FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1980260023> ?p ?o ?g. }

• W1980260023 endingPage "27135" @default.
• W1980260023 startingPage "27129" @default.
• W1980260023 abstract "Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxinN-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous sphingolipids as well as their changes in endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of ∼50% over a period of 3 h. Finally, treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells, suggesting that the two pathways do interact. Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxinN-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous sphingolipids as well as their changes in endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of ∼50% over a period of 3 h. Finally, treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells, suggesting that the two pathways do interact. N-methyl-N-nitro-N-nitrosoguanidine C2-ceramide C2-dihydroceramide myriocin sphingosine sphinganine minimal essential medium enzyme-linked immunosorbent assay high performance liquid chromatography Sphingolipids are emerging as an important group of signaling molecules involved in the cellular responses to stress (reviewed in Refs. 1Dbaibo G.S. Biochem. Soc. Trans. 1997; 25: 557-561Crossref PubMed Scopus (15) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar, 3Merrill Jr., A.H. Schmeltz E.-M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (566) Google Scholar). Recent research on sphingolipids has shown that they are involved in signal transduction pathways that mediate cell growth, differentiation, and death. For example, sphingosine and sphingosine 1-phosphate modulate cellular calcium homeostasis and cell proliferation (4Nakamura S. Kozutsumi Y. Sun Y. Miyake Y. Fujita T. Kawasaki T. J. Biol. Chem. 1996; 271: 1255-1257Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 5Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (818) Google Scholar, 6Olivera A. Zhang H. Carlson R.O. Mattie M.E. Schmidt R.R. Spiegel S. J. Biol. Chem. 1994; 269: 17924-17930Abstract Full Text PDF PubMed Google Scholar), and sphingosine induces cell cycle arrest and apoptosis (7Dbaibo G.S. Wolff R.A. Obeid L.M. Hannun Y.A. Biochem. J. 1995; 310: 453-459Crossref PubMed Scopus (22) Google Scholar, 8Hung W.C. Chuang L.Y. Biochem. Biophys. Res. Commun. 1996; 229: 11-15Crossref PubMed Scopus (39) Google Scholar, 9Spiegel S. Cuvillier O. Edsall L. Kohama T. Menzeleev R. Olivera A. Thomas D. Tu Z. Van Brocklyn J. Wang F. Biochemistry (Mosc). 1998; 63: 69-73PubMed Google Scholar, 10Sweeney E.A. Sakakura C. Shirahama T. Masamune A. Ohta H. Hakomori S. Igarashi Y. Int. J. Cancer. 1996; 66: 358-366Crossref PubMed Scopus (175) Google Scholar). Other studies have shown that ceramide, the structural backbone of sphingolipids, has multiple biological activities that are predominantly growth-suppressive. For example, exposure to short-chain, membrane-permeable ceramide analogs can cause cell cycle arrest or apoptosis (11Dbaibo G.S. Pushkareva M.Y. Jayadev S. Schwartz J.K. Horowitz J.M. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1347-1351Crossref PubMed Scopus (203) Google Scholar, 12Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 13Martin S.J. Newmeyer D.D. Mathias S. Farshchon D.M. Wang H. Reed J.C. Kolesnick R.N. Green D.R. EMBO J. 1995; 14: 5191-5200Crossref PubMed Scopus (241) Google Scholar, 14Vento R. Giuliano M. Lauricella M. Carabillo M. Di Liberto D. Tesoriere G. Mol. Cell Biochem. 1998; 185: 7-15Crossref PubMed Scopus (29) Google Scholar). A number of extra-cellular stimuli and reagents, including tumor necrosis factor, Fas ligand, interleukin-1, ionizing radiation, and chemotherapeutic drugs can cause an increase in the cellular ceramide level, resulting in either cell cycle arrest or apoptosis (15Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (598) Google Scholar, 16Dbaibo G.S. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar, 17Haimovitz-Friedman A. Kan C.C. Ehleiter D. Persanud R.S. McLaughlin M. Fuks Z. Kolesnick R.N. J. Exp. Med. 1994; 180: 525-535Crossref PubMed Scopus (855) Google Scholar, 18Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 20Wieder T. Orfanos C.E. Geilen C.C. J. Biol. Chem. 1998; 273: 11025-11031Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 21Xia P. Gamble J.R. Rye K. Wang L. Hii C.S.T. