Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+ homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+ pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2 treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]i in control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+ storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.
mitochondrial membrane potential; calcium ionophore; oxidative stress
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INTRODUCTION |
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THE ONCOPROTEIN Bcl-2 inhibits most types of apoptotic and nonapoptotic cell death. The mechanism by which Bcl-2 inhibits cell death is unknown. The distribution of Bcl-2 in intracellular membranes (8, 15, 17) has led to speculations that Bcl-2 interacts with electron transfer reactions and affects the formation of reactive oxygen intermediates (ROIs) (16). It has therefore been suggested that Bcl-2 has antioxidant properties and directly impairs oxidative stress, which is often observed in response to a variety of apoptotic stimuli (16, 20). In addition, it has been reported that Bcl-2 protected cells from H2O2- and menadione-induced cell death (16) and that Bcl-2 inhibited neural cell death by decreasing the generation of ROIs (20). Recent studies, however, show that Bcl-2 may also be protective against apoptosis in hypoxic conditions in which no ROIs are generated (18, 25, 33).
A role for Ca2+ in initiation of apoptosis has been inferred from several observations. These include 1) increases in intracellular Ca2+ concentration ([Ca2+]i) in thymocytes undergoing apoptosis by glucocorticoids (39), 2) the induction of Ca2+-dependent endonucleases in apoptotic thymocytes and other cells (11), 3) blockage of DNA fragmentation and prolongation of cell survival by treatment with Ca2+ chelators (23), and 4) induction of apoptosis by treatment of cells with Ca2+ ionophores (5). Mammalian cells normally maintain [Ca2+]i at ~100 nM, which is 10,000-fold lower than extracellular [Ca2+], sequestering Ca2+ for intracellular signaling. There exist two major types of intracellular Ca2+ stores (4): 1) the endoplasmic reticulum (ER), which functions as a high-affinity, low-capacity Ca2+ pool, and 2) mitochondria, which are low-affinity, high-capacity Ca2+ pools. It has been suggested that Bcl-2 may prevent cell death by regulating Ca2+ flux through ER (2, 13, 21) or mitochondria (24).
Mitochondria have the capacity to store Ca2+ as calcium phosphate and to buffer physiological increases in [Ca2+]i as long as the mitochondrial membrane potential is maintained. Mitochondria accumulate large loads of Ca2+ under conditions of rapid and prolonged entry of Ca2+ into the cytoplasm, which occurs during treatment with cytotoxic agents such as H2O2. Excess Ca2+ loads cause mitochondrial respiratory impairment, followed by a decrease in membrane potential and Ca2+ efflux from the mitochondria. In this context, it has been suggested that Bcl-2 prevents cell death in part by inhibiting the decrease in mitochondrial membrane potential caused by respiratory chain inhibitors (32), oxidant stress (28), and dexamethasone (6). In addition, Murphy et al. (24) showed that Bcl-2 increased the maximal Ca2+ uptake capacity of mitochondria in neuronal cells, indicating that Bcl-2 prevents the loss of mitochondrial membrane potential allowing excess Ca2+ storage in the mitochondria.
To better understand the mechanism of oxidant-induced cell death and its prevention by Bcl-2, we investigated the role of Ca2+ in this process. Here, we demonstrate that H2O2-induced cytotoxicity is Ca2+ dependent and that Bcl-2 inhibits cell death by increasing mitochondrial Ca2+ storage.
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MATERIALS AND METHODS |
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Cell line. A normal rat kidney (NRK) cell line (NRK-52E), obtained from the American Type Culture Collection (Manassas, VA), was maintained in DMEM supplemented with 10% fetal bovine serum.
