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W1977513244 abstract "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). 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). A number of studies have shown that ceramide (CER) 1The 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. 1The 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. plays an important role in transducing many effects of extracellular agents including apoptosis, differentiation, and proliferation (for review, see Ref. 1Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Google Scholar). 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 (2Gewirtz D.A. Biochem. Pharmacol. 1999; 57: 727-741Google Scholar, 3Chabner B.A. Myers C.E. Coleman C.N. Johns D.G. N. Engl. J. Med. 1975; 292: 1159-1168Google Scholar)), 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 (4Jaffrézou J.P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Google Scholar, 5Bezombes C. Plo I. Mansat-De Mas V. Quillet-Mary A. Negre-Salvayre A. Laurent G. Jaffrézou J.P. FASEB J. 2001; 15: 1583-1585Google Scholar, 6Datta R. Banach D. Kojima H. Talanian R.V. Alnemri E.S. Wong W.W. Kufe D.W. Blood. 1996; 88: 1936-1943Google Scholar).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. 7Ling V. Cancer (Phila.). 1992; 69: 2603-2609Google Scholar). 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. 8Lavie Y. Fiucci G. Czarny M. Liscovitch M. Lipids. 1999; 34: S57-S63Google Scholar). One glycolipid in particular is in the lime light, glucosylceramide (GlcCER).In 1996, Cabot and co-workers (9Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar) 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. 10Senchenkov A. Litvak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar). Furthermore, increasing CER glucosylation by transfecting wild-type MCF-7 cells with glucosylceramide synthase (GCS) led to an increase in Adriamycin resistance (11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar). 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 PROCEDURESDrugs and Reagents—DNR (cerubidine®) was supplied by Laboratoire Roger Bellon (Neuilly-sur-Seine, France) and 1-β-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) (12Durrieu F. Belaud-Rotureau M.A. Lacombe F. Dumain P. Reiffers J. Boisseau M.R. Bernard P. Belloc F. Cytometry. 1999; 36: 140-149Google Scholar). 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 × 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 (13Kapuscinski J. Skooczylas B. Anal. Biochem. 1977; 83: 252-257Google Scholar).Analysis of Exogenous CER Metabolism—5 × 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) (14Lipsky N.G. Pagano R.E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2608-2612Google Scholar).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 (4Jaffrézou J.P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Google Scholar). 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 × 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 × 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.RESULTSEffect 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) (4Jaffrézou J.P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Google Scholar). 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) (5Bezombes C. Plo I. Mansat-De Mas V. Quillet-Mary A. Negre-Salvayre A. Laurent G. Jaffrézou J.P. FASEB J. 2001; 15: 1583-1585Google Scholar). 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).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.Fig. 2Effect 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.View Large Image Figure ViewerDownload (PPT)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. 15Laurent G. Jaffrézou J.P. Blood. 2001; 98: 913-924Google Scholar). 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 (16Nicholson K.M. Quinn D.M. Kellett G.L. Warr J.R. Br. J. Cancer. 1999; 81: 423-430Google Scholar) 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 (17Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Google Scholar) to 1 week (18Mutoh T. Tokuda A. Inokuchi J. Kuriyama M. J. Biol. Chem. 1998; 273: 26001-26007Google Scholar)). These observations underline that exogenous NBD-CER is rapidly metabolized by GCS (and therefore easily blocked by PDMP) and evacuated from the cells (19Lipsky N.G. Pagano R.E. J. Cell Biol. 1985; 100: 27-34Google Scholar). However, the endogenous conversion of CER by GCS of our myeloid leukemic cells is not readily PDMP-sensitive.Fig. 3Effect of PDMP on DNR-induced CER generation. 