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (358) Google Scholar, 22Xu J. Yeh C.H. Chen S. He L. Sensi S.L. Canzoniero L.M. Choi D.W. Hsu C.Y. J. Biol. Chem. 1998; 273: 16521-16526Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In addition, ceramide can activate the stress-activated protein kinase/Jun-N-terminal kinase cascade, which is thought to play a central role in various stress responses (23Coroneos E. Wang Y. Panuska J.R. Templeton D.J. Kester M. Biochem. J. 1996; 316: 13-17Crossref PubMed Scopus (107) Google Scholar, 24Huang C. Ma W. Ding M. Bowden G.T. Dong Z. J. Biol. Chem. 1997; 272: 27753-27757Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25Jarvis W.D. Kolesnick R.N. Fornari F.A. Traylor R.S. Gewirtz D.A. Grant S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 73-77Crossref PubMed Scopus (484) Google Scholar, 26Reunanen N. Westermark J. Hakkinen L. Holmstrom T.H. Elo I. Eriksson J.E. Kahari V.M. J. Biol. Chem. 1998; 273: 5137-5145Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). One kind of stress frequently encountered by cells is DNA damage. Damage can be induced by exposure to UV- or γ-irradiation, environmental chemicals, or therapeutic reagents. Several signal transduction pathways involved in the cellular response to genotoxic stress have been identified, one of which is mediated by the tumor suppressor protein p53 (reviewed in Refs. 27Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1363) Google Scholar, 28Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar, 29Wang X. Ohnishi T. J. Radiat. Res. 1997; 38: 179-194Crossref PubMed Scopus (61) Google Scholar). After genotoxic stress, the cellular level of p53 is increased (30Fritsche M. Haessler C. Brandner G. Oncogene. 1993; 8: 307-318PubMed Google Scholar, 31Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 32Yang J. Duerksen-Hughes P. Carcinogenesis. 1998; 19: 1117-1125Crossref PubMed Scopus (85) Google Scholar). This protein then functions as a transcriptional modulator to activate or repress specific gene expression. One possible outcome is G1/S arrest, which allows repair of damaged DNA before replication, whereas a second possible outcome is apoptosis. In either case, replication of damaged DNA is prevented. The mechanism leading to this increased p53 protein level is stabilization of the protein, which is caused by changes in the phosphorylation and acetylation of p53 through the activation of several pathways, including the Jun-N-terminal kinase/stress-activated protein kinase pathways (33Chiarugi V. Cinelli M. Magnelli L. Oncology Res. 1998; 10: 55-57PubMed Google Scholar, 34Kubbatat M.H. Vousden K.H. Mol. Med. Today. 1998; 4: 250-256Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 35Meek D.W. Campbell L.E. Jardine L.J. Knippschild U. McKendrick L. Miline D.M. Biochem. Soc. Trans. 1997; 25: 416-419Crossref PubMed Scopus (24) Google Scholar, 36Milner J. Pathol. Biol. 1997; 45: 797-803PubMed Google Scholar). It has been proposed that many, if not all, genotoxic response pathways converge on p53, allowing it to serve as the “universal sensor of genotoxic stress” (37Liu Z.G. Baskaran R. Lea-Chou E.T. Wood L.D. Chen Y. Karin M. Wang J.Y. Nature. 1996; 384: 273-276Crossref PubMed Scopus (347) Google Scholar). However, there are also studies indicating the presence of a p53-independent pathway in the genotoxic stress response (38Strasser A. Harris A.W. Jacks T. Cory S. Cell. 1994; 79: 329-339Abstract Full Text PDF PubMed Scopus (679) Google Scholar). Recently, a role for ceramide in the genotoxic response has been proposed (1Dbaibo G.S. Biochem. Soc. Trans. 