic benefit to patients with cancer. The combination of G6PDH inhibitor along with other stressinducing stimuli (e.g., radiation and/or chemotherapy)
might prove to be beneficial.
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nonoxidative branch of the PPP may play a role in cell
survival as well as the oxidative branch.
We report here that G6PDH inhibition by DHEA and
6-ANAD enhanced cell death triggered by oxidative
stress and serum deprivation. The consequences of
enzyme inhibition are the lack of products catalyzed
through the enzymatic reaction. The activation of
G6PDH provides one molecule of NADPH. Subsequent
activation of the next enzyme in PPP, PGD, gives rise to
another NADPH. We previously demonstrated that
inhibition of G6PDH causes a decrease in intracellular
NADPH levels (36). Thus we conclude that lack of
NADPH due to G6PDH inhibition made the cells more
susceptible to oxidant damage, thus enhancing cell
death. Consistent with this conclusion, our results from
the stably transfected PC12 cells showed that overexpression of G6PDH made cells more resistant to oxidative stress-induced cell death. A previous study by
Costa Rosa et al. (6) appears to be in conflict with our
results concerning cell survival and the importance of
G6PDH. Although not specifically focused on cell survival, their study reported that epinephrine-induced
inhibition of G6PDH did not affect cell survival. A major
difference between the two studies is the incubation
time. Their experiments were all done within a 1-h
period that is likely too short a time to observe significant cell death, whereas exposure to inhibitors in our
experiments ranged from 3 to 48 h. Thus we believe
there is no conflict between the two studies.
Addition of substrates for G6PDH reduces ROS production in serum-deprived cells. Our interpretation of
this data is that the substrates G6P and NADP were
taken up by the cells and utilized by G6PDH to produce
NADPH and reduce ROS levels. It could be argued that
these substrates alone rather than G6PDH are able to
alter ROS levels. However, Fig. 3B shows that only
when both substrates for G6PDH were added was there
an effect on ROS levels. Neither substrate alone affected ROS levels, suggesting that the combination of
G6P and NADP is required to decrease ROS levels.
Although experiments discussed in Providing substrates for G6PDH to serum-deprived cells abrogates
intracellular ROS accumulation strongly suggest that
G6P and NADP are taken up by cells, it cannot be ruled
out that ectoenzymes convert G6P and NADP into
other compounds that are taken up by the cell. However, even if this occurs, it seems likely that the
ultimate effect is via G6PDH, as the effect on ROS
levels only occurs in the presence of both substrates.
Thus we conclude that the substrates are taken up by
the cell and utilized by G6PDH to produce NADPH,
leading to decreased ROS levels.
Inhibition of G6PDH leads to a loss of protein thiols.
We have found that G6PDH inhibition is closely associated with decreased protein thiols. A decrease in protein thiol content is a consequence of an increase in
intracellular oxidants. It is likely that an altered
thiol-to-disulfide ratio may have significant impact on
protein folding, conformation, and polymerization.
Based on their study on modulation of phosphofructokinase activity by the thiol-to-disulfide ratio, Gilbert and
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G6PDH ACTIVITY AFFECTS CELL DEATH
This work was supported in part by American Cancer Society
Grant BE-131A to R. C. Stanton. W.-N. Tian is a recipient of National
Institute of Diabetes and Digestive and Kidney Diseases National
Research Service Award DK-09265-02.
Address for reprint requests and other correspondence: R. C.
Stanton, Renal Division, Joslin Diabetes Center, 1 Joslin Place,
Boston, MA 02215 (E-mail: [email protected]).
Received 1 July 1998; accepted in final form 5 February 1999.
REFERENCES
1. Bank, K., E. Hutter, E. Colombo, N. J. Gonchoroff, and A.
Perl. Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J. Biol. Chem. 271:
32994–33001, 1996.
2. Barroso, M. P., C. Gomez-Diaz, G. Lopez-Lluch, M. M.
Malagnon, F. L. Crane, and P. Navas. Ascorbate and alphatocopherol prevent apoptosis induced by serum removal independent of Bcl-2. Arch. Biochem. Biophys. 343: 243–248, 1997.
3. Burdon, R. H., D. Alliangana, and V. Gill. Hydrogen peroxide
and the proliferation of BHK-21 cells. Free Radic. Res. 23:
471–486, 1995.