Preparation of stable transfectants expressing Bcl-2. The vector pCEP4 (14) containing the hygromycin resistance gene was obtained from Invitrogen. The vector pCEP-Bcl-2 containing the human Bcl-2 cDNA (10) under the control of the human cytomegalovirus promoter was obtained from Dr. Xin Wang [Laboratory of Human Carcinogenesis, National Cancer Institute (NCI), National Institutes of Health (NIH)]. Transfection of pCEP4 and pCEP-Bcl-2 into NRK cells was performed using the calcium phosphate precipitation procedure according to Chen and Okayama (7). Resistant clones were selected in medium containing 200 µg/ml hygromycin. After selection for 10 days, colonies were isolated and characterized for human Bcl-2 expression using immunoblotting with an anti-human Bcl-2 monoclonal antibody (DAKO). Bcl-2-expressing clones were maintained in medium containing 50 µg/ml hygromycin. Hygromycin was omitted in cultures plated for experiments.
The vector pD5neo is an adenovirus-derived expression vector containing the adenovirus 5 major late promoter, the simian virus 40 (SV40) enhancer, and the neomycin resistance gene under the control of the SV40 promoter. pD5neo was used for the construction of the expression vector pD5neo-Bcl-2 as described before (1). After transfection, cells were replated and grown in complete medium containing 800 µg/ml G418 for 10 days. Colonies were isolated and characterized for Bcl-2 expression as just described. Bcl-2-expressing clones were maintained in medium containing 200 µg/ml G418. G418 was omitted from cultures plated for experiments. The cell lines NRK-pCEP4 and NRK-pCEP4-Bcl-2 are referred to in the text as NRK control cells and NRK-Bcl-2 cells, respectively. All the experiments described here were done with the cell lines NRK-pCEP4 and NRK-pCEP4-Bcl-2 except the viability experiment (see Fig. 2B) and immunocytochemistry for Bcl-2 (see Fig. 1B), in which NRK-pD5neo and NRK-pD5neo-Bcl-2 cells were used.Western blot analysis and Bcl-2 immunostaining. For Western blot analysis, proteins were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane by electroblotting. The membrane was developed using a mouse monoclonal anti-human Bcl-2 antibody (DAKO).
For immunostaining, control and Bcl-2-transfected NRK cells were fixed and permeabilized with 2% paraformaldehyde for 10 min, followed by 20 min in methanol. After three washes in PBS-plus solution (0.15% glycine, 0.5% BSA in PBS), the fixed cells were incubated with mouse anti-Bcl-2 antibody (diluted 1:40 in PBS-plus) for 1 h at 37°C. After five washes in PBS, the slides were incubated with fluorescein-conjugated rabbit anti-mouse IgG (diluted 1:40 in PBS-plus) for 1 h at 37°C. The slides were mounted by adding 1 drop of VectaShield containing 0.5 µg/ml 4',6'-diamidine-2'-phenylindole dihydrochloride (DAPI). Photomicrographs were taken using an Olympus IMT-2 inverted microscope and a Bio-Rad MRC 600 confocal scanning laser imaging system. For rhodamine staining, control and Bcl-2-transfected NRK cells were treated with 2 µg/ml rhodamine 123 in DMEM for 15 min at 37°C. Then cells were washed twice with DMEM. Rhodamine 123 fluorescence was recorded using the confocal fluorescence microscope at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. After rhodamine staining, cells were fixed with paraformaldehyde-methanol, followed by Bcl-2 immunostaining as described above.Cytotoxicity assay.
Changes in cell viability were measured using the CytoFluor 2350 fluorescence measurement system (Millipore). Cell death was quantified
by propidium iodide fluorescence (excitation at 530 nm and emission at
645 nm). Control and Bcl-2 cells were trypsinized and plated on a
24-well plate at a density of 105
cells/well. The medium (DMEM supplemented with 10% fetal bovine serum)
was replaced 24 h later with medium containing 20 µM propidium iodide
and 1 mM
H2O2.
Fluorescence was measured at intervals of 1 h for up to 5 h. At the end
of the experiment, 100% cell death was assessed by permeabilizing
cells by addition of digitonin (375 µM) to each well and measuring
propidium iodide fluorescence as described above. Percent viability (V)
was calculated as V = 100(B X)/(B
A), where A is
initial fluorescence, B is fluorescence after addition of digitonin,
and X is fluorescence at any given time.