1997; 25: 557-561Crossref PubMed Scopus (15) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar). For example, Santana et al. (39Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar) showed that acid sphingomyelinase-deficient human lymphoblasts and mice were unable to generate ceramide and undergo apoptosis after exposure to ionizing radiation; these responses were restored after transfecting the cells with cDNA encoding human acid sphingomyelinase. Other reports also suggest that radiation can affect ceramide levels (24Huang C. Ma W. Ding M. Bowden G.T. Dong Z. J. Biol. Chem. 1997; 272: 27753-27757Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 40Brenner B. Koppenhoefer U. Weinstock C. Linderkamp O. Lang F. Gulbins E. J. Biol. Chem. 1997; 272: 22173-22181Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar,41Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1718) Google Scholar). However, whether this induction of ceramide is a general response to genotoxic stress or whether it is limited to ionizing or UV radiation has not been demonstrated. N-methyl-N-nitro-N-nitrosoguanidine (MNNG),1 a substance found in cigarette smoke, is a monofunctional alkylating agent. These chemicals target the cellular DNA and induce severe genotoxic stress to the cell that can result in chromosomal aberrations, sister chromatid exchanges, point mutations, and cell death. Therefore, most such alkylating agents, including MNNG, are considered genotoxic mutagens and carcinogens (42Smart R.C. Carcinogenesis. Appleton & Lange, East Norwalk, CT1994Google Scholar). MNNG treatment also induces apoptosis in cell culture, and this process can be inhibited by the Bcl-2 protein (43Hour T. Shiau S.L. Lin J. Toxicol. Lett. 1999; 110: 191-202Crossref PubMed Scopus (17) Google Scholar). Furthermore, the Jun-N-terminal kinase/stress-activated protein kinases and p38 kinase are activated in MNNG-treated cells (44Wilhelm D. Bender K. Knebel A. Angel P. Mol. Cell. Biol. 1997; 17: 4792-4800Crossref PubMed Scopus (225) Google Scholar), and our laboratory has previously shown that treatment with MNNG can activate the p53 pathway (32Yang J. Duerksen-Hughes P. Carcinogenesis. 1998; 19: 1117-1125Crossref PubMed Scopus (85) Google Scholar). Because of the ability of sphingolipids to mediate growth arrest and apoptosis and the growing evidence suggesting their possible role in responses to DNA damage, we wished to investigate whether sphingolipids such as ceramide might mediate cellular responses to DNA damage induced by genotoxins such as MNNG. We also wished to determine whether the sphingolipid- and p53-mediated pathways, both of which can lead to growth arrest and apoptosis, might interact with each other. C2-ceramide (Cer), C2-dihydroceramide (DHCer), ISP-1 (myriocin), sphingosine (So), sphinganine (Sa), and sphingosine 1-phosphate were obtained from Biomol (Plymouth Meeting, PA) and prepared following the manufacturer's instruction and Hannun et al. (45Hannun Y.A. Merrill A.H. Bell R.M. Methods Enzymol. 1991; 201: 316-328Crossref PubMed Scopus (61) Google Scholar). Briefly, Cer was dissolved in dimethyl sulfoxide (Me2SO) as a 60 mm stock solution; So was dissolved in ethanol as a 100 mm stock solution; DHCer was dissolved in methanol as a 15 mm stock solution; Sa was dissolved in Me2SO as a 10 mm stock solution; and sphingosine 1-phosphate was dissolved in Me2SO as a 5 mm stock solution. MNNG was obtained from Aldrich (Milwaukee, WI). LM mouse fibroblast cells were cultivated in minimum essential medium (MEM) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). LME6 cells were derived from LM cells that were cotransfected with pSV2neo and the human papilloma virus 16 E6 gene, which mediates the rapid degradation of p53 through the ubiquitin pathway. They express E6 mRNA, and their level of p53 is undetectable even after genotoxin treatment (46Duerksen-Hughes P.J. Yang J. Schwartz S.B. Virology. 1999; 264: 55-65Crossref PubMed Scopus (35) Google Scholar). They were cultured as were the LM cells with the exception that G418 (Life Technologies, Inc.) was present in the medium. LMD cells were derived from LM cells that were cotransfected with pSV2neo and a mutant version of the human papilloma virus 16 E6 gene (deleted for amino acids 106–110) that does not mediate the rapid degradation of p53 (Refs. 46Duerksen-Hughes P.J. Yang J. Schwartz S.B. Virology. 1999; 264: 55-65Crossref PubMed Scopus (35) Google Scholar and references therein). They were cultured as were the LME6 cells. The medium was changed to MEM with 2% fetal bovine serum or to serum-free Opti-MEM (Life Technologies, Inc.) before treatment with sphingolipids. The delivery of sphingolipids using the bovine serum albumin solution was carried out as described by Hannun et al. (45Hannun Y.A. Merrill A.H. Bell R.M. Methods Enzymol. 1991; 201: 316-328Crossref PubMed Scopus (61) Google Scholar). Cells were assayed for p53 by ELISA as described previously (32Yang J. Duerksen-Hughes P. Carcinogenesis. 