4. Butcher, R. G., and C. J. F. Van Noorden. Reaction rate
studies of glucose 6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts. Histochem. J. 17:
993–1008, 1985.
5. Cappell, R. E., J. W. Bremer, T. M. Timmons, T. E. Nelson,
and H. F. Gilbert. Thiol/disulfide redox equilibrium between
glutathione and glycogen debranching enzyme (amylo-1,6glucosidase/4-alpha-glucanotransferase) from rabbit muscle. J.
Biol. Chem. 261: 15385–15389, 1986.
6. Costa Rosa, L.-F. B. P., R. Curi, C. Murphy, and P. Newsholme. Effect of adrenaline and phorbol myristate acetate or
bacterial lipopolysaccharide on stimulation of pathways of macrophage glucose, glutamine and O2 metabolism. Biochem. J. 310:
709–714, 1995.
7. Dworkin, C. C., S. D. Gorman, L. L. Pashko, V. J. Cristofalo,
and A. G. Schwartz. Inhibition of growth of Hela and WI-38
cells by dehydroepianandrosterone and its reversal by ribo- and
deoxyribonucleosides. Life Sci. 38: 1451–1457, 1986.
8. Farquharson, C., J. Milne, and N. Loveridge. Mitogenic
action of insulin-like growth factor-I on human osteosarcoma
MG-63 cells and rat osteoblasts maintained in situ: the role of
glucose-6-phosphate dehydrogenase. Bone Miner. 22: 105–115,
1993.
9. Gardner, A. M., F. H. Xu, C. Fady, F. J. Jacoby, D. C. Duffey,
Y. Tu, and A. Lichtenstein. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radic. Biol. Med. 22:
73–83, 1997.
10. Gordon, G. B., L. M. Shantz, and P. Talalay. Modulation of
growth, differentiation, and carcinogenesis by dehydroepiandrosterone. Adv. Enzyme Regul. 26: 355–382, 1987.
11. Hockenbery, D. M., Z. N. Olvtviai, X. M. Yin, C. L. Miliman,
and S. J. Korsmeyer. Bcl-2 functions in an antioxidant pathway
to prevent apoptosis. Cell 75: 241–251, 1993.
12. Kaufmann, S. H., S. Desnoyers, Y. Ottaviano, N. E. Davidson, and G. G. Poirer. Specific proteolytic cleavage of poly(ADPribose) polymerase: an early marker of chemotherapy-induced
apoptosis. Cancer Res. 53: 3976–3985, 1993.
13. Kayalar, C., T. Ord, M. P. Testa, L. T. Zhong, and D. E.
Bredesen. Cleavage of actin by interleukin 1 beta-converting
enzyme to reverse DNase I inhibition. Proc. Natl. Acad. Sci. USA
93: 2234–2238, 1995.
14. Kletzien, R. F., P. K. W. Harris, and L. A. Foellmi. Glucose-6phosphate dehydrogenase: a ‘‘housekeeping’’ enzyme subject to
tissue-specific regulation by hormones, nutrients, and oxidant
stress. FASEB J. 8: 174–181, 1994.
15. Koopdonk-Kool, J. M., and C. J. F. Van Noorden. A novel
quantitative histochemical assay to measure endogenous substrate concentrations in tissue sections. Fundamental aspects.
Acta Histochem. 97: 409–419, 1995.
16. Kummer, J. L., P. K. Rao, and K. A. Heidenreich. Apoptosis
induced by withdrawal of trophic factors is mediated by p38
mitogen-activated protein kinase. J. Biol. Chem. 272: 20490–
20494, 1997.
17. Louvel, E., J. Hugon, and A. Doble. Therapeutic advances in
amyotrophic lateral sclerosis. Trends Pharmacol. Sci. 18: 196–
203, 1997.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 18, 2017
Increased cell death was associated with degradation
of G6PDH. Another interesting observation from this
study was the degradation of G6PDH during apoptosis.
It has been reported that protease activation may
function as the executioner upon apoptotic signal.
