Renal Division and Department of Medicine, Joslin Diabetes Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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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.
oxidative stress; pentose phosphate pathway; apoptosis
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INTRODUCTION |
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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 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 rate-limiting 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.
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In this paper, we show that inhibition of G6PDH potentiated H2O2-mediated cell death. Overexpression of G6PDH rendered the cells more resistant to H2O2-mediated cell death. Serum deprivation, another stimulator of cell death, caused a decrease in G6PDH activity and an increase in ROS. This serum deprivation-induced increase in ROS was almost completely abrogated 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.
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EXPERIMENTAL PROCEDURES |
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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 Eastman-Kodak. 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% penicillin-streptomycin 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 incubated with medium containing 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 2',7'-dichlorofluorescin diacetate (DCFDA) is freely permeable to cells. DCFDA is hydrolyzed to 2',7'-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 FLAG-G6PDH 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 epitope-tagged 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 4',6'-diamidine-2'-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).
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.
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RESULTS |
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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.
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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 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.
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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 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 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
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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.
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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
degradation 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.
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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.
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DISCUSSION |
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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.
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 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 factor-stimulated 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 stress-inducing stimuli (e.g., radiation and/or chemotherapy) might prove to be beneficial.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. ![]() |
ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. C. Stanton, Renal Division, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215 (E-mail: rstanton{at}bidmc.harvard.edu).
Received 1 July 1998; accepted in final form 5 February 1999.
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