Toxicological Sciences 53, 48-55 (2000)
Copyright © 2000 by the Society of Toxicology
Effect of Ceramide on Intracellular Glutathione Determines Apoptotic or Necrotic Cell Death of JB6 Tumor Cells
Department of
* Pathology and
Department of Epidemiology/Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland; and
Laboratory of Biochemical Physiology, National Cancer Institute, Frederick, Maryland
Received December 16, 1998; accepted April 15, 1999
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ABSTRACT |
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Selective induction of cell death is a means to remove unwanted cell populations from a tissue or organ. Understanding the signaling events responsible for mediating cell death by cytokines, such as tumor necrosis factor-alpha (TNF
) are key to the development of pharmacologic inducers of this response. Ceramide has been implicated as a secondary messenger for TNF-induced cell death, but many of the intracellular effects of ceramide are not fully understood. Recent reports suggest that ceramide signaling may involve oxidative stress. To explore the relationship between TNF sensitivity and ceramide signaling, two genetic variants of mouse JB6 RT101 epidermal tumor cells, one resistant and one sensitive to TNF- induced cytotoxicity, were treated with C2-ceramide. Treatment with 20 µM ceramide induced apoptosis and this was quickly followed by oncotic necrosis in the TNF-sensitive JB6 (TNFs) cells. The same concentration of ceramide induced apoptosis, but not oncotic necrosis of the TNF resistant JB6 (TNFr) cells. The basal level of glutathione was significantly higher in TNFr cells than in TNFs cells. Treatment with 20 µM ceramide decreased cellular glutathione in TNFs cells by 50%, in contrast to an insignificant decrease in the TNFr cells. A significant increase in reactive oxygen was noted in TNFs cells treated with 10 or 20 µM ceramide. Furthermore, pretreatment with the antioxidant N-acetylcysteine or with glutathione monoethylester delayed the onset of ceramide-induced oncotic necrosis, but did not inhibit apoptosis. Our results suggest that the severity of the decrease in glutathione appears to determine whether cells undergo just apoptosis or also oncotic necrosis. They also suggest that ceramide-induced oncotic necrosis is modulated by a decline in cellular glutathione and an elevation of reactive oxygen. These results suggest that a decrease in cellular redox potential determines susceptibility to ceramide-dependent killing pathways. Key Words: epidermal cells; cell death; ceramide; glutathione; apoptosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor necrosis factor alpha (TNF
) induces intracellular signals that mediate cell death. One major signal induced by TNF is activation of neutral and acidic sphingomyelinases which catalyze the degradation of sphingomyelin to ceramide (Hannun, 1996
; Testi, 1996). Although ceramide has been proposed to be an intracellular mediator for many of the early cellular responses elicited by TNF, including apoptosis (Cai et al., 1997; Schutze et al., 1994; Verheij et al., 1996; Westwick et al., 1995), the intracellular events that connect ceramide to these cellular responses remain unclear. However, alteration of nuclear factor kappa B (NF
B) expression of mitogen activated protein kinase (MAPK) activities and of protease activities are 3 of the potential intracellular events that may connect ceramide to cell death (Dbaibo, 1997; Hannun, 1996; Hartfield et al., 1997; Kitajima et al., 1996; Mizushima et al., 1996; Pronk et al., 1996; Shirakabe et al., 1997). Another important and immediate cellular effect of ceramide may be generation of reactive oxygen (ROS, which generally refers to hydroxyl anion, hydrogen peroxide, singlet oxygen, or other oxygen radicals). Indeed, many of the intracellular effects of ceramide may be attributable to modifications in thiols that act as direct targets for reactive oxygen. Moreover, the effect of ceramide on the major intracellular thiol, glutathione is of vital important since glutathione maintains the reduced state of most intracellular proteins.
