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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Key Words: epidermal cells; cell death; ceramide; glutathione; apoptosis.
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INTRODUCTION |
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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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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 1420002 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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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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DISCUSSION |
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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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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) 7068414. E-mail: mdavis{at}umaryland.edu.
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