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Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA
1Correspondence: Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932, USA. E-mail: taw{at}lsuhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis in undifferentiated cells • GSH/GSSG status and apoptosis • mitochondrial apoptosis signaling • diamide • NADPH • pentose phosphate pathway
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, because oxidants often elicit redox imbalance, it is difficult to distinguish the direct effects of cellular redox from that of oxidants, and a precise role for cellular redox in controlling cell apoptosis remains unresolved. Evidence in the literature supports a link between cellular glutathione (GSH) status and apoptosis (2)
, and various authors have speculated that early GSH loss may be an important early event in the cell death pathway. For example, decreases in cell GSH were observed in several cell types in which apoptosis was initiated by stimuli unrelated to reactive oxygen species (ROS) generation such as glucocorticoid-induced thymocyte apoptosis (3
, 4)
, serum withdrawal-induced fibroblast apoptosis (5)
, or dopamine-induced apoptosis in neuronal cells (6)
. In other studies, intracellular GSH status has been linked to Bcl-2 up-regulation (7
8
9
10)
and protection from apoptotic cell death. Other investigators have shown that the thiol oxidant diamide induces cell death and DNA fragmentation in various cell lines (11
, 12)
.
The dynamic process of cellular glutathione redox balance is achieved by maintenance of the thiol-disulfide status of GSH and its oxidized form, glutathione disulfide, GSSG (13)
. Oxidation reduction and thiol-disulfide exchange reactions during oxidant challenge can perturb redox balance by inducing GSH-GSSG redistribution, and this redox alteration can have deleterious consequences for metabolic regulation, cellular integrity, and organ homeostasis. Thus, the balance between GSH and GSSG can be used as an indicator of the redox balance of the cell (14)
. The intracellular GSH/GSSG homeostasis is maintained by reduced nicotinamide adenine dinucleotide phosphate (NADPH). Through the action of glucose-6-phosphate dehydrogenase (G6PD; 15
), the pentose phosphate shunt pathway uses glucose to maintain a supply of NADPH for the function of GSSG reductase in regenerating GSH from GSSG. Production of NADPH by the shunt is highly regulated. Thus, a decrease in glucose flux through the pathway by inhibition of G6PD activity or limiting glucose availability can inhibit the supply of NADPH for function of the glutathione redox cycle.
Greene and Tischler first characterized the pheochromocytoma PC12 cell model in 1976 that has since been used as a model to study cellular and molecular aspects of neuronal apoptosis when cells are induced to differentiate in culture with nerve growth factor (16)
. Because of the ease of growth and the ability to readily induce cell differentiation in culture, we used this cell line to investigate the possibility of differential vulnerability of cell stages (naive or differentiated) to the effects of oxidant and redox stress and apoptotic signaling. In the current study, we provide evidence for a direct role of cellular redox in mediating cell apoptosis in naive cells; studies are under way to define the apoptotic responses in differentiated cells. In this study, we have addressed the contribution of cellular redox to apoptosis after exposure of naive pheochromocytoma (PC-12) cells to a cell-permeant thiol oxidant, diamide. This approach allows for direct oxidation of GSH to GSSG, resulting in an imbalance of the GSH-to-GSSG ratio without the use of a ROS-generating oxidant. Since mitochondria have been widely implicated in the apoptotic cascade mediated by oxidative stress and redox imbalance, we investigated the mitochondrial pathway in diamide-induced PC12 apoptosis. Because NADPH is pivotal in the maintenance of cellular GSH/GSSG balance, the contribution of NADPH to the apoptotic process was examined by manipulation of NADPH availability from the pentose phosphate pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BH), 4'6-diamidino-2-phenylindole (DAPI), and dehydroisoandrosterone (DHEA). Fetal calf serum and horse serum were obtained from Atlanta Biologicals (Norcross, GA). Monoclonal antibodies against Bax, BcL-2 and CPP32 were acquired from Transduction labs (Lexington, KY); the monoclonal antibody against ß-actin was bought from Oncogene (Cambridge, MA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Life Technologies, Inc. (Grand Isle, NY). Nitrocellulose membranes and SDS-electrophoresis units were acquired from Bio-Rad Corporation (Hercules, CA). The enhanced chemiluminescence system for Western immunoblot and the hyperfilm were purchased from Amersham (Arlington Heights, IL). Fluorescent mounting media were obtained from DAKO Corporation (Carpinteria, CA). 12 mm circle No.1 glass coverslips used for DAPI staining were procured from Fisher (Pittsburgh, PA).
