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Originally published In Press as doi:10.1074/jbc.M314105200 on February 6, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18256-18261, April 30, 2004
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Cytoprotective Effect of Glucosylceramide Synthase Inhibition against Daunorubicin-induced Apoptosis in Human Leukemic Cell Lines*

Solène Grazide{ddagger}§, Anne-Dominique Terrisse{ddagger}§, Sandra Lerouge{ddagger}, Guy Laurent{ddagger}||, and Jean-Pierre Jaffrézou{ddagger}**

From the {ddagger}INSERM U563-Centre de Physiopathologie Toulouse Purpan, Institut Claudius Régaud, Toulouse 31052, and the ||Service d'Hématologie, Centre Hospitalier Universitaire Purpan, Toulouse 31059, France

Received for publication, December 23, 2003 , and in revised form, February 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have shown that ceramide (CER) glucosylation contributes to drug resistance in multidrug-resistant cells and that inhibition of glucosylceramide synthase sensitizes cells to various drug treatments. However, the role of glucosylceramide synthase has not been studied in drug-sensitive cancer cells. We have demonstrated previously that the anthracycline daunorubicin (DNR) rapidly induces interphasic apoptosis through neutral sphingomyelinase-mediated CER generation in human leukemic cell lines. We now report that inhibition of glucosylceramide synthase using D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) protected U937 and HL-60 cells from DNR-induced apoptosis. Moreover, blocking CER glucosylation did not lead to increased CER levels but to increased CER galactosylation. We also observed that pretreating cells with galactosylceramide (GalCER) significantly inhibited DNR-induced apoptosis. Finally, we show that GalCER-enriched lymphoblast cells (Krabbe's disease) were significantly more resistant to DNR- and cytosine arabinoside-induced apoptosis as compared with normal lymphoblasts, whereas glucosylceramide-enriched cells (Gaucher's disease) were more sensitive. In conclusion, this study suggests that sphingomyelin-derived CER in itself is not a second messenger but rather a precursor of both an apoptosis second messenger (GD3) and an apoptosis "protector" (GalCER).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of studies have shown that ceramide (CER)1 plays an important role in transducing many effects of extracellular agents including apoptosis, differentiation, and proliferation (for review, see Ref. 1). On the basis of these studies, our group investigated the role of the sphingomyelin-CER (SM-CER) pathway in daunorubicin- (DNR-) and Ara-C-induced apoptosis. Clinically relevant DNR and Ara-C concentrations (1 and 40 µM, respectively (2, 3)), which induced rapid (<6 h) interphasic apoptosis, stimulated an early (5–10 min) SM cycle (hydrolysis and resynthesis) and subsequent CER generation in both U937 and HL-60 cells through the stimulation of neutral sphingomyelinase (4–6).

Mammalian cells selected in vitro for resistance to a chemotherapeutic drug are frequently cross-resistant to a wide variety of cytotoxic agents (multidrug resistance (MDR)) (for review, see Ref. 7). These cells present an altered pharmacology of drugs in relationship to the overexpression of the gene product of MDR1, an integral membrane protein termed P-glycoprotein, which acts as a drug efflux pump, thereby preventing them from accumulating in the tumor cell. MDR cells have also been shown to present altered lipid composition such as elevated cholesterol and SM as well as glycolipids (for review, see Ref. 8). One glycolipid in particular is in the lime light, glucosylceramide (GlcCER).

In 1996, Cabot and co-workers (9) first demonstrated that drug-resistant MCF-7-AdrR breast cancer cells accumulated GlcCER in comparison with wild-type MCF-7 cells. This study was followed by a series of studies that clearly demonstrated that by blocking GlcCER synthesis (such as with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)) one could sensitize MDR cells to chemotherapeutic drugs (for review, see Ref. 10). Furthermore, increasing CER glucosylation by transfecting wild-type MCF-7 cells with glucosylceramide synthase (GCS) led to an increase in Adriamycin resistance (11). The gist of these studies provided ample evidence that GlcCER contributes greatly to drug resistance. However, the role of GCS in drug-sensitive cells has largely been overlooked.