[Ca2+]i measurement following treatment with H2O2 and Ca2+-mobilizing compounds. NRK control and NRK-Bcl-2 cells were plated on 6-cm dishes with glass coverslips mounted in the bottom of the dish. Two days later, cells were preloaded with 5 µM fluo 3-AM (Molecular Probes), which has an excitation wavelength of 490 nm and an emission wavelength of 525 nm, in DMEM for 1 h at room temperature. The cells were washed three times with modified Hanks' balanced salt solution [HBSS; in mM: 137 NaCl, 5.37 KCl, 0.81 MgSO4, 0.44 KH2PO4, 0.37 Na2HPO4, 10 HEPES (pH 7.2), and 5.56 glucose, with or without 1.37 CaCl2]. Nominally Ca2+-free HBSS was prepared without added Ca2+ and contained 2-5 µM Ca2+. Then the cells were treated with 10 mM H2O2 or 5 µM ionomycin in Ca2+-containing HBSS or with 2 µM thapsigargin or 4 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) in nominally Ca2+-free HBSS. With confocal microscopy, images of [Ca2+]i were recorded every 2-20 s for up to 20 min. The measurement system consisted of a Bio-Rad MRC 600 confocal laser imaging system with an Olympus IMT-2 inverted microscope and an IBM-compatible computer. [Ca2+]i is expressed as emission intensity of fluo 3 at 525 nm. For data presentation, at least 10 cells were chosen per experiment, and changes in intensity corresponding to fluo 3 fluorescence were calculated for each cell.
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RESULTS |
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Overexpression of Bcl-2 in NRK cells. As shown on the Western blot in Fig. 1A, NRK-Bcl-2 clone 4 cells show high expression of transfected Bcl-2, which appears as a band at 26 kDa. In contrast to Bcl-2-transfected NRK cells, pCEP4-transfected NRK cells do not express Bcl-2. The expression level for Bcl-2 in NRK-pD5neo-Bcl-2 cells was similar to that in NRK-Bcl-2 cells (data not shown).
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Bcl-2 protects NRK cells from oxidant-induced cytotoxicity. To examine the effect of Bcl-2 expression on oxidant-induced cell killing, we performed viability assays measuring propidium iodide uptake after treatment with 1 mM H2O2. As shown in Fig. 2, A and B, Bcl-2-expressing cells were more resistant to H2O2-induced cytotoxicity than control cells. After 5 h of H2O2 treatment, viability of control cells was reduced by 80%, whereas loss of viability in Bcl-2-expressing cells was only 20-30%. These results clearly show that Bcl-2 protects cells against oxidant-induced loss of viability. In Fig. 2A, cell viability in H2O2-treated cells (up to 2 h of treatment) in the presence or absence of EGTA-AM appears to exceed 100%. This apparent paradox can be explained by photobleaching of propidium iodide, which would decrease the fluorescence signal of propidium iodide at any given time during incubation compared with the initial fluorescence.
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Oxidant-induced cell death is dependent on intracellular Ca2+. Because oxidants have been shown to damage membranes by lipid peroxidation (37) and as a consequence to affect intracellular Ca2+ homeostasis (19, 22), we hypothesized that an increase in [Ca2+]i contributes to the cytotoxic effect of oxidants. To test this hypothesis, we measured H2O2-induced cytotoxicity in the absence of intracellular Ca2+ by pretreating cells with the Ca2+ chelator EGTA-AM. Figure 2 shows that EGTA-AM protected both control and Bcl-2 cells from H2O2-induced toxicity, indicating that oxidant-induced toxicity in NRK cells is dependent on the availability of intracellular Ca2+. To answer the question of whether the intracellular Ca2+ mediating H2O2 toxicity in NRK cells is derived from the intracellular Ca2+ stores or from the extracellular Ca2+ pool, we compared the viability of control and Bcl-2-expressing cells after H2O2 treatment in PBS containing 0.9 mM Ca2+ and in nominally Ca2+-free PBS ([Ca2+] 2-5 µM). As shown in Fig. 3, both control and Bcl-2-expressing cells were more resistant to oxidant-induced toxicity in nominally Ca2+-free PBS, indicating that extracellular Ca2+ taken up by the cell is responsible for oxidant-induced toxicity.