Cell culture and incubations
PC-12, a generous gift from Dr. Nikki Holbrook (National Institute on Aging, Baltimore, MD), were cultured in DMEM containing 10% heat-inactivated fetal bovine serum, 5% heat-inactivated horse serum, penicillin (100 units/mL), streptomycin (100 µg/ml), amphotericin B (25 µg/ml), gentamicin (1 ml/L), and 2 mM glutamine. In our experimental cell model, we have used DMEM media that contained 25 mM glucose (hereafter designated standard glucose DMEM) to support optimal propagation, proliferation, and growth of PC-12 cells and to minimize cell differentiation. The cells were cultured at 37°C in a 5% CO2 humidified environment/95% air at 37°C. The culture medium was changed every 2 days. For all experiments, PC12 cells were plated in standard glucose DMEM at a specified density the day before the experiment was performed. On the day of the experiment before addition of redox agents, the culture media were replaced with either fresh standard glucose DMEM (i.e., 25 mM glucose) or DMEM containing 5 mM glucose (hereafter designated reduced glucose DMEM) to investigate the effect of decreased glucose on redox imbalance-induced PC12 apoptosis. Whenever present, reagents were added to cell cultures at the following final concentrations: diamide, 200 µM; tert-butyl hydroperoxide, 100 µM; NAC, 5 mM; DHEA, 100 nM.
Detection of apoptosis by DAPI staining
DAPI staining was performed according to the method of Wang et al. (17)
. Briefly, 1 x 105 PC12 cells were grown on 12 mm circular glass coverslips in 24-well plates. Cells were treated with 100 µM diamide for 24 h, washed with PBS, and fixed with cold 4% paraformaldehyde for 15 min. After washing with PBS, cells were fixed with cold 70% ethanol at -20°C for at least 1 h and stained with 1 µg/ml DAPI for 30 min in the dark. The slides were washed three times with PBS and mounted using DAKO fluorescent mounting fluid. Cells were viewed and counted using a fluorescent Olympus Bx50 microscope with a 20x objective. At least six fields of total and apoptotic cells were counted on each slide; 200 cells were counted.
Measurements of GSH and GSSG
Cellular glutathione (GSH and GSSG) was determined by the high-performance liquid chromatography method of Reed et al. (18)
. 2 x 106 cells were cultured in 100 mm culture plates and exposed to diamide in 10 ml of standard or reduced glucose DMEM. In some experiments, cells were concomitantly treated with the thiol modifying agent NAC (5 mM) and diamide at time zero. At time points ranging from 0 to 6 h, cells on culture plates were harvested by scraping in ice-cold 5% trichloroacetic acid (TCA). Floaters were collected by centrifugation and added to TCA suspension. The combined cell suspensions were then centrifuged to remove TCA-insoluble proteins. The acid supernatant was derivatized with 6 mM iodoacetic acid and 1% 2,4-dinitorphenyl fluorobenzene to yield the S-carboxymethyl and 2,4-dinitrophenyl derivatives of GSH and GSSG. Separation of GSH and GSSG derivatives was performed on a 250 mm x 4.6 mm Alltech Lichrosorb NH2 10 micron column. Cellular GSH and GSSG contents were quantified by comparison to standards derivatized in the same manner.
Preparation of cell lysates for Western analyses
Analyses of CPP32, BAX, and BCL-2
PC12 cells (2x106) were plated per T-25 dish. After treatment with diamide for 0–6 h, cells were ruptured with 300–600 µL of lysis buffer containing 300 mM NaCl, 50 mM Tris-HCl, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1.8 mg/ml iodoacetamide for 30 min at 4°C. Adherent cells were scraped and combined with floaters. Whole cell extracts were prepared by homogenization and stored at -20°C until Western analyses.
