Biochemical Mechanisms of the Generation of Endogenous Long Chain Ceramide in Response to Exogenous Short Chain Ceramide in the A549 Human Lung Adenocarcinoma Cell Line
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m110699200
ISSN1083-351X
AutoresBesim Öğretmen, Benjamin J. Pettus, Michael J. Rossi, Rachel E. Wood, Julnar Usta, Zdzisław M. Szulc, Alicia Bielawska, Lina M. Obeid, Yusuf A. Hannun,
Tópico(s)Polysaccharides and Plant Cell Walls
ResumoTreatment of A549 cells with C6-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C6-ceramides, [sphingosine-3-3H]d-erythro-, and N-[N-hexanoyl-1-14C]d-erythro-C6-ceramide. The results demonstrated that 3H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the 14C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C6-ceramide is due to recycling of the sphingosine backbone of C6-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C6-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced byd-erythro- but notl-erythro-C6-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced byd-erythro-C6-ceramide, and notl-erythro-C6-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses. Treatment of A549 cells with C6-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C6-ceramides, [sphingosine-3-3H]d-erythro-, and N-[N-hexanoyl-1-14C]d-erythro-C6-ceramide. The results demonstrated that 3H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the 14C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C6-ceramide is due to recycling of the sphingosine backbone of C6-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C6-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced byd-erythro- but notl-erythro-C6-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced byd-erythro-C6-ceramide, and notl-erythro-C6-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses. poly(ADP-ribose) polymerase fumonisin B1 myriocin diacylglycerol phosphate kinase mass spectrometry high performance liquid chromatography 3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide telomere repeat amplification protocol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The sphingolipid ceramide has been shown to mediate anti-proliferative responses such as apoptosis, growth arrest, differentiation, and senescence (1.Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar). Ceramide is generated in response to various agents including Fas ligand, tumor necrosis factor-α, ionizing radiation, and chemotherapeutic agents (2.Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Crossref PubMed Scopus (157) Google Scholar, 3.Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (297) Google Scholar). Although some of these agents cause early and reversible generation of ceramide, they can also cause a delayed and sustained generation of ceramide, which, in most cases, is associated with its downstream biological activities. Ceramide generation occurs via agonist activation of either sphingomyelinase, which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine (4.Kim M.Y. Linardic C. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar, 5.Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 6.Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar), or through the de novo pathway of sphingolipid biosynthesis (7.Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google Scholar, 8.Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Several studies have utilized inhibitors of the de novo pathway to establish a role for ceramide in apoptosis induced by several agents including daunorubicin, etoposide, tumor necrosis factor, camptothecin, IgM, and angiotensin II (7.Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google Scholar, 8.Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 9.Suzuki A. Iwasaki M. Kato M. Wagai N. Exp. Cell Res. 1997; 233: 41-47Crossref PubMed Scopus (78) Google Scholar, 10.Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar, 11.Kroesen B-J. Pettus B. Luberto C. Busman M. Sietsma H. de Leij L. Hannun Y.A. J. Biol. Chem. 2001; 276: 13606-13614Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 12.Lehtonen J.Y. Horiuchi M. Daviet L. Akishita M. Dzau V.J. J. Biol. Chem. 1999; 274: 16901-16906Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In many malignant cell lines (such as leukemia and breast carcinoma cells), ceramide rapidly and specifically induces apoptosis, whereas dihydroceramide and other related lipids are inactive (13.Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1618) Google Scholar). Ceramide has been shown to activate proteases of the ICE family (caspases), especially caspase-3, a PARP1-cleaving protease (14.Smyth M.J. Perry D.K. Zhang J. Poirier G.G. Hannun Y.A. Obeid L.M. Biochem. J. 1996; 316: 25-28Crossref PubMed Scopus (198) Google Scholar,15.Mizushima N. Koike R. Kohsaka H. Kushi Y. Handa S. Yagita H. Miyasaka N. FEBS Lett. 1996; 395: 267-271Crossref PubMed Scopus (124) Google Scholar). Importantly, activation of caspase-3 by ceramide and induction of apoptosis are inhibited by overexpression of bcl-2(14.Smyth M.J. Perry D.K. Zhang J. Poirier G.G. Hannun Y.A. Obeid L.M. Biochem. J. 1996; 316: 25-28Crossref PubMed Scopus (198) Google Scholar), and Bcl-2 does not reduce the levels of ceramide produced in response to extracellular agents (16.Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (294) Google Scholar). Taken together, these results suggest that the generation of ceramide occurs upstream of the execution phase of apoptosis. In addition to its role in apoptosis, ceramide has been shown to be involved in cell cycle arrest (17.Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazarie F. Pushkareva M. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Ceramide induces a G0/G1 cell cycle arrest, and this was mechanistically shown to be due to the induction of dephosphorylation of the retinoblastoma gene product (Rb) (17.Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazarie F. Pushkareva M. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). It has been reported also that the treatment of NIH 3T3 cells with a specific inhibitor of glucosylceramide synthase, which results in the accumulation of ceramide, causes a G2/M cell cycle arrest, possibly mediated by ceramide-induced inhibition of the cyclin-dependent p34cdc2 and Cdk2 kinases (18.Rani C.S. Abe A. Chang Y. Rosenzweig N. Saltiel A.R. Radin N.S. Shayman J.A. J. Biol. Chem. 1995; 270: 2859-2867Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). More recent studies have shown that ceramide specifically inactivates the cyclin-dependent kinase Cdk2, but not Cdk4, through activation of a phosphatase (19.Lee J.Y. Bielawska A.E. Obeid L.M. Exp. Cell Res. 2000; 261: 303-311Crossref PubMed Scopus (66) Google Scholar). Recently, we have demonstrated that both exogenous (with C6-ceramide treatment) and endogenous ceramides (generated in response to bacterial sphingomyelinase overexpression or daunorubicin treatment) inhibit mRNA synthesis of telomerase reverse transcriptase and telomerase activity via inactivation of c-Myc transcription factor in the A549 human lung adenocarcinoma cell line (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21.Ogretmen B. Kraveka J.M. Schady D. Usta J. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2001; 276: 63506-63514Google Scholar). Moreover, this inhibition of telomerase activity mediated by ceramide has been shown to be independent of apoptosis but correlated with cell cycle arrest at G0/G1 (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These results add to a growing body of literature implicating ceramide in senescence of human cells and aging yeast (22.Venable M.E. Obeid L.M. Biochim. Biophys. Acta. 1999; 1439: 291-298Crossref PubMed Scopus (55) Google Scholar, 23.Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). It has been shown previously that the addition of short chain C2- and C6-ceramides to cells mimics many of the biological responses of agonists in mammalian and yeast cells, and this approach has been employed to suggest possible roles for endogenous ceramide in these processes (24.Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). In addition, there are results suggesting a mere direct relationship between exogenous and endogenous ceramides. It has been shown that treatment of Madin-Darby canine kidney cells with N-octanoyl-sphingosine liposomes caused about 10-fold increase in the total ceramide levels (25.Abe A. Wu D. Shayman J.A. Radin N.S. Eur. J. Biochem. 