1998; 19: 1117-1125Crossref PubMed Scopus (85) Google Scholar). Briefly, cells were removed from the plate by trypsinization, concentrated by centrifugation (10 min, 1,000 rpm), and suspended in phosphate-buffered saline/TDS (50 mmNa2HPO4, 17 mmNaH2PO4, 68 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4, with 1% aproteinin added before use). After lysis, lysates were stored at −80 °C for no more than 1 week before analysis. The ELISA analysis used pAB122 (hybridoma was obtained from ATCC, and antibodies were purified from the culture medium using protein-A Sepharose) as the primary or capture antibody, biotinylated anti-p53 (Roche Molecular Biochemicals) as the detection antibody, and glutathione S-transferase (GST)-p53 (Santa Cruz Biotechnology, Santa Cruz, CA) as a standard. Each point was measured in triplicate, and results were normalized to the amount of protein present in each sample to control for any variability in the number of cells recovered from each plate. Protein concentration was determined by the bicinchoninic acid assay (BCA assay) (Pierce). The cellular levels of the Bax protein were determined by Western blot as described previously (32Yang J. Duerksen-Hughes P. Carcinogenesis. 1998; 19: 1117-1125Crossref PubMed Scopus (85) Google Scholar), with the exception that anti-Bax was used as the primary antibody rather than anti-p53. Briefly, after electrophoresis, proteins were transferred to a nitrocellulose membrane (Micron Separations Inc., Westborough, MA) and incubated with anti-Bax (2 µg/ml) (Oncogene, Cambridge, MA), and the Bax protein was detected using the Kirkegaard and Perry Laboratories (Gaithersburg, MD) chemiluminescent horseradish peroxidase system following the manufacturer's instructions. Apoptosis was measured by the cell death detection ELISA (Roche Molecular Biochemicals). The assay is based on the quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysate. The cell death detection ELISA was conducted as described by the manufacturer. Briefly, lysates were added to wells to which the anti-histone antibody had been fixed adsorptively. Nucleosomes in the sample bound via their histone components to the immobilized anti-histone antibody. Anti-DNA peroxidase, which binds to the DNA portion of the nucleosomes, was then added. The amount of bound peroxidase was determined photometrically by measuring absorbance at 405 nm after the addition of 2,2′-azino-di(3-ethylbenzthiazolin sulfonate) (ABTS) as substrate. The CellTiter 96Aqueous One Solution cell proliferation assay (Promega, Madison, WI) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. It contains a tetrazolium compound, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), which is bioreduced by viable cells into a colored formazan product that is soluble in tissue culture medium. It was preformed according to the manufacturer's instruction. Briefly, 1 × 104 cells were seeded into a 96-well culture plate (Corning Glass, Corning, NY) and treated with the indicated concentrations of sphingolipids or genotoxins for the specified times. 20 µl of the CellTiter solution was added directly to each culture well and incubated at 37 °C for 2 h, and the absorbance at 490 nm was determined in a Bio-Rad microplate reader. 1 × 105 cells were seeded into 12-well plates (Corning) and grown in 3 ml of media. After treatment, cells were harvested with 0.25% trypsin, and the number of Trypan blue-excluding cells was determined using a hemacytometer. Determination and quantification of intracellular ceramide content were performed as described (22Xu J. Yeh C.H. Chen S. He L. Sensi S.L. Canzoniero L.M. Choi D.W. Hsu C.Y. J. Biol. Chem. 1998; 273: 16521-16526Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) with some modifications. 1 × 106 cells were cultured in MEM (10% fetal bovine serum) with 10 µCi of [3H]palmitate (Sigma) for 16–24 h before the addition of MNNG. After further incubation for the times indicated (allowing the labeled palmitate to remain in the culture medium), cells were harvested by scraping into a glass tube and pelleted by centrifugation. Lipid was extracted as described (47Merrill A.H. Wang E. Mullins R.E. Jamison W.C.L. Nimkar S. Liotta D. Anal. Biochem. 