Analyses of cytochrome c
PC12 cells were plated at a density of 2 x 106 cells and harvested 0–6 h after diamide treatment. Culture media were removed and cells were washed twice with PBS. Cells were harvested in 1 ml PBS by scraping, transferred to Eppendorf tubes, and collected by centrifugation at 800 g for 10 min. The cell pellets were suspended in 0.5 ml of cold lysis buffer, incubated for three minutes on ice, and homogenized with 10 up and down strokes using a Glas-Col homogenizer. The suspension was centrifuged at 750 g for 15 min at 4°C to collect the mitochondrial fractions (pellet). Mitochondrial extracts were stored at -80°C until further use in Western analyses.
Western blot analyses
Given the ample amount of protein available for analyses, the expression of pro-caspase-3, Bax, and BcL2 was routinely determined in whole cell extracts. Total cellular protein (20 µg) was resolved on 12% acrylamide gels (100 V, 90 min) and blotted onto nitrocellulose membranes. The membranes were individually probed with anti-CPP32, Bax, or BcL-2 (1:500–1:1000). The secondary antibody corresponded to the primary antibody (goat, mouse, etc.) and was conjugated to horseradish peroxidase (1:1000). We elected to determine the loss of mitochondrial cytochrome c as an indicator of activation of mitochondrial apoptotic signaling because Western blots run with mitochondrial proteins consistently gave cleaner bands of cytochrome c expression with significantly lower background than those run with cytosolic proteins. To detect cytochrome c, 20 µg mitochondrial protein was run for 6 h in a 12% SDS-PAGE gel at 50V and 4°C. Protein was transferred onto nitrocellulose membranes overnight at 20V and 4°C. Membranes were probed with primary and secondary cytochrome c antibodies (1:500, 1:5000). Detection of chemiluminescence was performed with an ECL Western blotting detection reagent according to the manufacturer’s recommendation. After exposure to one antibody, each membrane was stripped (6.25 mM Tris pH 6.7, 2% SDS, and 100 mM mercaptoethanol) and probed again for ß-actin to verify equal protein loading in each lane.
Determination of cellular oxidant production
Oxidant formation was measured using the oxidant-sensitive nonfluorescent probe dihydrorhodamine 123 (DHR). Previous studies have used DHR to detect reactive oxygen species in cells (19)
. Because intracellular oxidation of DHR is mediated by biological oxidants like peroxynitrite and a variety of secondary hydrogen peroxide-dependent intracellular reactions that includes H2O2-cytochrome c and H2O2-Fe2-+ (19
, 20)
, the detection of increased oxidation of this probe is a useful marker of a change in general cellular oxidant production. Fluorometric determination of rhodamine from DHR oxidation has been used to determine the interactions between superoxide and nitric oxide (21)
. Intracellularly, DHR is oxidized by two electrons that result in the formation of rhodamine 123, which possesses high molar absorptivity and is fluorescent. DHR can be oxidized in different compartments throughout the cell, but the oxidation product rhodamine 123 will preferentially accumulate in mitochondria according to the trans-mitochondrial potential. Since excitation of rhodamine at 500 nm results in light emission at 536 nm, measurement of the amount of rhodamine at these excitation/emission wavelengths provides a reasonable quantification of overall cellular oxidant production.
DHR was prepared as a 25 mM stock solution in nitrogen-purged dimethyl formamide (DMF) and stored in the dark at -20°C. On the day of each experiment, stock DHR was diluted fresh with DMF and added to cells at a final concentration of 5 µM. Cells grown in standard or reduced glucose DMEM were exposed to diamide for 30 min, then washed twice with PBS. The cells were harvested with a scraper into 2 ml of PBS and sonicated using a Braun-Sonic sonicator (B. Braun Biotech Int., Allentown, PA). Rhodamine123 accumulation was quantified using an AMINCO Bowman Series 2 luminescence spectrophotometer (Thermo Spectronic, Rochester, NY) at excitation and emission wavelengths of 500 and 536 nm, respectively. Results were expressed as relative fluorescence unit/mg protein.