In this study, we elected to investigate the effect of blocking GCS in our highly drug-sensitive leukemic models. Surprisingly, we observed that by using the classic GCS inhibitors PDMP and PPMP, we completely blocked DNR- and Ara-C-induced apoptosis. Moreover, we demonstrated that by inhibiting the glucosylation of CER, galactosylation is increased and that this is associated with DNR and Ara-C resistance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Reagents—DNR (cerubidine®) was supplied by Laboratoire Roger Bellon (Neuilly-sur-Seine, France) and 1-{beta}-D-arabinofuranosylcytosine (Ara-C) by Upjohn (Paris, France). Silica gel 60 thin-layer aluminum sheet chromatography plates were from Merck. 12-(N-methyl-N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl])-labeled CER (NBD-CER) was purchased from Molecular Probes (Eugene, OR). GalCER, which was purified from bovine brain, was generously obtained from Prof. Thierry Levade (INSERM U466, Toulouse, France). All other drugs and reagents were purchased from Sigma, Carlo Erba (Rueil-Malmaison, France), or Prolabo (Paris, France).

Cell Culture—The human leukemic cell lines U937 and HL-60 were purchased from the ATCC (Manassas, VA). The multidrug-resistant U935-DR cell line was generously provided by Dr. H. Morjani (CNRS Unité Mixte de Recherche 6142, Reims, France) (12). Human Epstein-Barr virus-transformed peripheral blood lymphocytes were derived from control subjects (cell lines Dau, Cha, Gha, and Lel), from a patient affected with Gaucher's disease (cell lines Cas, Vio, Cuc, and Tre), or from patients with Krabbe's disease (cell lines Nun, Den, and GM6805). These cell lines were kindly provided by Prof. Thierry Levade (INSERM U466, Toulouse, France). Cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 µg/ml penicillin (all from Eurobio, les Ulis, France) at 37 °C and 5% CO2.

DNA Analysis—Apoptotic nuclei were visualized after the cells were fixed in 3% paraformaldehyde for 15 min, washed in 150 mM phosphate-buffered saline, pH 7.4, stained by the DNA-intercalating fluorescent probe DAPI (0.1 µg/ml in (pH 7.0) Tris/EDTA/NaCl (10:10:100 v/v)), and mounted in Fluoprep for fluorescence microscopy (Leica model Diaplan). In each experiment, the presence or absence of apoptotic nuclei in samples of 300 cells was scored by two independent observers. For quantitative DNA fragmentation, cells were allowed to lyse for 15 min in 500 µl of lysis buffer (5 g/liter Triton X-100, 20 mM EDTA, and 5 mM Tris, pH 8.0) and were then ultracentrifuged for 30 min at 20,000 x g to separate the chromatin pellet from cleavage products. The pellet (resuspended in 500 µl of 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA) and the supernatant were assayed for DNA determination by the spectrofluorometric DAPI procedure (13).

Analysis of Exogenous CER Metabolism—5 x 106 cells were preincubated with 5 µM NBD-CER for 45 min at 4 °C. Cells were then incubated in kinetic experiments at 37 °C. At each time point, cells were washed, and lipids were extracted and resolved by thin-layer chromatography developed in chloroform/methanol/water (100:42:6, by volume) up to two-thirds of the plate and then in chloroform/methanol/acetic acid (70:30:5, by volume). NBD-labeled lipid products were visualized under a UV light, scraped, and eluted in chloroform/methanol (2:1). NBD fluorescence (emitted at 536 nm) was quantitated by fluorometry (excitation at 466 nm) (14).

Metabolic Cell Labeling and Quantitation of CER and Metabolites— Total cellular CER and CER metabolite quantitation was performed by labeling cells to isotopic equilibrium with 1 µCi/ml [9,10-3H]palmitic acid (53.0 Ci/mmol, Amersham Biosciences) for 48 h in complete medium as described previously (4). Cells were then washed and resuspended in serum-free medium for kinetic experiments. Lipids were extracted and resolved by thin-layer chromatography developed in chloroform/methanol/acidic acid/formic acid/water (65:30:10:4:2, by volume) up to two-thirds of the plate and then in chloroform/methanol/acetic acid (94:5:5, by volume). CER and CER metabolites were scraped and quantitated by liquid scintillation spectrometry. Lipid standards were used to identify the various metabolic products.

Caspase-3 Activity Assay—Caspase-3 colorimetric activity assay (R&D Systems, Abingdon, United Kingdom) was performed according to the manufacturer's recommendations. Briefly, 2 x 107/ml cells were washed and resuspended in lysis buffer collected by centrifugation. Lysis buffer was added on the cell pellet, incubated on ice for 10 min, and centrifuged at 10,000 x g for 10 min. For each lysate, 10 µl of supernatant was incubated with caspase-3 colorimetric substrate for 2 h at 37 °C. Cleavage of the substrate by caspase-3 was quantified spectrophotometrically at a wavelength of 405 nm.