Despite identification of several proteases, the substrate list for these proteases is far from extensive. The
few substrates for proteases identified so far, however,
are very critical proteins implicated in the apoptotic
cascade. For instance, the cleavage of lamin B1 leads to
the collapse of nuclear matrix. (21). The cleavage of
poly(ADP-ribose) polymerase inhibits most DNA repair
activity (12). Proteolytic cleavage of actin may destroy
its ability to inhibit DNase I and its association with
fordrin, thus leading to some apoptotic changes in
membranes and cytoskeleton (13). The degradation of
any enzyme in metabolic pathways has not yet been
reported. Given the critical role of G6PDH to provide
NADPH, the cleavage of it during apoptosis may be an
important event in programmed cell death by inactivating an important antioxidant protein.
Inhibition of G6PDH alters phosphorylation patterns
of MAP kinase. Our results also show that G6PDH
inhibition is associated with alteration in phosphorylation of p42 and p44 MAP kinase (Fig. 8). The MAP
kinase cascade has been implicated in cell growth, cell
death, and cell differentiation. Recent work by Stevenson et al. (33) has shown that oxidants such as H2O2 can
stimulate phosphorylation of p42 and p44 MAP kinase.
It is likely that the activation of these kinases is a
cellular protective response to oxidative stress (29, 44).
In contrast, oxidant-induced activation of p38 kinase,
another member of the MAP kinase family, promotes
cell death (16). Our data showed that H2O2, menadione,
and DHEA stimulated phosphorylation of p42 and p44
MAP kinase in cells not exposed to growth factors. Our
data indicated that DHEA alone has a modest effect on
MAP kinase similar to H2O2 (Fig. 8, top). Interestingly,
when cells were preincubated with DHEA and then
exposed to EGF, the EGF-induced phosphorylation of
p42 and p44 MAP kinase was greatly decreased. H2O2
displayed similar but lower reduction of EGF-stimulated MAP kinase phosphorylation. Therefore, G6PDH
inhibition seemed to cause an effect on phosphorylation
similar to that of the oxidants (H2O2 and menadione).
Last, use of an inhibitor of the MAP kinases, PD-98059,
synergistically enhanced DHEA-induced cell death (data
not shown). This result suggests that the increase in
p42 and p44 MAP kinase phosphorylation seen after
DHEA, H2O2, and menadione is a protective response
to increased oxidative stress. Taken together, these
results add further support to the hypothesis that
G6PDH is important for cell death regulation by controlling intracellular redox.
In summary, we have found that G6PDH plays an
important role in cell death by regulating the intracellular redox status.
G6PDH ACTIVITY AFFECTS CELL DEATH
33. Stevenson, M. A., S. S. Pollock, C. N. Coleman, and S. K.
Calderwood. X-irradiation, phorbol esters, and H2O2 stimulate
mitogen-activated protein kinase activity in NIH-3T3 cells
through the formation of reactive oxygen intermediates. Cancer
Res. 54: 12–15, 1994.
34. Street, J. C., U. Mahmood, D. Ballon, A. A. Alfieri, and J. A.
Koutcher. 13C and 31P NMR investigation of effect of 6-aminonicotinamide on metabolism of RIF-1 tumor cells in vitro. J. Biol.
Chem. 271: 4113–4119, 1996.
35. Sundaresan, M., Z. X. Yu, V. J. Ferrans, K. Irani, and T.
Finkel. Requirement for generation of H2O2 for platelet-derived
growth factor signal transduction. Science 270: 296–299, 1995.
36. Tian, W.-N., L. D. Braunstein, J. Pang, K. Stuhlmeier, Q.-C.
Xi, X. Tian, and R. C. Stanton. Importance of glucose 6-phosphate dehydrogenase activity for cell growth. J. Biol. Chem. 273:
10609–10617, 1998.
37. Tian, W.-N., J. N. Pignatare, and R. C. Stanton. Signal
transduction proteins that associate with the platelet-derived
growth factor (PDGF) receptor mediate the PDGF-induced release of glucose 6-phosphate dehydrogenase. J. Biol. Chem. 269:
14798–14805, 1994.
38. Ursini, M. V., A. Parella, G. Rosa, S. Salzano, and G.
Martini. Enhanced expression of glucose 6-phosphate dehydrogenase in human cells sustaining oxidative stress. Biochem. J.
323: 801–806, 1997.
39. Varnes, M. E. Inhibition of pentose cycle of A549 cells by
6-aminonicotinamide: consequences for aerobic and hypoxic radiation response and for radiosensitizer action. NCI Monogr. 6:
199–203, 1988.