Measurement of cellular NADPH
Cells grown in standard or reduced glucose DMEM were treated with diamide with or without DHEA for 0–2 h. Cell extracts were prepared and cellular NADPH concentrations were measured using the protocol of Zhang et al. (22)
. Cells were collected by scraping, washed twice with PBS, resuspended in 0.15 ml extraction buffer containing 0.1M Tris-HCl, pH 8.0, 0.01M EDTA and 0.05%(v/v) Triton-X-100, and transferred to 1.5 ml Eppendorf tubes. Cell suspensions were sonicated on ice for 2 min with 30 s intervals and centrifuged at 5000 rpm for 5 min at 4°C. The supernatants were collected and immediately analyzed for NADPH. An aliquot (50 µL) of the extract (representing total NADPH and NADH) in a volume of 1 ml was read spectrophotometrically at 340 nm (A1). A similar aliquot (50 µL) was incubated in a reaction mixture containing 0.1M phosphate buffer, pH 7.6, 0.05M GSSG, and 5 IU with glutathione reductase at 25°C for 5 min to convert NADPH to NADP+ (A2). The difference in absorbance between the two readings (A1-A2) represented the amount of NADPH in the sample. NADPH was quantified by comparison to standard NADPH.
Protein assay
Protein was measured using Bio-Rad Protein Assay kit (Bio-Rad) according to the manufacturer’s protocol.
Statistical analysis
Results are expressed as mean ± SE. Data were analyzed using a 1-way ANOVA with Bonferroni corrections for multiple comparisons or a Student’s t test. P values of <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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summarizes results on the effect of thiol agents on apoptosis of naive PC12 cells. A 24 h culture of control cells typically resulted < 10% cell death exhibiting characteristics of apoptosis, consistent with normal cell turnover. Treatment of cells with the thiol oxidant diamide resulted in 30% apoptosis, suggesting an association between the oxidation of cellular thiols, such as GSH, with enhanced cell apoptosis. To test this suggestion, we pretreated cells with the glutathione precursor NAC, concomitant with diamide exposure. The presence of NAC protected the cells against diamide-induced apoptosis and brought the level of apoptosis to control values (Fig. 1)
.
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Kinetic changes of intracellular redox status induced by diamide and the effect of NAC
To determine the effect of diamide on intracellular redox, we measured the kinetics of diamide-induced changes in cellular GSH and GSSG at different times after treatment of PC12 cells with 200 µM diamide with or without NAC (5 mM). The results in Fig. 2
show that the GSH/GSSG status in untreated cells remained constant throughout the 4 h incubation. Treatment of cells with diamide induced a loss in cellular GSH by 30 min and remained below baseline levels (Fig. 2A
). Correspondingly, diamide caused an early and significant surge in GSSG at 15 to 30 min that returned to near baseline levels at 1 h (Fig. 2B
). These results are consistent with an initial rapid diamide-induced oxidation of GSH to GSSG and subsequent reduction of GSSG through the action of GSSG reductase. The initial loss of GSH and the corresponding increase in GSSG resulted in a marked decrease in the GSH-to-GSSG ratio shortly after diamide challenge (within 30 min; Fig. 2C
). Concomitant treatment of PC12 cells with diamide and NAC resulted in significant increases in cellular GSH levels for up to 2 h (Fig. 1A
), indicating that NAC not only preserved the cellular GSH pool, but also caused GSH synthesis. The diamide-induced early surge in GSSG was abrogated by NAC (Fig. 2B
). Indeed, NAC treatment decreased cellular GSSG to levels below that of controls. Accordingly, NAC eliminated the imbalance in cellular GSH/GSSG ratio caused by diamide and increased the GSH-to-GSSG ratio 10-fold above control (Fig. 2C
).