1992; 210: 765-773Crossref PubMed Scopus (48) Google Scholar). Treatment of U937 human myeloid leukemia cells with 25 μm cell-permeable C6-ceramide triggered a sustained endogenous ceramide generation at 24 h, which was inhibited by fumonisin B1 (26.Jaffrezou J-P. Maestre N. De Mas-Mansat C.B. Levade T. Laurent G. FASEB J. 1998; 12: 999-1006Crossref PubMed Scopus (67) Google Scholar). Also, it has been demonstrated that C6-ceramide triggers neutral sphingomyelinase activation, sphingomyelin hydrolysis, and total ceramide accumulation in HL-60 and U937 cells at 10–30 min (26.Jaffrezou J-P. Maestre N. De Mas-Mansat C.B. Levade T. Laurent G. FASEB J. 1998; 12: 999-1006Crossref PubMed Scopus (67) Google Scholar). Furthermore, it has been shown recently that treatment of human skin fibroblasts and peripheral blood mononuclear cells with C6-ceramide induces acid sphingomyelinase expression at the mRNA level that correlated with apoptotic cell death (27.Deigner H-P. Claus R. Bonaterra G.A. Gehrke C. Bibak N. Blaess M. Cantz M. Metz J. Kinscherf R. FASEB J. 2001; 15: 807-814Crossref PubMed Scopus (82) Google Scholar). Thus, there appear to be multiple mechanisms by which exogenous ceramide influences ceramide metabolism. These observations also raise the important question of whether downstream biological effects are mediated by short chain ceramide per se or if they are dependent on the generation of endogenous ceramide. Therefore, in this study, we determined if exogenous short chain ceramide induces the generation of endogenous long chain ceramide in the A549 human adenocarcinoma cell line. We focused on identifying the mechanisms of this effect and its possible role in mediating cell responses. We show here, for the first time, that the sustained generation of long chain endogenous ceramide requires the biochemical recycling of the sphingosine backbone of C6-ceramide, which involves deacylation and reacylation of ceramide for the generation of endogenous long chain ceramide (mainly C16:0- and C24:1-ceramides), most likely by CoA-dependent ceramide synthase, which is inhibited by fumonisin B1. Moreover, the generation of long chain endogenous ceramide in response to C6-ceramide treatment is stereospecific, as shown by increased long chain ceramide generation byd-erythro-C6-ceramide but notl-erythro-C6-ceramide. In addition, evidence is provided that in A549 cells the generation of long chain endogenous ceramide mediates the effects of exogenous C6-ceramide on growth inhibition, cell cycle arrest, and the modulation of telomerase activity. Therefore, this study provides new insights into the mechanisms of generation of endogenous ceramide and its downstream biological roles in response to exogenous short chain ceramide. The A549 human lung carcinoma cells were obtained from Dr. Alice Boylan (Medical University of South Carolina, Charleston, SC). The HL-60 human acute myeloid leukemia and MCF-7 human breast cancer cell lines were obtained from the ATCC (Manassas, VA). Cells were maintained in growth medium containing 10% fetal calf serum and 100 ng/ml each of penicillin and streptomycin (Invitrogen) at 37 °C in 5% CO2. Cell-permeable and biologically active short chain ceramides and their radioactively labeled analogs were obtained from the Synthetic Lipid Core at the Department of Biochemistry and Molecular Biology (Medical University of South Carolina). Fumonisin B1 (FB1) was obtained from Alexis. Myriocin (MYR) was obtained from Sigma. Exogenous short chain ceramides were dissolved in ethanol at a concentration of 100 mm and then directly added to the medium containing 10% fetal calf serum to obtain final concentration of 1–20 μm. The final volume of ethanol in the medium was 0.02%, which had no effect on cell growth and/or survival. Total endogenous ceramide levels were measured using the diacylglycerol phosphate kinase (DGK) method as described previously (28.Perry D.K. Bielawska A. Hannun Y.A. Methods Enzymol. 