1988; 171: 373-381Crossref PubMed Scopus (305) Google Scholar). Briefly, CH3OH (2 ml) and CHCl3 (0.5 ml) were added to the cells, followed by H2O (3 ml), and the mixture was vortexed and centrifuged to separate the phases. After transfer of the organic phase to a clean tube, residual proteins were removed by an additional extraction with H2O, then the organic phase was transferred to a sodium sulfate column to remove residual H2O. The eluate was vacuum-dried and resuspended in 40 µl of chloroform:CH3OH (20:1, v/v). Half was used to determine the total counts of the labeled lipids, whereas the other half was analyzed by thin layer chromatography (TLC) (Panvera, Madison, WI) using chloroform:acetone:methanol:acetic acid:H2O (50:20:15:10:5) as the solvent system. Lipids were visualized by iodine vapor, and the radioactive spots corresponding to ceramide were scraped off and quantitated by liquid scintillation counting. The ceramide standard used was obtained from Matreya, Inc. (Pleasant Gap, PA). Determination and quantification of intracellular sphinganine and sphingosine contents was performed as described (47Merrill A.H. Wang E. Mullins R.E. Jamison W.C.L. Nimkar S. Liotta D. Anal. Biochem. 1988; 171: 373-381Crossref PubMed Scopus (305) Google Scholar). Briefly, lipids were extracted as above except that the cells were not radiolabeled. The acylglycerolipids were cleaved by incubation with 0.1 m KOH in CH3OH and chloroform (2:1, v/v). The free long-chain bases were then recovered in the chloroform phase and derivatized witho-phthalaldehyde (Sigma). The HPLC analyses were performed using an Isco (Lincoln, NE) model 2300 pump with a Waters (Milford, MA) Nova Pak C18 column (5 µm, 3.9 × 150) and a Nova Pak C18 guard column (Waters). Sphingosine and C20-sphingosine standards (Matreya, Inc.) were prepared by dissolving them in EtOH to make 5 mm stocks. They were then serially diluted to 10 µm. 10 µl of each of the standards was derivatized with o-phthalaldehyde, and the final volume was made up to 1 ml with CH3OH, 5 mm potassium phosphate, pH 7.0 (90:10, v/v), yielding a final standard concentration of 100 pm. LM is a mouse fibroblast cell line that expresses p53, whereas LME6 is a cell line derived from LM cells transfected with the human papilloma virus 16 E6 protein. In contrast to the parental LM cells, LME6 cells express no detectable p53 either before or after genotoxin treatment (46Duerksen-Hughes P.J. Yang J. Schwartz S.B. Virology. 1999; 264: 55-65Crossref PubMed Scopus (35) Google Scholar). They are therefore able to serve as a system in which to study genotoxin-triggered apoptotic pathways other than those mediated by p53. As shown in Fig. 1, LME6 cells were killed by MNNG treatment to an extent and with kinetics essentially equivalent to that of the parental LM cells, as measured by either the cell death ELISA (Fig. 1 A) or a cell viability assay (CellTiter assay) (Fig. 1 B). The ability of MNNG to induce cell death in the absence of p53 indicated that these cells possess a p53-independent pathway to apoptosis. Because of the well documented ability of sphingolipids such as ceramide and sphingosine to cause cell cycle arrest and apoptosis (25Jarvis W.D. Kolesnick R.N. Fornari F.A. Traylor R.S. Gewirtz D.A. Grant S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 73-77Crossref PubMed Scopus (484) Google Scholar, 48Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1618) Google Scholar, 49Dickson R.C. Nagiec E.E. Skrzpek M. Tillman P. Wells G.B. Lester R.L. J. Biol. Chem. 1977; 272: 30196-30200Abstract Full Text Full Text PDF Scopus (220) Google Scholar, 50Jenkins G.M. Richards A. Wahl T. Mao C. Obeid L. Hannun Y. J. Biol. Chem. 1997; 262: 32566-32572Abstract Full Text Full Text PDF Scopus (261) Google Scholar, 51Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 52Skrzpek M.S. Nagiec M.M. Lester R.L. Dickson R.C. J. Bacteriol. 