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Effect of Bcl-2 on intracellular Ca2+ homeostasis. To study the effect of Bcl-2 overexpression on intracellular Ca2+ homeostasis, we measured changes in [Ca2+]i after treatment with H2O2 and the Ca2+-mobilizing compounds ionomycin, thapsigargin, and the uncoupler CCCP. Ca2+ images were obtained using the Ca2+ dye fluo 3 and confocal microscopy. In these images, brightness of the cells is proportional to [Ca2+]i and is expressed as increase in brightness over time, in multiples of the brightness at time 0. Figure 4A shows the kinetics of increase in [Ca2+]i in control and Bcl-2-transfected cells following treatment with 10 mM H2O2. It is apparent that in Bcl-2 cells, the rise in [Ca2+]i was delayed compared with control cells. Treatment with 5 µM ionomycin (Fig. 4B), a Ca2+ ionophore (29), rapidly increased [Ca2+]i in both NRK and NRK-Bcl-2 cells. However, NRK-Bcl-2 cells showed a much faster decrease in [Ca2+]i after peak than did control cells, indicating stronger intracellular Ca2+ buffering by either the ER or the mitochondria in conditions of Ca2+ overload. Treatment of cells with 2 µM thapsigargin (an inhibitor of ER ATPase) (3) increased [Ca2+]i transiently, with no difference seen between control and Bcl-2-expressing cells (Fig. 5A).
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DISCUSSION |
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Although changes in Ca2+ regulation have been shown to occur in cells undergoing either apoptotic or necrotic cell death induced by various agents (2, 30), the role of intracellular Ca2+ homeostasis in oxidant-induced cell death is unknown. Using NRK cells as a model for cell death, we obtained evidence that oxidant-induced cell death is mediated by intracellular Ca2+, that Bcl-2 protects NRK cells from oxidant-induced cell death, and that in Bcl-2-expressing NRK cells the capacity of mitochondria to store Ca2+ is increased compared with control cells.
We chose NRK cells as an in vitro model system to study apoptosis and necrosis in proximal tubular epithelial cells of the kidney. NRK cells have been shown to undergo apoptosis or necrosis induced by a variety of agents (12). To answer the question of whether oxidant-induced cell death in NRK cells is necrotic or apoptotic, we examined nuclear morphology using DAPI staining in control and Bcl-2-transfected cells either treated with H2O2 or untreated (data not shown). In addition, overall morphological changes in NRK cells as a consequence of oxidant treatment were examined (data not shown). Examining nuclear morphology, untreated cells contained regularly shaped nuclei. After treatment with H2O2, 40% of NRK cells showed condensed chromatin, but only a few cells contained fragmented nuclei characteristic of apoptosis. NRK-Bcl-2 cells treated under the same conditions showed fewer cells with condensed chromatin or fragmented nuclei than control cells. Electron microscopic analysis revealed morphological changes typical of necrosis, such as dilated ER, condensed mitochondria with calcification, dissociation of two layers of nuclear membranes, and disrupted plasma membranes with relatively well-preserved nuclei, but not apoptosis. These changes occurred in a dose-dependent manner in NRK as well as in NRK-Bcl-2 cells, albeit at a lower frequency in Bcl-2-transfected than in control NRK cells. From these observations it can be concluded that oxidant-induced cell death in NRK cells is necrotic and not apoptotic.