2000; 312: 22-31Crossref PubMed Google Scholar). In short, after total cellular lipids were extracted using the standard Bligh and Dyer protocol, they were dried under N2. The dried lipids were then resuspended and used for phosphate measurements and the Escherichia coli diacylglycerol kinase assay as modified for ceramide. This assay depends on the phosphorylation of ceramide and diacylglycerol, generating ceramide phosphate and phosphatidic acid, respectively, by diacylglycerol kinase in the presence of radiolabeled ATP. The radiolabeled products were visualized by thin layer chromatography, and the phosphorylated products of ceramide and diacylglycerol were identified by comparison with known standards run on the same plate. In addition, the amounts of products that were scraped from the plates were quantitated by scintillation counting and normalized to internal phosphate levels. The radiolabeled sphingolipids after treatments with [3-3H]d-erythro-C6-ceramide or N-[1-14C]d-erythro-C6-ceramide were extracted by the Bligh and Dyer method and separated by thin layer chromatography using a solvent system containing chloroform, methanol, and 15 mm CaCl2 (7.5:4.4:1). The levels of endogenous ceramide species were analyzed by MS utilizing normal phase high performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization. Separations were performed using a ThermoFinnigan (Foster City, CA) LCQ ion trap mass spectrometer. 2B. J. Pettus, M. Busman, P. D. R. Moeller, B. J. Kroesen, Z. M. Szulc, A. Bielawska, and Y. A. Hannun, unpublished data. Cells (1 × 105), grown in six-well plates, were treated in the presence of 1 μCi/ml [3H]palmitate (Amersham Biosciences) with or without 20 μm short chain ceramides at various time points. The lipids were extracted using Bligh and Dyer protocol and separated by thin layer chromatography (TLC) using a solvent system containing chloroform, methanol, and 2 nNH4OH (40:10:1), as described (29.Jenkins G.M. Hannun Y.A. J. Biol. Chem. 2001; 276: 8574-8581Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Various concentrations of total ceramide obtained from rat brain were used as controls. The effects of the inhibition of endocytosis on the generation of endogenous long chain ceramide in response to C6-ceramide were determined by palmitate labeling and the diacylglycerol kinase (DGK) assays as described above. Cells (1 × 105) grown in six-well plates were pretreated with known inhibitors of endocytosis such as nystatin (25 μg/ml), genistein (200 μm), and chlorpromazine (6 μg/ml) (30.Puri V. Watanabe R. Singh R.D. Dominguez M. Brown J.C. Wheatley C.L. Marks D.L. Pagano R.E. J. Cell Biol. 2001; 154: 535-547Crossref PubMed Scopus (278) Google Scholar) and also with brefeldin A (10 μg/ml), which causes the disassembly of the Golgi (31.Fukunaga T. Furuno A. Hatsuzawa K. Tani K. Yamamoto A. Tagaya M. FEBS Lett. 1998; 435: 237-240Crossref PubMed Scopus (14) Google Scholar, 32.Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar), for 30–60 min as described previously. Then cells were treated with 20 μmC6-ceramide for 60 min in the presence (for palmitate labeling) or absence (for the DGK assay) of 1 μCi/ml [3H]palmitate as described above. Ceramide measurements were performed as described above. The concentrations ofd-erythro-C6-ceramide or its stereoisomers that inhibited cell growth by 50% (IC50) were determined from cell survival plots obtained by MTT or trypan blue exclusion assays as described (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In short, cells (5 × 103 cells/well) were plated into 96-well plates containing 100 μl of the growth medium in the absence or presence of increasing concentrations of C6-ceramide or its streioisomers at 37 °C in 5% CO2 for 72 h. They were then treated with 25 μl of MTT for 4–5 h. After lysing the cells in 100 μl of the lysis buffer, the plates were read in a microplate reader (Dynatech, Chantilly, VA) at 570 nm. After that, the IC50 concentrations of the compounds were determined from cell survival plots as described (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Triplicate wells were used for each treatment. The final concentration of ethanol (a solvent for ceramide analogs) in the growth medium was less than 0.1% (v/v), which had no effect on cell growth and survival. For trypan blue exclusion analysis (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), cells (100 × 103 cells/well) were grown in six-well plates with 4 ml of media in the absence or presence of increasing concentrations of C6-ceramide or its stereoisomers for 24 h. Then cells were trypsinized and then diluted in PBS. The floating dead cells in the medium and cells attached to the plates were then counted using a hematocytometer in the presence of trypan blue solution at a 1:1 ratio (v/v) (Sigma) as