Statistical Analysis—Student's t test was used for statistical analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PDMP and PPMP on DNR-induced Apoptosis in U937 Cells—The effect of GCS inhibition was evaluated in drug-sensitive myeloid leukemia cells. Treatment of U937 cells for 6 h with 1 µM DNR presented significant apoptosis (~50%) as estimated by morphological analysis (Fig. 1). Apoptosis was also confirmed by DNA laddering and poly(ADP-ribose)polymerase cleavage (data not shown) (4). However, when the cells were pretreated for 15 h with 20 µM PDMP or 20 µM PPMP, DNR-induced apoptosis was significantly reduced. Similar results were observed with Ara-C and on U937 and HL-60 cells (data not shown) (5). This surprising observation led us to confirm whether, in an MDR version of our cell model (U937-DR), PDMP could sensitize this cell line to DNR. DNR alone presented little cytotoxic effect on U937-DR cells after 6 h of incubation. However, in the presence of PDMP, we observed a ~4-fold increase in apoptosis (2.2% versus 8.9%) (data not shown).



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  		FIG. 1.
Effect of PDMP and PPMP on DNR-induced apoptosis. 5 x 105/ml U937 cells were preincubated with or without 20 µM PDMP or 20 µM PPMP for 15 h and then treated with 1 µM DNR for 6 h. Representative fluorescence microscopy analyzing 200 cells/sample (A) and quantitative analysis by DNA fragmentation from five independent experiments (±S.E.) (B and C) are shown. *, p < 0.01. Cont, control.

 
Effect of PDMP on DNR-induced CER Generation—To confirm the efficacy of GCS inhibition by PDMP, a dose-effect study was performed using NBD-CER. As shown in Fig. 2A, PDMP at 20 µM almost completely blocked, as expected, NBD-CER conversion to NBD-GlcCER (>90% inhibition). Higher concentrations were cytotoxic (data not shown). Moreover, we observed that concomitant with this inhibition was a pronounced increase in NBD-GalCER generation. From these observations, it appears that NBD-CER glucosylation is compensated by NBD-CER galactosylation. However, in U937-DR cells, 20 µM PDMP also significantly decreased NBD-GlcCER levels (although less efficiently); however, we observed little NBD-GalCER formation (Fig. 2B). It is noteworthy that in the parental U937 cells, the metabolism of NBD-CER to NBD-GlcCER and -GalCER appears comparable in the absence of PDMP, whereas in the MDR U937-DR cells, NBD-GalCER formation is very limited.



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  		FIG. 2.
Effect of PDMP on NBD-CER metabolism. U937 cells (A) were incubated with increasing concentrations of PDMP for 15 h followed by a 3-h incubation with 5 µM NBD-CER. Similarly, U937-DR cells (B) were incubated with or without 20 µM PDMP. Fluorescent lipids were extracted and resolved by thin-layer chromatography and photographed under UV light (365 nm). Lipids were then scraped and quantitated by fluorometry as described under "Experimental Procedures." Results are representative of two independent experiments.

 
Since PDMP appeared to redirect CER metabolism toward galactosylation, we investigated the effect of PDMP on drug-induced endogenous CER generation. Indeed, apoptosis induced by DNR in myeloid leukemia cells has been correlated with early SM-derived CER generation (for review, see Ref. 15). As shown in Fig. 3, PDMP did not affect DNR-induced CER generation (~20–30% increase at 8 min). Moreover, PDMP had no significant effect on basal endogenous CER and GlcCER levels (Fig. 3, inset). A previous study (16) showed modest if any effect of 75 µM PDMP on CER and GlcCER levels in the drug-sensitive human carcinoma cell line KB-3-1 after 24 h. Significant decrease in GlcCER and increase in CER have only been observed for longer time periods (72 h (17) to 1 week (18)). These observations underline that exogenous NBD-CER is rapidly metabolized by GCS (and therefore easily blocked by PDMP) and evacuated from the cells (19). However, the endogenous conversion of CER by GCS of our myeloid leukemic cells is not readily PDMP-sensitive.



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  		FIG. 3.
Effect of PDMP on DNR-induced CER generation. 5 x 105/ml U937 cells were preincubated with (black circles) or without (white circles) 20 µM PDMP for 15 h and then treated with 1 µM DNR for the time intervals indicated. CER levels were determined as described under "Experimental Procedures." Inset, basal CER and GlcCER levels in U937 cells treated with (white bars) or without (gray bars) PDMP. Results are representative of three independent experiments performed in triplicate. *, p < 0.01.