To study the effect of Bcl-2 on oxidant-induced cell death, we constructed the cell lines NRK-Bcl-2 and NRK-pD5neo-Bcl-2, which contain the episomal expression vector pCEP-Bcl-2 and the stably integrated plasmid pD5neo-Bcl-2, respectively. Our Western blot data (Fig. 1A) show that control NRK cells do not express Bcl-2, whereas Bcl-2-transfected NRK cells produce large amounts of the Bcl-2 oncoprotein. Immunocytochemistry reveals that transfected Bcl-2 is localized to the cytoplasm, with the strongest staining in the perinuclear area (Fig. 1B). This observation is in agreement with published results showing that Bcl-2 is found primarily in membranes of the ER, the nucleus, and, for the most part, the mitochondria (8, 15, 17). To demonstrate that in Bcl-2-transfected NRK cells Bcl-2 localizes to the mitochondria, we performed colocalization studies using the mitochondrion-specific dye rhodamine 123 in combination with anti-Bcl-2 immunostaining. As shown in Fig. 1Bc, rhodamine 123 staining overlaps with Bcl-2 immunostaining, indicating colocalization. This observation suggests that Bcl-2 protects NRK cells against oxidant-induced cell death by preventing those mitochondrial changes that are characteristic of the induction of cell death, such as mitochondrial permeability transition (40), loss of mitochondrial membrane potential (32), and mitochondrial Ca2+ overload (24).
To study the role of intracellular Ca2+ in oxidant-induced cell death and its prevention by Bcl-2, we first compared the viability of NRK and NRK-Bcl-2 cells after treatment with oxidants in the presence or absence of the intracellular Ca2+ chelator EGTA-AM. Our results in Figs. 2 and 3 show that Bcl-2 protects against H2O2-induced toxicity and that buffering of [Ca2+]i by intracellular EGTA and the removal of extracellular Ca2+ render NRK as well as NRK-Bcl-2 cells resistant to oxidant toxicity. To examine whether Bcl-2 protects against cell death induced by oxidants other than H2O2, we compared the viability of NRK and NRK-Bcl-2 cells treated with menadione (data not shown). The quinone compound menadione undergoes redox cycling intracellularly, thereby generating superoxide. As with H2O2-induced cell death, Bcl-2 protected against menadione-induced cell death, suggesting a more generalized effect of Bcl-2 on oxidant-induced cell death.
Although the exact mechanism through which Ca2+ mediates cell death induced by oxidants is not known, several hypotheses have been proposed. 1) Oxidant-induced changes in Ca2+ homeostasis lead to cytoskeletal changes such as Ca2+-induced loss of actin binding capacity and proteolytic cleavage of actin by Ca2+-activated proteases (27). Both processes lead to cell surface blebbing, which is an early event in cell death. 2) Ca2+-activated phospholipases are widely distributed in mammalian cells, and it has been suggested that an enhanced rate of phospholipid hydrolysis may result in irreversible cell damage (9). 3) Intracellular protein degradation is also known to be stimulated by a rise in cytosolic Ca2+, and a group of cytosolic proteases, whose activity depends on Ca2+, seems to be involved in the process of cell death (26). 4) A third catabolic process found to be sensitive to a nonphysiological elevation of cytosolic Ca2+ concentration during oxidative stress is that of an endogenous endonuclease. Ca2+-dependent endonuclease activation has been shown to be responsible for the occurrence of DNA laddering, which is a hallmark of apoptosis (35, 38).