described by the manufacturer. The degradation of PARP protein levels in cells was detected by Western blot analysis. In short, total proteins (50 μg/lane) were separated by 4–15% SDS-PAGE and blotted onto an Immobilon membrane, and PARP protein was detected using 1 μg/ml rabbit polyclonal anti-PARP antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and peroxidase-conjugated secondary anti-rabbit antibody (1:2500) as described (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The effects of 20 μm C6-ceramide on the cell cycle profiles of A549 cells at 24 h were analyzed in the presence of DNase-free RNase and propidium iodine by flow cytometry as described (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Untreated cells were used as controls. Telomerase activity in cell extracts was measured by the PCR-based telomere repeat amplification protocol (TRAP) using the TRAPeze kit (Intergen, Gaithersburg, MD), which includes a 36-bp internal control to allow quantification of activity as described by the manufacturer. Briefly, the cells, grown in six-well plates, were washed in phosphate-buffered saline and homogenized in 1× CHAPS lysing buffer for 30 min on ice. Then 50–100 ng of protein from each cell extract was analyzed in the TRAP reaction, as described (20.Ogretmen 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: 24901-24910Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21.Ogretmen B. Kraveka J.M. Schady D. Usta J. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2001; 276: 63506-63514Google Scholar). The cell extracts were added directly to the TRAP reaction mixture containing dNTPs, TS primer (6 × 105 cpm), reverse primer mix, and Taqpolymerase. Then the extended telomerase products were amplified by two-step PCR (94 °C for 30 s, 60 °C for 30 s) for 27 cycles. The telomerase activity in each sample was quantitated by measuring the ratio of the 36-bp internal standard to the extended telomerase products as described by the manufacturer using ChemiImager (Alpha Innotech Corp., San Leandro, CA). In order to examine the generation of endogenous long chain ceramide in response to exogenous C6-ceramide, A549 cells were grown in the absence or presence of 20 μm C6-ceramide for 24 h, and then total ceramide levels were measured by the DGK assay as described under "Experimental Procedures." Interestingly, as seen in Fig. 1A, total endogenous long chain ceramide levels were significantly increased (about 6-fold) in cells treated with C6-ceramide (lane 2) compared with that of untreated controls (lane 1). In order to determine the biochemical pathway(s) that are involved in the generation of endogenous long chain ceramide in response to C6-ceramide, A549 cells were treated with C6-ceramide (20 μm) in the presence or absence of 50 μm FB1, an inhibitor of CoA-dependent dihydroceramide/ceramide synthase, or 50 nm MYR, a specific inhibitor of serine palmitoyltransferase, and total ceramide levels were measured by the DGK assay. The results showed that FB1 prevented the generation of long chain endogenous ceramide levels in response to C6-ceramide, whereas MYR did not have any effect (Fig. 1A, lanes 3 and 4, respectively). The levels of C6-ceramide and diacylglycerol (DAG) were equal in these samples (Fig. 1A,lanes 2–4, lower and upper bands, respectively). The activity of MYR (50 nm) was confirmed in independent experiments in which it blocked the generation of endogenous ceramide in A549 cells after exposure to the chemotherapeutic agent gemcitibine. 3B. Ogretmen, M. J. Rossi, and R. Wood, unpublished data. Also, measurement of the activity of neutral sphingomyelinase in response to C6-ceramide (20 μm) at 10 min to 24 h showed no significant changes in these cells (data not shown). Similar results were obtained when total endogenous ceramide levels were measured by HPLC/MS in response to C6-ceramide treatment, in which long chain ceramide levels were significantly elevated (around 5-fold) in the presence of C6-ceramide (20 μm for 24 h) compared with that of untreated controls (Fig. 1B). This elevation was blocked completely by 50 μm FB1 but not by MYR (Fig. 1B). Moreover, the analysis of subspecies of endogenous ceramide levels generated in response to C6-ceramide by HPLC/MS revealed that C6-ceramide treatment resulted in increased levels