The objective of this study was to examine the relationship between TNF sensitivity, ceramide signaling, and ceramide-induced oxidative stress. We hypothesized that the cytotoxic effects of exogenous ceramide are attributable to ceramide-induced oxidative stress. To connect the effect of ceramide on cellular redox to TNF sensitivity, we used variants of mouse JB6 epidermal cells (Singh et al., 1995) that show differential sensitivity to TNF-induced cell death. The JB6 tumor cell variant RT101 TNFr, passage 
200 cells is relatively insensitive to TNF-induced cell death, while the other variant, RT101 TNFs, passage 
90, is highly sensitive to TNF cytotoxicity (Singh et al., 1995; Sun et al., 1992). If ceramide via ROS is a mediator of TNF killing, then the TNFr and the TNFs variants should show differential responses to exogenous ceramide and this response to ceramide should be mediated by changes in cellular redox status. We report here that the TNFs variants are more sensitive to ceramide-induced cytotoxicity than the TNFr variants. The TNFr cells have a higher basal level of GSH than the TNFs cells, which may protect these cells from the acute cytotoxic effects of ceramide. The results suggest that increased ROS and decreased GSH mediate the cytotoxic effects of and cellular susceptibility to ceramide.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and treatments.
The JB6-derived transformed mouse RT101 epidermal cell variants with differential sensitivity to TNF
-induced cell death used in these studies have been previously described (Singh et. al., 1995; Sun et. al., 1992). Sixty mm dishes containing 4 x 105 cells were maintained in Eagles minimal essential medium (EMEM) containing 5% fetal bovine serum, 200 µM glutamine, penicillin (100U/ml), amphotericin B (0.25 µg/ml) and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air. Treatment of TNFr and TNFs cells was carried out in EMEM (Paragon) containing 0.25% serum minus antibiotics, in the same environment. Four 60-mm dishes of cells per treatment group were treated with 10 or 20 µM N-acetyl-N-sphingosine (C2-ceramide, Biomol with purity and stability [assessed by thin layer chromatography]) for specified time points. In some experiments, cells were pre-treated with 200 µM N-acetyl-cysteine (NAC, Fluka) or 2 mM 
-glutamylcysteinylethyl ester (-GCE, glutathione ester; BACHEM) in EMEM containing 0.25% serum for 6 h. Cell extracts were prepared for GSH analysis as described in the methods. Three independent experiments were performed for GSH analysis with each pre-treatment, with indicated concentrations of ceramide.
Glutathione (GSH) Analysis.
Cell extract preparation and HPLC analysis of thiols were carried out as outlined in Faris and Reed (1987). Briefly, cells in 60-mm dishes were washed with Hanks Balanced Salt solution (HBSS) without magnesium or calcium and collected in 10% perchloric acid containing baptho-phenathrolinedisulfonic acid (BPDS, metal chelator). Cells were then lysed and acid extract prepared by sonication, freezing at –70°C, and thawing. Five hundred µl of the acid extract was used to derivatize amino acids with 1-fluoro-2,4-dinitrobenzene, as outlined in the referenced method. A Beckman System Gold Nouveau® Chromatography System and software with a SulpelcosilTM NH2 HPLC column (Sulpelco) were used to separate and analyze the derivatized thiols. Following injection of 50 µl of the derivatized sample, a mobile phase gradient consisting of Mobile phase A (80% methanol) and Mobile phase B (0.5-M sodium acetate in 64% methanol) was applied. The gradient conditions were: 80% A:20% B for 5 min followed by a linear gradient to 99% B for 10 min, which was maintained for 10 min, followed by equilibration to 80% A:20% B for 10 min. The eluted peaks were detected and measured at 360 nm with a Beckman System Gold® UV detector.
Morphologic Evaluations and Cytotoxicity Assays
Phase-contrast microscopy.
Live cells were observed and photographed during the course of each experiment using a Nikon Diaphot inverted microscope and a 40x objective. Cells were photographed using Kodak-TMax 100 film.
Cytotoxicity assays.
Cells grown in 60-mm culture dishes or cells in 12-well multiwell plates were used to measure uptake of propidium iodide (PI) using a Cytofluor 2300-plate reader (PerSeptive Biosystems, Foster City, CA). PI is excluded from live cells with an intact cell membrane and PI is fluorescent when bound to DNA. The Cytofluor quantitates the number of cells that have nuclear PI staining as a measure of loss of cell membrane integrity or oncotic necrosis. Cells (1 x 104 per well) were treated with ceramide at the indicated times and concentrations. In some wells, PI was added at a final concentration of 20 µM and in other wells, DNA was isolated. Ceramide-induced cytotoxic effects were measured by Cell Death Detection ELISA (Apoptosis induction, Boeringer Mannheim) after 2-h treatment with 20 µM ceramide using the manufacturers protocol. The 2-h time point was chosen so that pre-necrotic changes were measured in the TNFs cells, because these cells show a measurable amount of oncotic necrosis by 3 h. Histone-associated DNA fragments were detected and measured by photometric enzyme immunoassay absorbance A405-A490 with a Wallac VictorTM 1420–002 multilabel microplate counter (EG & G Wallach, Finland).