The results in Figs. 2 and 3 showing that H2O2-induced cytotoxicity is mediated through intracellular Ca2+ could indicate that the decreased sensitivity of NRK-Bcl-2 cells compared with NRK cells toward H2O2-induced cytotoxicity is due to decreased Ca2+ mobilization in Bcl-2-expressing cells. To test this hypothesis, we compared oxidant-induced Ca2+ mobilization in NRK and NRK-Bcl-2 cells. Figure 4A shows that the increase in [Ca2+]i following H2O2 treatment is delayed in NRK-Bcl-2 cells compared with control cells, suggesting either decreased uptake of Ca2+ from the extracellular space and/or increased sequestration of Ca2+ into the intracellular Ca2+ stores, ER, and mitochondria. To distinguish between these possibilities, we employed ionomycin, thapsigargin, and the uncoupler CCCP as specific inducers of Ca2+ mobilization from the extracellular space, the ER, and the mitochondria, respectively. The Ca2+ ionophore ionomycin allows Ca2+ influx from the extracellular space as well as the release of Ca2+ from intracellular stores. Thapsigargin is a specific releaser of Ca2+ from the ER pool, including the inositol 1,4,5-trisphosphate-sensitive Ca2+ compartment. Treatment of cells with an uncoupler of oxidative phosphorylation, such as CCCP, leads to the release of Ca2+ from mitochondrial stores. In Fig. 4B, our results show that ionomycin increased [Ca2+]i in both NRK and NRK-Bcl-2 cells. However, after reaching a peak, [Ca2+]i decreased faster in NRK-Bcl-2 cells than in NRK cells. This observation suggests that in NRK-Bcl-2 cells excess Ca2+ entering the cell from outside as a result of ionomycin treatment is quickly reabsorbed into intracellular pools, thereby reestablishing normal [Ca2+]i. In contrast to NRK-Bcl-2 cells, the [Ca2+]i peak in control cells decreases very slowly, reequilibrating at a Ca2+ concentration that is three times higher than in Bcl-2-transfected cells. These results suggest that Bcl-2 facilitates intracellular Ca2+ buffering after Ca2+ influx from the extracellular milieu. To test the hypothesis that Bcl-2 prevents cell death by increasing the Ca2+-buffering activity of the intracellular Ca2+ stores, ER, and mitochondria, we first examined the effect of thapsigargin treatment on intracellular Ca2+ mobilization in NRK and NRK-Bcl-2 cells. In Fig. 5A, we show that thapsigargin treatment leads to a transient increase in [Ca2+]i; no difference is seen between control and Bcl-2-expressing cells, suggesting that Bcl-2 does not affect the ER Ca2+ pool in our system. This observation is in contrast to previous reports showing that Bcl-2 inhibits the release of Ca2+ from the ER (21) following thapsigargin treatment and that Bcl-2 inhibits H2O2-induced ER Ca2+ pool depletion (13). To estimate the amount of Ca2+ stored in the mitochondria, we measured mitochondrial Ca2+ release induced by the uncoupler CCCP. As shown in Fig. 5B, in the absence of extracellular Ca2+, CCCP treatment leads to a rise in [Ca2+]i in both control and Bcl-2-expressing NRK cells. The increase in [Ca2+]i observed in NRK-Bcl-2 cells is about twice the increase seen in NRK control cells, indicating a greater steady-state capacity for Ca2+ in mitochondria of NRK-Bcl-2 cells than in those of control cells. In this context, the possibility of release of Ca2+ from nonmitochondrial Ca2+ pools because of compromised ATP production as a consequence of CCCP treatment cannot be ruled out. However, CCCP-induced Ca2+ release in NRK cells was almost immediate in our experiments, making the chance of a secondary Ca2+ mobilization from nonmitochondrial stores remote.
Our results are in agreement with a previous report showing that Bcl-2 potentiates the maximal Ca2+ uptake capacity of neural cell mitochondria (24). The reason for increased Ca2+ storage in Bcl-2-expressing cells could be that Bcl-2 is maintaining the mitochondrial membrane potential, thereby inhibiting apoptosis and necrosis under conditions of Ca2+ overload. Such a mechanism has been suggested in recent reports (28, 31, 32) showing that Bcl-2 prevented the decrease in mitochondrial membrane potential as well as apoptosis and necrosis induced by oxidative stress.
In summary, we show that oxidant-induced cell death is dependent on the availability of Ca2+ in the cytosol and that Bcl-2 inhibits cell death by augmenting the Ca2+ storage capability of the mitochondria.
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ACKNOWLEDGEMENTS |
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We thank Dr. Xin Wang (Laboratory of Human Carcinogenesis, NCI, NIH) for the pCEP-Bcl-2 plasmid.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15440 and a School of Medicine Intramural Grant Competition from the University of Maryland.
Address for reprint requests: P. A. Amstad, Dept. of Pathology, Room 7-23, MSTF, 10 South Pine St., Baltimore, MD 21201.
Received 15 December 1997; accepted in final form 20 May 1998.
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