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Kinetics of diamide-induced changes in mitochondrial proteins (cytochrome c, Bax, BcL-2) and caspase-3
Changes in Bax, BcL-2, and cytochrome c
To demonstrate the role of the mitochondria in signaling in diamide-induced PC-12 apoptosis, we examined the central and common pathway for mitochondria-mediated apoptotic signaling, namely, the control and release of mitochondrial cytochrome c and subsequent activation of the death protease caspase-3 (1)
. Mitochondrial release of cytochrome c has been linked to the gatekeeper function of two mitochondrial proteins, Bax and BcL-2, which form heterodimers (1
, 23)
in the mitochondria. BcL-2 has been shown to block mitochondrial cytochrome c release into the cytoplasm. To determine whether diamide-induced redox imbalance and subsequent cell apoptosis were associated with changes in the function of these proteins, we measured the expression of Bax and BcL-2 by Western blot. Results in Fig. 3
A show that the Bax protein was essentially absent in control cells. Diamide treatment caused an early rise in Bax protein levels at 30 min, remained high for 2 h, and returned to baseline levels at 4 to 6 h postdiamide treatment (Fig. 3A
). The BcL-2 protein level was high in control cells and did not markedly change over the same period after diamide treatment (Fig. 3B
). The ratio of Bax to BcL-2 was significantly increased between 30 min and 2 h (Fig. 3C
), consistent with a favoring of initiation of apoptotic signaling as early as 30 min postdiamide treatment of PC12 cells. Correspondingly, the kinetic shift in expression in favor of Bax corresponded to the time course of the loss of mitochondrial cytochrome c content within 30 min after diamide treatment (Fig. 3D
). The mitochondrial cytochrome c content began to increase at 4 h, consistent with a resynthesis of the cytochrome.
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Caspase-3 activation
Figure 3E
illustrates the results of activation of caspase-3 from its precursor procaspase-3 form CPP32. Expression of procaspase-3 was essentially absent in control cells. After diamide treatment, proenzyme levels increased significantly at 1 h, followed by a decrease at 2 to 6 h, consistent with cleavage and activation of procaspase-3 to active caspase-3. Kinetically, activation of caspase-3 was preceded by the induction of redox imbalance, up-regulation of Bax, and the release of mitochondrial cytochrome c. (see Fig. 2
and Fig. 3A, C
, respectively), thus correlating loss of redox balance with mitochondrial initiation of PC-12 apoptosis.
Diamide does not enhance cellular ROS production
To verify that the effect of diamide on PC12 apoptosis was mediated by redox shift independent of the production of oxygen radicals secondary to decreased cellular GSH, we quantified ROS production by measuring the oxidation of DHR. PC12 cells cultured in standard or reduced glucose DMEM were preloaded with 5 µM DHR and exposed to diamide. ROS formation was measured as the increase in fluorescence of rhodamine 123 (see Materials and Methods). Figure 4
A summarizes the results of intracellular rhodamine 123 accumulation at 30 min with and without diamide treatment. The data show no significant difference in rhodamine fluorescence between control cells and diamide-treated cells grown in 25 mM glucose, indicating that diamide at 200 µM with an unlimited glucose source did not increase cellular ROS production. Cells grown in 5 mM glucose alone did not induce ROS generation, but treatment with diamide resulted in a threefold increase in rhodamine fluorescence, indicating an increase in ROS generation by concomitant decreased glucose availability and oxidant challenge. To confirm that ROS generation can be measured by DHR oxidation, we exposed cells grown in standard glucose DMEM to tert-butyl hydroperoxide (BH), a model hydroperoxide, for 30 min and quantified the DHR oxidation. The results (Fig. 4B
) show that BH resulted in a threefold increase in rhodamine fluorescence. Moreover, addition of NAC blocked the oxidation of DHR in BH-treated cells. These results are consistent with the generation of ROS in the presence of an oxidant like BH but not typically in the presence of a thiol agent like diamide under conditions of unlimited glucose availability.
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Decreased glucose availability exacerbates diamide-induced apoptosis
Our kinetic data are consistent with the notion that PC-12 apoptosis was initiated by a rapid and abrupt rise in GSSG or a fall in the GSH/GSSG ratio within the first 30 min after diamide challenge. Once initiated, the recovery of redox balance did not prevent the progression of apoptosis to its biological end point at 24 h. To explore the importance of cellular GSSG in apoptotic cell death, we designed experiments to manipulate cellular NADPH, which is pivotal in the reduction of GSSG and regeneration of GSH. Because the pentose phosphate shunt is a major pathway for NADPH supply, we sought to alter the shunt function by decreasing glucose availability in two ways. First, media glucose concentrations were reduced from 25 mM to 5 mM to limit substrate supply; second, cells were exposed to DHEA, an inhibitor of glucose 6-phosphate dehydrogenase, the rate-limiting enzyme in the pathway. Figure 5
summarizes the results on the effect of decreased glucose availability on diamide-induced PC-12 apoptosis. The data show that diamide exposure caused significant apoptosis at 6 h in cells grown in media containing 5 mM glucose (26% vs. 12% in normal glucose, Fig. 5A
). The kinetics of apoptotic cell death occurred more quickly (in 6 h) in cells cultured under reduced glucose conditions compared with the time it took for the same degree of apoptosis to be achieved in cells cultured under standard glucose conditions (30% at 24 h; see Fig. 1
). Cell apoptosis was further enhanced when cells grown in 5 mM glucose were treated with DHEA. In contrast, at 6 h diamide caused a smaller increase in cell apoptosis in cells grown in 25 mM glucose that was not potentiated by DHEA treatment. However, DHEA treatment did result in an exacerbation of diamide-induced PC-12 apoptosis in glucose sufficient cells at 24 h (35%). A significant number of cells were detached from the coverslips under conditions of reduced glucose with or without DHEA treatment at 24 h, which precluded quantitative evaluation of apoptotic cell number. Collectively, these results are consistent with a role for the pentose phosphate shunt in cell preservation after challenge with the thiol oxidant.