 
Effect of DNR on CER Metabolism—Since PDMP protected U937 cells from DNR-induced apoptosis but did not affect DNR-induced CER generation, we investigated the effect of DNR on endogenous CER metabolism. As shown in Fig. 4, treatment of U937 cells with DNR led to a rapid time-dependent decrease in [3H]palmitic acid-labeled GlcCER. GlcCER was rapidly metabolized to form [3H]lactosylceramide, followed by [3H]GM3 and finally [3H]GD3, which accumulated at 4 h. It is at this time that we showed previously that the initial phases of the execution step of apoptosis are initiated (20). Indeed, GD3 has been shown previously to be a potent apoptotic mediator directly targeting mitochondria and disrupting mitochondrial transmembrane potential (21–23).



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  		FIG. 4.
Effect of DNR on endogenous CER metabolism. U937 cells were incubated with 1 µM DNR for the time intervals indicated. The quantitation of CER metabolites was determined as described under "Experimental Procedures." Results are representative of two independent experiments performed in triplicate. *, significantly different from control (p < 0.01). Basal GlcCER, lactosylceramide (LacCER), GM3, and GD3 were 109 ± 46, 42 ± 12, 86 ± 14, and 67 ± 17 pmol/mg protein, respectively.

 
Effect of GalCER on DNR-induced Apoptosis—In our cell model, PDMP blocked DNR-induced apoptosis and prevented the metabolism of NBD-CER to NBD-GlcCER while increasing the formation of NBD-GalCER (Fig. 2A). Furthermore, PDMP had little effect on DNR-induced SM-derived CER generation (Fig. 3). We investigated whether PDMP and PPMP could reproduce these effects on endogenous [3H]palmitic acid-labeled CER. As shown in Fig. 5, DNR alone had no significant effect on GalCER levels in U937 cells, but when cells were pretreated with PDMP or PPMP, DNR induced a significant increase in [3H]GalCER. Moreover, PDMP and PPMP alone induced a drastic increase in basal [3H]GalCER levels (~3-fold increase), confirming what was already observed with NBD-CER.



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  		FIG. 5.
Effect of PDMP and PPMP on GalCER levels in DNR-treated U937 cells. 3 x 105 U937 cells were preincubated in the absence ({square}) or in the presence ({blacksquare}) of 20 µM PDMP or in the presence of 20 µM PPMP () for 15 h and then treated with 1 µM DNR for the time intervals indicated. GalCER levels were determined as described under "Experimental Procedures." Results are representative of three independent experiments performed in triplicate. *, significantly different from control (p < 0.01).

 
To examine whether this increase in GalCER could present an antiapoptotic effect, we investigated whether exogenous GalCER could influence DNR-induced apoptosis. As shown in Fig. 6, pretreatment of U937 cells with 5 µM GalCER presented a significant inhibition of DNR-induced apoptosis. However, 10 µM GalCER alone presented cell toxicity, which precluded its use (data not shown). Experiments were also performed using psychosine (the immediate metabolite of GalCER), but no cytoprotective effect was observed (data not shown). Finally, we investigated whether sphingosine-1-phosphate could contribute to the observed cytoprotective effect of PDMP. Cells were co-incubated with 10 µM dimethylsphingosine (an inhibitor of sphingosine kinase) and PDMP for 15 h, followed by an additional 6 h with 1 µM DNR. In this study, we did not observe any significant effect of dimethylsphingosine on cell survival (data not shown).



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  		FIG. 6.
Effect of GalCER on DNR-induced apoptosis. U937 cells were either untreated (white bars) or preincubated in the presence of 1 µM (gray bars) or 5 µM (black bars) GalCER for 18 h and then treated with 1 µM DNR for 6 h. A, percent of apoptotic cells was determined by morphological analysis using DAPI staining. B, caspase-3 activity was measured using a colorimetric assay. Results are mean ± S.E. of three independent experiments. *, significantly different compared with DNR-treated U937 cells in the absence of GalCER (p < 0.01).