1999; 181: 1134-1140Crossref PubMed Google Scholar, 53Wells G.B. Dickson R.C. Lester R.L. J. Biol. Chem. 1998; 273: 7235-7243Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), their possible involvement in this p53-independent cytolytic pathway was examined. If these sphingolipids are important in mediating MNNG-triggered apoptosis in LME6 cells, one prediction was that the addition of these compounds exogenously should also cause cell death in LME6 cells. LM and LME6 cells treated with ceramide at different concentrations (Fig. 2 A, top panel) or for different times (Fig. 2 B, top panel) experienced a loss of viability in a time- and dose-dependent manner as measured by the CellTiter assay. Cells undergoing cell death also demonstrated the budding morphologically associated with apoptosis. Additionally, flow cytometry results using propidium iodide staining of fixed cells showed the characteristic apoptotic peak of cells containing less than 2N DNA in ceramide-treated cells but not control cells (data not shown). Sphingosine, another member of the sphingolipid family, had a similar effect on both LM and LME6 cells (Fig. 2, A andB, middle panel). However, DHCer, a closely related molecule considered to be largely biologically inactive, had little or no effect on the cell viability at a similar concentration, although it did demonstrate cytotoxic effects at much higher concentrations (Fig. 2, A and B, bottom panel). The effects noted with DHCer indicate that it may not be completely biologically inactive. When observed over a period of days, untreated cells proliferated vigorously, as assessed by a direct cell count (Fig. 3). Ceramide and sphingosine treatment, on the other hand, resulted in an inhibition of LM and LME6 proliferation that was essentially comparable for the two cell types, whereas DHCer treatment did not inhibit proliferation (Fig. 3). These results indicated that ceramide and sphingosine can induce cell death regardless of the presence or absence of p53.Figure 3Cell proliferation measured by direct cell count for LM and LME6 cells treated with Cer (25 µm), So (25 µm), or DHCer (20 µm) for a 3-day period. Trypan blue-excluding cells were counted. Points are the means of three culture dishes, and error bars represent the S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) If the MNNG-triggered cytotoxic response were indeed mediated by sphingolipids such as ceramide, their level would be expected to increase after MNNG treatment. To test this prediction, LME6 cells were treated with MNNG, and their cellular ceramide levels were measured. As shown in Fig. 4, the ceramide level rose in LME6 cells after treatment with MNNG. An increase of ∼50% was achieved within the first 3 h, and this increase was transient, since the ceramide level began to drop after that point, returning to the control level by 6 h. There was no further change through 24 h post-treatment (data not shown). In an effort to identify a possible role for other sphingolipids in this response, the cellular levels of sphingosine and sphinganine were also measured by HPLC. Our results demonstrated that there was no significant change in either the sphingosine or sphinganine level after MNNG treatment (data not shown), excluding their involvement in the response. To determine whether MNNG treatment causes the accumulation of ceramide in cells expressing p53 as well as in those lacking p53, the parental LM cells were treated with the genotoxin, and their cellular ceramide levels were measured. As shown in Fig. 4, ceramide did not accumulate in MNNG-treated LM cells. Later measurements (up to 24 h) also showed no accumulation of ceramide (data not shown). These results suggest that ceramide does not play an important role in mediating MNNG-triggered apoptotic signals in LM cells. However, LM cells did activate their p53 pathway after MNNG treatment (Fig. 5). Within 2 h post-treatment, p53 levels began to rise, reaching a maximum value 12 h post-treatment (panel A). The levels of Bax, a p53-regulated protein involved in apoptosis (54Miyashita T. Krajewski S. Krajewska M. Wang H.G. Lin H.K. Liebermann D.A. Hoffman B. Reed J.C. Oncogene. 1994; 9: 1799-1805PubMed Google Scholar), were also observed to rise, showing a detectable increase 12 h post-treatment (panel B), and these events were followed by an increase in cell death which was maximal 16 h post-treatment (panel C). In contrast, neither p53 levels nor those of Bax increased after treatment of LME6 cells with MNNG (data not shown). The difference in the ceramide response of LM and LME6 cells could be due either to the fact that LM cells express p53, whereas LME6 cells do not, or to the fact that LME6 cells express the viral E6 protein, whereas LM cells do not. To address this question, a third cell line was tested for its" @default.