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Decreased glucose availability for phosphate shunt activity enhances GSH/GSSG imbalance
Figure 6
shows the kinetics of changes in cellular GSH and GSSG in PC-12 cells grown in 25 mM glucose in the presence of DHEA (Fig. 6A, B
) or in cells grown in 5 mM glucose (Fig. 6C, D
). Exposure of DHEA-treated cells to diamide resulted in a decrease in cellular GSH at 15 min that fell below levels exhibited by treating cells with diamide alone (Fig. 6A
). Moreover, the GSH pool remained low and failed to recover to control levels over the 4 h incubation. Correspondingly, combined treatment of cells with DHEA and diamide resulted in a rapid and significant rise in cellular GSSG that peaked at 15–30 min and remained elevated for 2 h (Fig. 6B
). Peak GSSG levels achieved under these conditions were at least twofold greater than those obtained with diamide treatment alone, indicating a potentiation of cellular redox imbalance when the pentose phosphate shunt activity was inhibited. In cells cultured under reduced glucose conditions, treatment with diamide alone led to a significant decrease in cellular GSH at 15–30 min, which recovered to control levels by 4 h (Fig. 6C
). The addition of DHEA resulted in greater and irreversible losses of cellular GSH. Cellular GSSG under reduced glucose conditions was elevated and peaked at 3 nmol/mg protein (open circle, Fig. 6D
), levels higher than those in cells cultured in 25 mM glucose (1.5 nmol/mg protein; open circle, Fig. 6B
). Moreover, this elevation in GSSG levels in cells under reduced glucose conditions was sustained from 15 min to 2 h whereas GSSG levels in cells grown in 25 mM glucose returned to control levels within 1 h (Fig. 6D, B
, respectively). Inhibition of G6PD with DHEA in cells with reduced glucose caused a further increase in GSSG (Fig. 6D
). Collectively, these results are consistent with the notion that at a given diamide dose, the intracellular oxidized state is heightened and sustained longer within cells cultured in reduced glucose DMEM than cells grown in standard glucose DMEM.