of mainly C16:0-ceramide and to a lesser extent C24:1-ceramide in these cells (Fig. 1C). Importantly, there were no significant differences in the levels of dihydroceramide species in the absence or presence of C6-ceramide (Fig. 1D). The absolute mass of endogenous ceramide was about 0.8 and 5.1 pmol/nmol Pi in the absence and presence of 20 μm C6-ceramide at 24 h, respectively, in A549 cells as measured using the DGK method. Therefore, these results show that the measurement of the levels of endogenous ceramide by the HPLC/MS method is highly comparable with the DGK assay. Taken together, these results also show that treatment of A549 cells with C6-ceramide results in a significant increase in the generation of endogenous long chain ceramide, via a pathway that is distinct from the de novosynthesis (resistance to myriocin) or hydrolysis of sphingomyelin (no change in the activity of neutral sphingomyelinase and sphingomyelin levels). To examine the kinetics of the generation of long chain endogenous ceramide in response to C6-ceramide, A549 cells were grown in the presence of 1 μm[sphingosine-3-3H]d-erythro-C6-ceramide (1 μCi/ml) at 0, 1, 3, 6, and 24 h, and then the labeled lipids were analyzed as described under "Experimental Procedures." Results showed that the incorporation of the 3H label into the long chain endogenous ceramide started to be detected slightly at 1 h and reached its maximum levels at 6 h, and this increased ceramide generation was sustained at 24 h (Fig. 2A, lanes 1–5). There was also a marked increase in the levels of labeled short chain glucosylceramide, lactosylceramide, and sphingomyelin. The 3H label was also detected in long chain glucosylceramide, lactosylceramide, and sphingomyelin at 24 h (Fig. 2A, lane 5). These results show that the 3H label in the sphingosine backbone was initially incorporated into long chain ceramides followed by incorporation into complex sphingolipids. The results also show that C6-ceramide served as a direct substrate for SM synthase and glucosylceramide synthase. Total endogenous long chain ceramide levels in response to 20 μm unlabeled ceramide at various time points were also measured by HPLC/MS. The accumulation of total long chain endogenous ceramides (mainly C16:0 and C24:1) in response to C6-ceramide was observed at 1 h, reached its maximum levels at 6 h, and was still detectable at 24 h (Fig. 2B). These results were highly comparable with the results obtained using 1 μm[sphingosine-3-3H]d-erythro-C6-ceramide as shown in Fig. 2A. To determine the biochemical mechanisms by which C6-ceramide caused increased generation of endogenous long chain ceramide, A549 cells were grown in the presence of 1 μm[sphingosine-3-3H]-d-erythro-C6-ceramide (1 μCi/ml) for 24 h, and then labeled lipids were analyzed by thin layer chromatography following Bligh and Dyer extraction as described under "Experimental Procedures." As Fig. 3A shows, 3H label was incorporated into the newly synthesized long chain ceramides (lane 1). Interestingly, the presence of FB1 caused a significant decrease in the incorporation of the3H label into the long chain ceramide and caused some inhibitory effect on the incorporation of the label into the long chain glucosylceramide, lactosylceramide, and sphingomyelin (Fig. 3A, lane 2). The pretreatment of cells with MYR, however, did not have any significant effect on the generation of ceramide (Fig. 3A, lane 3). As seen in Fig. 3A, the majority (about 86%) of the [sphingosine-3-3H]d-erythro-C6-ceramide was metabolized to long chain derivatives after 24 h (lane 1). The major derivatives were long chain sphingomyelin, glucosylceramide, lactosylceramide, and ceramide (35, 32, 9.8, and 9.2%, respectively). More importantly, when these cells were treated with 1 μmN-[N-hexanoyl-1-14C]d-erythro-C6-ceramide (1 μCi/ml) with the label on the fatty acid chain, the14C label was not incorporated into the long chain endogenous ceramide, glucosylceramide, lactosylceramide, or sphingomyelin at 24 h, whereas it was readily detectable in their short chain forms in the absence or presence of FB1 (Fig. 3B, lanes 2 and 3). Taken together, these results show that the generation of long chain endogenous ceramide in response to C6-ceramide treatment is due to bioche
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