 
Effect of DNR on Gaucher's and Krabbe's Disease Lymphocytes—Since our observations indicated that the metabolism of CER toward GD3 is an important apoptotic pathway whereas the conversion of CER toward GalCER is potentially antiapoptotic, we investigated the sensitivity to both DNR and Ara-C on cells that constitutively present deficient catabolism of GlcCER or GalCER, Gaucher and Krabbe cells, respectively (24, 25). As shown in Fig. 7 compared with four normal immortalized lymphocytic cell lines, the four Gaucher cell lines were significantly more sensitive to DNR-induced apoptosis, whereas the three Krabbe cell lines were significantly more resistant. Since basal apoptotic levels were observed to be similar, one could speculate that GlcCER and GalCER levels are only implicated in stress-induced cell signaling. Although this is highly indirect evidence, these observations underline the potential role of GalCER as an antiapoptotic lipid.



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  		FIG. 7.
Effect of DNR on Gaucher's and Krabbe's disease lymphoblasts. Four normal (white circles), four Gaucher (black circles), and three Krabbe (gray circles) lymphoblast cell lines were either untreated or treated for 6 h with either 1 µM DNR or 40 µM Ara-C. The percent of apoptotic cells was determined by morphological analysis using DAPI staining. Results are the mean of triplicate determinations ± S.E. Bars, mean. *, significantly different compared with drug-treated normal lymphoblast cell lines (p < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated, using DNR and Ara-C, which induce rapid interphasic apoptosis at the clinically optimal concentrations of 1 and 40 µM, respectively, the role of early CER metabolism (<20 min) in U937 and HL-60 leukemic cells (4, 5). The initial generation of CER has been shown to be implicated in apoptosis signaling in several human leukemia cells, as well as in a number of acid sphingomyelinase-deficient models (4, 26–28). We and others (20, 26, 29) have shown that only this initial CER generation but not the later delayed CER accumulation (which is at least partially fumonisin B-inhibitable) is correlated with apoptosis signaling.

Surprisingly, pretreatment of U937 cells with PDMP or PPMP completely blocked DNR-induced apoptosis, while it sensitized the MDR U937-DR model (data not shown). Moreover, blocking GCS did not lead to an increase in DNR-induced CER generation but to an increase in GalCER content, which was not observed in the MDR model. It has been demonstrated previously that in drug-sensitive Jurkat cells, retrovirally transduced with GlcCER, CER generated by ligation of the death receptor CD95, or etoposide, or {gamma}-radiation was not glycosylated by GCS (30). In contrast, de novo synthesized CER as well as an exogenously supplied cell-permeable CER analog was efficiently glycosylated. The authors (30) concluded that GCS, located at the Golgi, is topologically segregated from CER produced in the plasma membrane. Therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of CER accumulation is limited. To determine the potential role of GalCER in "protecting" U937 cells from DNR, we preincubated the cells with 5 µM GalCER before drug treatment. Our study shows that exogenous GalCER protected U937 cells from DNR- and Ara-C-induced apoptosis. Finally, to further underline the role of GlcCER and GalCER in cell response to DNR and Ara-C, we treated Krabbe and Gaucher cells, which are inherently defective in GalCER and GlcCER metabolism, respectively, with both drugs. Interestingly, compared with normal lymphoblasts, cells originating from Krabbe patients were more resistant to both DNR- and Ara-C-induced apoptosis, whereas Gaucher cells presented significantly greater sensitivity. The observation that basal apoptotic levels were similar in all cell types suggests that endogenous GalCER and GlcCER levels only play a role in modulating stress-induced cell signaling (survival versus cell death).

CER has long been considered a proapoptotic mediator (for review, see Ref. 1), and inhibition of GlcCER synthesis leading to increased CER content has been considered to be the mechanism by which PDMP sensitizes MDR cells (10). However, there are several studies that show that apoptosis induction by CER requires its conversion to GD3 (21–23). Since the first step in this conversion is the glucosylation of CER, one would expect that cells enriched in GlcCER would present higher sensitivity to apoptosis. Indeed, Tepper et al. (30) did notice a modest sensitization of retrovirally transduced Jurkat cells with GCS to CD95 ligation.