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Reduced cellular NADPH levels are associated with decreased glucose availability and decreased flux through the pentose phosphate pathway
To verify that the exacerbation of redox imbalance caused by decreased glucose flux through the shunt was due to decreased NADPH production, we quantified cellular NADPH levels under the different treatment conditions. Results are presented in Fig. 7
. NADPH levels in diamide-treated cells grown in 25 mM glucose remained essentially constant for 2 h after treatment, indicating glucose availability was not limiting in maintaining a steady supply of NADPH at this level of diamide stress. However, in the presence of DHEA, diamide caused a time-dependent decrease in cellular NADPH consistent with a progressive inhibition of glucose flux through the pentose phosphate shunt. Diamide treatment of cells grown under decreased glucose conditions resulted in a 50% decrease in NADPH at 30 min, which remained unchanged at this concentration over 2 h, consistent with reduced glucose availability to support maximal NADPH production. Not surprisingly, the addition of DHEA to cells grown in 5 mM glucose caused a greater initial loss of NADPH at 30 min (vs. other treatment groups) and remained low over the 2 h period. Taken together, these results suggest that a reduced flux of glucose through the pentose phosphate pathway leads to decreased cellular NADPH that contributes to an exaggerated redox imbalance and an increased oxidative state.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and a loss of cellular GSH through active efflux has been proposed as an initiating apoptotic signal in several cell types (24)
. In contrast, GSH increases were associated with BcL2 expression that preserved the nuclear redox status (10)
. The investigation of a direct role for GSSG or increased GSSG-to-GSH in apoptosis signaling is lacking. In a recent study we provided unequivocal evidence for redox mediated cell arrest in an intestinal cell line independent of ROS generation (25)
. In the current study, we have used a dose of diamide to change GSH-to-GSSG balance without the involvement of ROS (26, Fig. 4
). Our results have demonstrated a direct relationship between induction of an early and rapid elevation in cellular GSSG and the initiation of apoptosis in PC12 cells. Several lines of evidence support this conclusion. We have found that oxidation of GSH to GSSG by diamide at 15–30 min resulted in PC12 apoptosis at 24 h, which was blocked by NAC in association with an attenuated GSSG level and a restored cellular GSH/GSSG status. The kinetics of GSH/GSSG imbalance preceded induction of the mitochondrial apoptotic signaling. Enhanced cellular ROS production that was prevented by exogenous NAC was elicited by treatment of PC12 cells with the oxidant tert-butyl hydroperoxide but not with the thiol oxidant diamide under conditions of unlimited glucose availability. However, diamide challenge in the face of a reduced glucose source did promote ROS formation. Our results show that decreasing the media concentration of glucose from 25 to 5 mM without the accompanying diamide stress did not elicit ROS generation, in contrast to a recent study demonstrating attenuated ROS production by endothelial cells grown in 5 mM glucose even in the absence of an imposed stress (27)
. Finally, the centrality of GSSG in determining PC12 apoptosis initiation was verified by studies that blocked NADPH-dependent GSSG reduction. Specifically, we found that the blockade of NADPH production by the pentose phosphate pathway exacerbated diamide-induced apoptosis that was linked to highly elevated and sustained levels of GSSG.
An interesting observation in the present study is the finding that apoptosis in PC-12 cells was mediated by increases in GSSG levels (relative to GSH) rather than changes in GSH levels per se. Distinguishing between regulation by GSH and regulation by GSSG-to-GSH (i.e., redox) is a relevant mechanistic issue. We earlier found that NF
B activation was uniquely induced by changes in GSSG-to-GSH ratio but not by changes in GSH alone (28)
. Furthermore, we found it was the change in cellular GSH-to-GSSG ratio rather than in GSH that specifically mediated apoptosis in an intestinal cell line and that this redox imbalance induced apoptosis was preceded by caspase-3 activation (29)
. In the current study, attenuation of diamide-induced apoptosis by exogenous NAC directly correlated with abrogation of the GSSG surge and restoration of the GSH/GSSG redox status, consistent with a role for redox modulation of apoptosis. Mechanistically, NAC could function in one of two ways. 1) A direct interaction of diamide with NAC could spare the oxidation of GSH, thereby preserving the cellular GSH pool. 2) NAC could serve as a cysteine precursor for de novo GSH synthesis. That NAC treatment caused a net increase in the total intracellular GSH pool (GSH+GSSG, Fig. 2
) indicates that the major role of NAC was in the synthesis of new GSH, although we cannot rule out a direct role of NAC as a reductant in diamide elimination. The mechanism by which redox regulates apoptosis signaling is unclear. The observation that mitochondrial permeability transition is under redox control is important. Functional divalent thiol-reactive agents were found to induce apoptosis via formation of disulfide cross-links that blocked the gatekeeping function of BcL2 in preventing mitochondrial permeability transition (30)
. The active site cysteine in caspases is prone to oxidation or thiol alkylation (1)
. Limited evidence in the literature indicates that the mitochondrial permeability transition may also be directly modulated by NADPH/NADP+ (31)
.