Measurement of reactive oxygen (ROS) generation.
Dihydrorhodamine123 (DHR123) is a cell-permeable fluorescent indicator for reactive oxygen species generation, mainly H2O2 and O2- (Emmendorffer et al., 1990). The oxidation of the non-fluorescent DHR123 by generated reactive oxygen species results in an ionic species of DHR123, which is sequestered by the mitochondria. Cells were grown in 12-well plates at 3 x 105 cells/well. Two dishes were used for each treatment protocol and 2 columns of wells were used for each treatment. Ceramide was added to 2-day cultures at concentrations of 10 and 20 µM. Treatment of cells with 1-mM H2O2 was used a positive control. At various time points, cells were loaded with 20 µM DHR123 in medium for 30 min. After loading, the cells were washed twice with medium and fluorescence was measured with the Cytofluor system described above, with an excitation of 490 nm and emission of 520 nm. Three independent experiments were used for statistical evaluation.
Statistical analysis.
In some cases, data were analyzed with a paired or unpaired, 2-tailed Student's t-distribution using QuattroTM Pro V. 6.0 for Windows. In experiments in which multiple comparisons were made, ANOVA was used. Data are expressed as mean ± SE for n = 3 independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential Cytotoxicity to TNFr and TNFs Cells of Ceramide
Treatment with 10 µM ceramide caused cell death of the TNFs cells but not TNFr cells after 24 h (Fig. 1A
). When treated with 20 µM ceramide, TNFs cells showed a significant increase in propidium iodide (PI) uptake at 6 h, followed by a major increase in PI uptake at 24-h post treatment, indicating an oncotic/necrotic response (Fig. 1B). There was no significant increase in PI uptake in TNFr cells when treated with 10 or 20 µM ceramide at any time point measured (Figs. 1A and 1B). TNFs cells treated with 20 µM ceramide for 6 h showed morphologic changes of oncotic/necrotic cell death, such as swelling and large fluid-filled blebs by phase contrast microscopy (Fig. 2A, panel c). However, TNFr cells treated with 20 µM ceramide showed morphologic changes consistent with apoptosis (such as cell shrinkage and small dense blebs with little loss in cell membrane integrity) 6 h after treatment (Fig. 2A, panel f). Treatment with 10 µM ceramide for 24 h induced a mixture of oncotic necrosis and apoptotic morphology in the TNFs cells (Fig. 2A, panel b) but no apparent morphologic change in the TNFr cells (Fig. 2A, panel c). Thus, while both TNFs and TNFr cells are sensitive to ceramide-induced apoptosis, only TNFs cells are sensitive to ceramide-induced oncotic necrosis.
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Since the morphologic alterations induced by 20 µM ceramide differed in the two cell types, we used a cell-death assay which measures nucleosome-bound DNA, as a means to quantitate the induction of apoptosis. The results of this assay (Fig. 2B
) confirm that TNFr variants undergo apoptosis after treatment with 20 µM ceramide. It appears from the results of this assay that internucleosomal DNA fragmentation also occurs in TNFs cells in response to 20 µM ceramide, although they rapidly undergo oncotic necrosis. These results suggest two possibilities: one is that a subpopulation of TNFs cells undergoes apoptosis and the rest undergo oncotic necrosis. A second possibility is that apoptotic cells in culture are not phagocytosed, lose cell-membrane integrity, and swell (showing oncotic necrosis) after apoptotic morphology is exhibited. The TNFr cells showed only an apoptotic response.
Ceramide Causes an Early Decrease in Intracellular Glutathione Which is Inhibited by NAC Pre-treatment
Ceramide decreased intracellular reduced glutathione in TNFs, but not TNFr cells (Fig. 3). Ceramide treatment (20 µM for 6 h) caused a small, statistically insignificant decrease in glutathione (10%) in TNFr cells, whereas it caused a significant decrease in GSH in TNFs cells (48%). Treatment with a lower concentration of ceramide (10 µM for 6 h) did not induce a measurable decrease in GSH in either cell type, but did decrease GSH levels in TNFs cells by 40% after longer treatment times of 16 h (not shown). The basal level of GSH was significantly higher in the TNFr cells than the TNFs cells, suggesting a possible protective effect against oncotic necrosis. The higher basal GSH concentration may contribute to the decreased susceptibility of the TNFr cells to undergo necrosis. N-acetyl-cysteine (NAC) functions as a general free-radical scavenger supplying the cell with cysteine. Pre-treatment of either cell variant with 100 µM of the anti-oxidant NAC for 6 h abrogated the ceramide-induced decline in GSH (Fig. 3). These results imply that the ceramide-mediated decrease in GSH may be modulated by an increase in ROS.