A close look at the literature suggests that blocking GlcCER synthesis does not necessarily lead to increased apoptosis. Indeed, okadaic acid-induced CER elevation, while markedly potentiated by blocking GlcCER synthesis, did not result in increased apoptosis in human CHP-100 neuroepithelioma cells (31). Such discrepancies may be explained by different basal levels of GlcCER and GalCER. For example, HepG2 hepatoma cells displayed only a modest apoptotic response to doxorubicin treatment (32). Since liver cells are rich in GalCER (33), one could speculate that in such cells the high GalCER levels provide a protective effect against certain effectors. Moreover, MDR and MRP cells present elevated GalCER as well as GlcCER (34, 35). We propose that the relative balance (or imbalance) of basal GalCER and GlcCER levels could represent a predisposition for sensitivity or resistance to rapid interphasic SM-derived CER-mediated apoptosis. Finally, it has also been demonstrated that in MDR cells, gangliosides are important P-glycoprotein regulators perhaps through their capacity to modulate P-glycoprotein phosphorylation and that PDMP-induced ganglioside depletion plays a significant role in PDMP-mediated MDR reversal (36).

These observations question the role of CER as an apoptotic mediator. Indeed, CER generation has also been implicated in cell proliferation of quiescent fibroblasts (37). It would be of interest to determine whether in such a model the SM-derived CER is converted to GalCER rather than GlcCER. Furthermore, one of the main arguments for linking CER to apoptosis has been the use of exogenous cell-permeable analogs. However, we have demonstrated previously that such analogs act as stress inducers leading to endogenous SM-derived CER (20).

Taken together, our results show that in a rapid interphasic apoptotic model, where early SM-derived CER is rapidly converted to GD3, blocking the glucosylation of CER leads not to sensitization but rather to protection from apoptosis. The rerouting of CER metabolism toward GalCER appears to be implicated in cell death protection. Previously, studies have demonstrated that upon ceramidase stimulation, CER can be catabolized into sphingosine, which, in turn, can be converted into sphingosine-1-phosphate through sphingosine-1-kinase. Sphingosine-1-phosphate has emerged as a potent regulator of apoptosis. Indeed, sphingosine-1-phosphate inhibits apoptosis induced by CER and other effectors (38, 39). The mechanism by which sphingosine-1-phosphate interferes with CER-induced apoptotic signaling is not fully understood. However, it has been reported that sphingosine-1-phosphate inhibits CER-induced c-Jun NH2-terminal kinase stimulation and caspase activities (39, 40). From these studies, one can speculate that sphingosine-1-kinase stimulation may contribute to DNR resistance. However, in our study we observed that the sphingosine-1-kinase inhibitor dimethylsphingosine did not affect the cytoprotective effect of PDMP in DNR- or Ara-C-treated cells (data not shown). Moreover, we reported previously that DNR induced both CER production and apoptosis in cells derived from Farber's disease, which are genetically deficient for lysosomal ceramidase, the major component of cellular ceramidase activity (41). Hence, although we cannot totally exclude the implication of extralysosomal ceramidases (42), these results suggest that sphingosine-1-phosphate plays little if any role in DNR-induced apoptosis in myeloid or lymphoid cells.

Finally, our study strongly suggests that SM-derived CER in itself is not a second messenger but rather a precursor of both an apoptosis second messenger (GD3) and an apoptosis protector (GalCER). Furthermore, our study suggests that cell sensitivity to drug-induced apoptosis is related to the basal GalCer and GlcCER levels. Hence the balance between GalCER versus GlcCER levels may represent a novel "rheostat" that determines cell sensitivity to drug-induced apoptosis. Further studies are ongoing in our laboratory using small interfering RNA-based technology and UDP-galactose-ceramide galactosyltransferase transfection to further evaluate the role of GalCER in stress-induced cell signaling and apoptosis.


   FOOTNOTES
 
* This work was supported by la Ligue Nationale Contre le Cancer and les Comités Départementaux du Gers, de l'Aveyron, de la Haute-Garonne (to J.-P. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

Recipient of a grant from la Fondation pour la Recherche Médicale. Back

** To whom correspondence should be addressed: INSERM U563-CPTP, Institut Claudius Régaud, 20 rue du Pont-St. Pierre, Toulouse 31059, France. Tel.: 33-5-61-42-41-73; Fax: 33-5-61-42-46-06; E-mail: jaffrezou{at}icr.fnclcc.fr.

1 The abbreviations used are: CER, ceramide; SM, sphingomyelin; DNR, daunorubicin; Ara-C, cytosine arabinoside; MDR, multidrug resistance; GlcCER, glucosylceramide; GalCER, galactosylceramide; PDMP, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; GCS, glucosylceramide synthase; PPMP, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; NBD, 12-(N-methyl-N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]); DAPI, 4',6-diamidino-2-phenylindole. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Thierry Levade (INSERM U566) for critical comments and expert technical advice as well as for providing us with the normal, Gaucher, and Krabbe cell lines.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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