Regardless of the mechanism of redox regulation, the present study provided evidence that redox-mediated apoptosis in naive PC12 cells occurred through mitochondrial signaling and activation of caspase-3, a pathway of apoptosis commonly attributed to oxidant-mediated apoptotic death (1)
. Our results support a temporal link between the loss of mitochondrial cytochrome c with enhanced expression of Bax, a proapoptotic member of the BcL-2 family of proteins. Expression of the anti-apoptotic family member BcL-2 remained unaltered. This lack of change in BcL-2 expression with diamide treatment is intriguing because it has been reported that BcL-2 up-regulation protects cells from various apoptotic insults by up-regulating or maintaining GSH levels in cells (7
8
9
10)
. One possible explanation of our data was that the rapid oxidation of cellular GSH to GSSG shortly after diamide insult rendered the BcL-2 protein unable to respond to protect cells from the apoptotic challenge. Because the balance between expression of Bax/BcL-2 controls the release of cytochrome c and dictates whether a cell initiates apoptosis, our results indicate that apoptosis was favored under diamide stress, as evidenced by a significant increase in the Bax-to-BcL2 ratio between 30 min and 2 h after diamide exposure (Fig. 2B
). GSSG increases, release of mitochondrial cytochrome c at 15–30 min, and apoptosis at 24 h support our conclusion that a rapid loss of redox homeostasis is a critical upstream event in mediating apoptosis signaling at the level of the mitochondria. This early loss of cytochrome c from the mitochondria to the cytosol allowed for formation of the proapoptotic apoptosome, which can process pro-caspase-3 (1)
. Activation of caspase-3, which occurred between 2 and 4 h after diamide treatment, was downstream of redox shift and cytochrome c release, in agreement with our earlier observations (29)
. The recovery of redox homeostasis past 1 h postdiamide challenge did not prevent the final apoptotic outcome 24 h later. Taken together, results from these kinetic studies are consistent with our notion that the response of mitochondrial signaling to an abrupt shift in redox is a rapid process shortly after exposure to the apoptotic stimuli and that sustained disruption of redox balance is not necessary to effect the ultimate apoptotic outcome.
Another notable observation is the finding that diamide-induced apoptosis of PC-12 cells was exacerbated by decreased glucose availability in the culture media and inhibition of pentose phosphate shunt function. Previous studies in our laboratory have shown that NADPH levels were governed by glucose availability and that a reduction of NADPH led to impaired regeneration of GSH by the GSH redox cycle (32
, 33)
. Glucose flux through the pentose phosphate pathway has been shown to be important in cellular NADPH production (33
, 34)
. In our current study, direct measurements of NADPH agree with a decreased production of this reductant in cells cultured in reduced glucose media and cells treated with DHEA, the inhibitor of the pentose phosphate pathway. Moreover, determination of cellular GSH/GSSG changes under these conditions indicated that glucose availability and the function of G6PD are important factors contributing to redox homeostasis via NADPH supply. For instance, treatment of cells with diamide plus DHEA resulted in a slower regeneration of cellular GSH from GSSG than treatment of cells with diamide alone in accordance with reduced NADPH levels. Similarly, diamide-induced elevation in GSSG in cells grown in reduced glucose was sustained longer in this oxidized state, which was directly correlated with decreased NADPH. The combined DHEA inhibition of glucose flux through the pentose phosphate pathway and limitation in media glucose led to the most dramatic loss in NADPH (Fig. 7)
and the most severely compromised GSH pool (Fig. 6C
) among the different treatment conditions.
In summary, we have shown that induction of an early and transient loss of cellular redox balance, independent of ROS production, initiated apoptosis in naive PC-12 cells through mitochondrial signaling and activation of caspase-3. The cellular status of GSSG was an important determinant of cell apoptosis. The addition of exogenous NAC protected well against redox imbalance-induced cell apoptosis by attenuating the rise in GSSG and restoring cellular GSH/GSSG homeostasis. The supply of cellular NADPH was critical to the maintenance of the GSH/GSSG status through the pentose phosphate pathway. The current finding of a temporal relationship between an early induction of cellular redox imbalance and initiation of mitochondrial apoptotic signaling will be relevant to understanding the oxidative mechanism of cell loss in the undifferentiated and mitotic competent stage of the cell. Our ongoing studies are investigating the role of redox in mediating cell apoptosis at the differentiated stage of the cell; the results should provide novel information about apoptosis regulation between mitotic competent and terminally differentiated, quiescent cells.
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ACKNOWLEDGMENTS |
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Received for publication October 17, 2001.
Revision received January 29, 2002.
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