5 × 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.View Large Image Figure ViewerDownload (PPT)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 (20Jaffrézou J.P. Maestre N. Mansat-Demas V. Bezombes C. Levade T. Laurent G. FASEB J. 1998; 12: 999-1006Google Scholar). Indeed, GD3 has been shown previously to be a potent apoptotic mediator directly targeting mitochondria and disrupting mitochondrial transmembrane potential (21DeMaria R. Lenti L. Malisan F. d'Agostino F. Tomassini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Google Scholar, 22Garcia-Ruiz C. Colell A. Paris R. Fernandez-Checa J.C. FASEB J. 2000; 14: 847-858Google Scholar, 23Garcia-Ruiz C. Colell A. Morales A. Calvo M. Enrich C. Fernandez-Checa J.C. J. Biol. Chem. 2002; 277: 36443-36448Google Scholar).Fig. 4Effect 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.View Large Image Figure ViewerDownload (PPT)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.Fig. 5Effect of PDMP and PPMP on GalCER levels in DNR-treated U937 cells. 3 × 105 U937 cells were preincubated in the absence (□) or in the presence (▪) 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).View Large Image Figure ViewerDownload (PPT)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).Fig. 6Effect 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).View Large Image Figure ViewerDownload (PPT)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 (24Hardy B. Teitelman-Weissman B. Chazan S. Neri A. Biomed. Pharmacother. 1987; 41: 40-44Google Scholar, 25O'Brien J.S. Bernett J. Veath M.L. Paa D. Arch. Neurol. 1975; 32: 592-599Google Scholar). 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.Fig. 7Effect 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).View Large Image Figure ViewerDownload (PPT)DISCUSSIONIn 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 (4Jaffrézou J.P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Google Scholar, 5Bezombes C. Plo I. Mansat-De Mas V. Quillet-Mary A. Negre-Salvayre A. Laurent G. Jaffrézou J.P. FASEB J. 2001; 15: 1583-1585Google Scholar). 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 (4Jaffrézou J.P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Google Scholar, 26Cuvillier O. Edsall L. Spiegel S. J. Biol. Chem. 2000; 275: 15691-15700Google Scholar, 27Bourteele S. Hausser A. Doppler H. Horn-Muller J. Ropke C. Schwarzmann G. Pfizenmaier K. Muller G. J. Biol. Chem. 1998; 273: 31245-31251Google Scholar, 28Bezombes C. Segui B. Cuvillier O. Bruno A.P. Uro-Coste E. Gouaze V. Andrieu-Abadie N. Carpentier S. Laurent G. Salvayre R. Jaffrézou J.P. Levade T. FASEB J. 2001; 15: 297-299Google Scholar). We and others (20Jaffrézou J.P. Maestre N. Mansat-Demas V. Bezombes C. Levade T. Laurent G. FASEB J. 1998; 12: 999-1006Google Scholar, 26Cuvillier O. Edsall L. Spiegel S. J. Biol. Chem. 2000; 275: 15691-15700Google Scholar, 29Ogretmen B. Schady D. Usta J. Wood R. Kraveka J.M. Luberto C. Birbes H. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2001; 276: 24901-24910Google Scholar) 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 γ-radiation was not glycosylated by GCS (30Tepper A.D. Diks S.H. van Blitterswijk W.J. Borst J. J. Biol. Chem. 2000; 275: 34810-34817Google Scholar). In contrast, de novo synthesized CER as well as an exogenously supplied cell-permeable CER analog was efficiently glycosylated. The authors (30Tepper A.D. Diks S.H. van Blitterswijk W.J. Borst J. J. Biol. Chem. 2000; 275: 34810-34817Google Scholar) 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. 1Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Google Scholar), and inhibition of GlcCER synthesis leading to increased CER content has been considered to be the mechanism by which PDMP sensitizes MDR cells (10Senchenkov A. Litvak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar). However, there are several studies that show that apoptosis induction by CER requires its conversion to GD3 (21DeMaria R. Lenti L. Malisan F. d'Agostino F. Tomassini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Google Scholar, 22Garcia-Ruiz C. Colell A. Paris R. Fernandez-Checa J.C. FASEB J. 2000; 14: 847-858Google Scholar, 23Garcia-Ruiz C. Colell A. Morales A. Calvo M. Enrich C. Fernandez-Checa J.C. J. Biol. Chem. 2002; 277: 36443-36448Google Scholar). 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. (30Tepper A.D. Diks S.H. van Blitterswijk W.J. Borst J. J. Biol. Chem. 2000; 275: 34810-34817Google Scholar) 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 (31Di Bartolomeo S. Spinedi A. Biochem. Biophys. Res. Commun. 2001; 288: 269-274Google Scholar). 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 (32Di Bartolomeo S. Spinedi A. Biochem. Pharmacol. 2001; 61: 851-856Google Scholar). Since liver cells are rich in GalCER (33Nilsson O. Svennerholm L. J. Lipid R" @default.
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W1977513244 title "Cytoprotective Effect of Glucosylceramide Synthase Inhibition against Daunorubicin-induced Apoptosis in Human Leukemic Cell Lines" @default.
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