40. Walters, D. W., and H. F. Gilbert. Thiol/disulfide exchange
between rabbit muscle phosphofructokinase and glutathione.
Kinetics and thermodynamics of enzyme oxidation. J. Biol.
Chem. 261: 15372–15377, 1986.
41. Weber, G. Strongly conserved segment of gene expression in
cancer cells. In: Modulation of Liver Cell Expression, edited by W.
Reutter, H. Popper, I. M. Arias, P. C. Heinrich, D. Keppler, and L.
Landmann. Lancaster: MTP, 1987, p. 303–314. (Falk Symp. vol.
43)
42. Wyllies, A. H. Cell death: a new classification separating
apoptosis from necrosis. In: Cell Death in Biology and Pathology,
edited by I. D. Bowen. London: Chapman & Hall, 1981, p. 9–34.
43. Xia, Z., M. Dickens, J. Raingeaud, R. J. Davis, and M. E.
Greenberg. Opposing effects of ERK and JNK-p38 MAP kinases
on apoptosis. Science 270: 1326–1331, 1995.
44. Yan, C. Y. I., and L. A. Greene. Prevention of PC12 cell death by
N-acetylcysteine requires activation of the Ras pathway. J.
Neurosci. 18: 4042–4049, 1998.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 18, 2017
18. Marshall, M. S. Ras target proteins in eukaryotic cells. FASEB
J. 9: 1311–1318, 1995.
19. Martini, G., and M. V. Ursini. A new lease of life for an old
enzyme. Bioessays 18: 631–637, 1996.
20. Mazurek, S., A. Michael, and E. Eigenbrodt. Effect of extracellular AMP on cell proliferation and metabolism of breast
cancer cell lines with high and low glycolytic rates. J. Biol. Chem.
272: 4941–4952, 1997.
21. Neamati, N., A. Fernandez, S. Wright, J. Kiefer, and D. J.
McConkey. Degradation of lamin B1 precedes oligonucleosomal
DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J. Immunol. 154: 3788–3795, 1995.
22. Orrenius, S. Apoptosis: molecular mechanisms and implications for human disease. J. Intern. Med. 237: 529–536, 1995.
23. Ortiz, A., F. N. Ziyadeh, and E. G. Neilson. Expression of
apoptosis-regulatory genes in renal proximal tubule epithelial
cells exposed to high ambient glucose and in diabetic kidneys. J.
Investig. Med. 45: 50–56, 1997.
24. Pandolfi, P. P., F. Sonati, R. Rivi, P. Mason, F. Grosveld, and
L. Luzzatto. Targeted disruption of the housekeeping gene
encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is
dispensable for pentose synthesis but essential for defense
against oxidative stress. EMBO J. 14: 5209–5215, 1995.
25. Rosenstraus, M., and L. Chaisin. Isolation of mammalian cell
mutants deficient in glucose 6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyl transferase. Proc.
Natl. Acad. Sci. USA 72: 493–497, 1975.
26. Schwartz, A. G., J. M. Whitcomb, J. N. Nyce, M. L. Lewbart,
and L. L. Pashko. Dehydroepiandrosterone and structural
analogs: a new class of cancer chemopreventive agents. Adv.
Cancer Res. 51: 391–424, 1988.
27. Simonian, N. A., and J. T. Coyle. Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 36: 83–106,
1996.
28. Slekar, K. H., D. J. Kosman, and V. C. Culotta. The yeast
copper/zinc superoxide dismutase and the pentose phosphate
pathway play overlapping roles in oxidative stress protection. J.
Biol. Chem. 271: 28831–28836, 1996.
29. Stadheim, T. A., and G. L. Kucera. Extracellular signalregulated kinase (ERK) activity is required for TPA-mediated
inhibition of drug-induced apoptosis. Biochem. Biophys. Res.
Commun. 245: 266–271, 1998.
30. Stanton, R. C., and J. L. Seifter. Epidermal growth factor
rapidly activates the hexose monophosphate shunt in kidney
cells. Am. J. Physiol. 254 (Cell Physiol. 23): C267–C271, 1988.
32. Stanton, R. C., J. L. Seifter, D. C. Boxer, E. Zimmerman,
and L. C. Cantley. Rapid release of bound glucose-6-phosphate
dehydrogenase by growth factors. Correlation with increased
enzymatic activity. J. Biol. Chem. 266: 12442–12448, 1991.
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