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Ceramide Causes Generation of Reactive Oxygen Species
Dihydrorhodamine 123 (DHR123) is a cell-permeable fluorogenic marker, which is specifically used to monitor intracellular reactive oxygen generation (Emmendorffer et al., 1990
). We observed a significant increase in reactive oxygen generation after treatment with 10- or 20 µM ceramide in TNFs cells (Fig. 4). Pretreatment of cells with NAC decreased the generation of reactive oxygen species (data not shown). These results show that ceramide causes an increase in ROS, which may cause the observed decrease in GSH.
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Glutathione Monoethylester Protects against Secondary Oncotic Necrosis but Not Apoptosis
To examine the effect of cellular GSH on ceramide-induced cell death, we pretreated the cells with glutathione monoethylester, which releases GSH intracellularly when cleaved by esterases. Glutathione monoethylester pre-treatment produced a notable decrease in oncotic necrosis (Figs. 5A and 6
). The ceramide-induced decrease in cellular GSH appears to mediate the onset of oncotic necrosis of the TNFs cells and appears to augment ceramide-induced cytotoxicity. The phase-contrast micrographs of the TNFs cells in Figure 6 demonstrate the protective effects of glutathione monoethylester on ceramide-induced cytotoxicity. The data also imply that the decrease in GSH occurs after ROS production since glutathione monoethylester specifically increased GSH and did not decrease ROS generation (not shown). NAC pre-treatment resulted in a similar decrease in the onset of oncotic necrosis. However, glutathione monoethylester pre-treatment did not protect the TNFs or TNFr cells from ceramide-induced apoptosis (Fig. 5B). Ceramide-decreased GSH occurred in TNFs cells not TNFr cells, thus correlating with sensitivity to ceramide.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results outlined above established that ceramide-induced cell death is mediated by a pathway that includes ROS generation and decreased GSH. The results of the present study show that cellular susceptibility to ceramide parallels that of TNF
. The TNFr cells were resistant to ceramide-induced oncotic necrosis and underwent apoptosis when treated with 20 µM ceramide, while the TNFs cells underwent rapid oncotic necrosis. The intracellular responses of TNFr and TNFs cells also differed. Ceramide caused an elevation in ROS and a significant decrease in GSH in TNFs but not in the TNFr variants. In this model, preventing a decrease in GSH appears to inhibit the onset of oncotic necrosis but does not appear to inhibit apoptosis. The TNFr cells have a higher basal level of GSH, which may be protective against the acute induction of ceramide-induced oncotic necrosis. It appears, however, that the induction of apoptosis can occur when the intracellular GSH concentration is not significantly decreased. Furthermore, a decrease in the reducing capacity of the cell may mediate the genetic susceptibility to ceramide seen in the sensitive variants.
Although a role for ceramide in regulating cell cycle arrest, cell death, and cell senescence has been suggested, the identity of the specific secondary messengers for ceramide in these responses remains unclear. In the mouse JB6 variants, the ceramide-mediated cell-death response is likely due to a depletion of GSH, which potentiates the elevation in ROS such as H2O2. Since, N-acetyl-L cysteine functions as a general free-radical scavenger, its observed effects on ceramide-induced ROS may occur independently of glutathione depletion. The fact that GSH monoethylester also protected TNFs cells from oncotic necrosis suggests that the intracellular effects of ceramide can be directly modulated by a decrease in GSH. The protective effects of GSH may involve an increased supply of cysteine through disulfide exchange and maintenance of cell membrane integrity. It has been reported that GSH inhibits neutral magnesium-dependent sphingomyelinase, but not acid-sphingomyelinase in some cell types, implying that depletion of cellular GSH can result in the hydrolysis of SM and additional generation of ceramide (Liu and Hannun, 1997). Thus, loss of GSH may provide a positive feedback loop between ceramide generated from the acid-sphingomyelinase pool and the neutral sphingomylinase. Ceramide has also been reported to activate the transcription factor NFKB, which is also regulated by an intracellular redox state (Dbaibo et al., 1993). However, the acute loss of cell viability (onset of oncotic necrosis) is likely to occur independent of transcriptional regulatory pathways, while apoptotic pathways can be mediated by transcription factors. Cellular redox may be the primary determinant of which cellular response path the cell will take. The distinction between oncotic necrosis and apoptosis is also of great importance in vivo because the pathway of death will determine the extent of tissue damage and inflammation.
A decrease in GSH in organelle pools may be more specifically related to differential sensitivity to ceramide. A large decrease in the cytosolic pool of GSH, such as the one observed in the TNFs cells, will likely decrease GSH in the mitochondria. A decrease in mitochondrial GSH will cause alterations in mitochondrial Ca2+ homeostasis and loss of activity of respiratory chain complex IV (Casini et al., 1987; Richter et al., 1995). Recently, Garcia-Ruiz et al. (1997) have reported a direct effect of ceramide on mitochondrial electron transport in isolated rat hepatocytes, which was potentiated by depletion of GSH. Therefore, our observed decrease in cellular GSH may be part of a series of intracellular responses to ceramide-induced oxidative stress that involves mitochondrial dysfunction. Indeed, Quillet-Mary and coworkers (1997) reported that C6 ceramide induced H2O2 production in human myeloid leukemia cells, which appeared to be generated at the ubiquinone site of the mitochondrial respiratory chain. C2-ceramide was shown to increase H2O2 generation and decrease the function of rat liver mitochondria (Arora et al., 1997; Cai et al., 1997; Garcia-Ruiz et al., 1997; Gomez et al., 1996). Considering these reports and our observed GSH decrease in TNFs cells, the difference in the observed sensitivity of the TNFs and TNFr cells to ceramide may lie at the level of the mitochondria.
Exogenous short-chain ceramides have been used to examine the multiplicity of intracellular responses elicited by ceramide generation. The exogenous ceramide used in this study is equivalent to endogenous ceramide generated in the acidic (lysosomal/endosomal) compartment, which is released by acidic sphingomyelinase (Higuchi et al., 1996; Santana et al., 1996), and not the neutral sphingomyelinase-generated ceramide (cytosol/inner and outer leaflet of cell membrane; Zhang et al., 1997). The distinct pool of acidic sphingomyelinase-released ceramides has been implicated as the "Cell Death Pool" of endogenous ceramide (Pena et al., 1997). The reason for two distinct biological responses to the alternate sphingomyelinase pools is not clear. As implied by our results, loss of GSH may determine the pathway of death for the cell (apoptosis vs. oncotic necrosis) and may contribute to the mechanism of these distinct biological responses. Furthermore, access of generated ceramides to cellular GSH pools may also explain the distinct biological responses to alternate sphingomyelinase pools. Distinct GSH pools can be found in the mitochondria, nucleus, cytoplasm, and endoplasmic reticulum (Smith et al., 1996). Depletion of these pools will have different consequences for the cell (Mithofer et al., 1992; Reed and Fariss, 1984). Therefore, the effect of ceramide on each of the distinct pools of GSH will be important to determine.
In summary, our observations demonstrate the coupling of cellular redox potential to ceramide-activated cell death. Genetic variants sensitive to ceramide killing show elevated ROS and decreased GSH while the sensitive cells show neither. Since the redox potential differs between cell types and because GSH contributes to the regulation of Redox potential, it follows that susceptibility to ceramide-induced cell killing will be regulated in part by cellular glutathione levels. Indeed, specific elevation of GSH decreases both the ROS generation and the cell killing induced by ceramide. The effect of accumulating ceramide during systemic stress in disease, tissue injury, or ischemia may be to potentiate the cytotoxic effects of inflammatory cells. Depending on the cells microenvironment (i.e., solid vs. soft tissue or lymphatic tumor, oxygen tension, endogenous metabolism, GSH levels) ceramide may have a major impact on cell survival in vivo.
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NOTES |
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Portions of this work were presented in preliminary form at the American Association for Cancer Research Meeting, 1997, San Diego, CA.
1 To whom correspondence should be addressed at the Department of Pathology, University of Maryland School of Medicine, 10 S. Pine Street, Baltimore, MD 21201. Fax: (410) 706–8414. E-mail: mdavis{at}umaryland.edu. 
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