• W1980260023 created "2016-06-24" @default.
• W1980260023 creator A5042474689 @default.
• W1980260023 creator A5042664406 @default.
• W1980260023 date "2001-07-01" @default.
• W1980260023 modified "2023-09-30" @default.
• W1980260023 title "Activation of a p53-independent, Sphingolipid-mediated Cytolytic Pathway in p53-negative Mouse Fibroblast Cells Treated with N-Methyl-N-nitro-N-nitrosoguanidine" @default.
• W1980260023 cites W1491611834 @default.
• W1980260023 cites W1582592433 @default.
• W1980260023 cites W1963681578 @default.
• W1980260023 cites W1969158591 @default.
• W1980260023 cites W1969436183 @default.
• W1980260023 cites W1973800195 @default.
• W1980260023 cites W1975456426 @default.
• W1980260023 cites W1986010238 @default.
• W1980260023 cites W1991494214 @default.
• W1980260023 cites W1995391501 @default.
• W1980260023 cites W1996192399 @default.
• W1980260023 cites W1998166016 @default.
• W1980260023 cites W2003474943 @default.
• W1980260023 cites W2007604755 @default.
• W1980260023 cites W2014170738 @default.
• W1980260023 cites W2014245712 @default.
• W1980260023 cites W2017191638 @default.
• W1980260023 cites W2017729786 @default.
• W1980260023 cites W2024830340 @default.
• W1980260023 cites W2027151723 @default.
• W1980260023 cites W2031296027 @default.
• W1980260023 cites W2032072281 @default.
• W1980260023 cites W2032361307 @default.
• W1980260023 cites W2032770280 @default.
• W1980260023 cites W2042223647 @default.
• W1980260023 cites W2042415030 @default.
• W1980260023 cites W2042868752 @default.
• W1980260023 cites W2049108649 @default.
• W1980260023 cites W2053858732 @default.
• W1980260023 cites W2054773029 @default.
• W1980260023 cites W2057839760 @default.
• W1980260023 cites W2060527795 @default.
• W1980260023 cites W2060836077 @default.
• W1980260023 cites W2063364589 @default.
• W1980260023 cites W2065298161 @default.
• W1980260023 cites W2069496353 @default.
• W1980260023 cites W2092330976 @default.
• W1980260023 cites W2101950406 @default.
• W1980260023 cites W2113070607 @default.
• W1980260023 cites W2118385360 @default.
• W1980260023 cites W2126544407 @default.
• W1980260023 cites W2135687585 @default.
• W1980260023 cites W2136046634 @default.
• W1980260023 cites W2136953396 @default.
• W1980260023 cites W214713915 @default.
• W1980260023 cites W2155368714 @default.
• W1980260023 cites W2156299289 @default.
• W1980260023 cites W2157395014 @default.
• W1980260023 cites W2274605276 @default.
• W1980260023 cites W2280929046 @default.
• W1980260023 cites W2319955241 @default.
• W1980260023 cites W2397164225 @default.
• W1980260023 cites W76409271 @default.
• W1980260023 cites W969257698 @default.
• W1980260023 doi "https://doi.org/10.1074/jbc.m100729200" @default.
• W1980260023 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11369765" @default.
• W1980260023 hasPublicationYear "2001" @default.
• W1980260023 type Work @default.
• W1980260023 sameAs 1980260023 @default.
• W1980260023 citedByCount "29" @default.
• W1980260023 countsByYear W19802600232012 @default.
• W1980260023 countsByYear W19802600232013 @default.
• W1980260023 countsByYear W19802600232014 @default.
• W1980260023 countsByYear W19802600232015 @default.
• W1980260023 countsByYear W19802600232016 @default.
• W1980260023 countsByYear W19802600232018 @default.
• W1980260023 countsByYear W19802600232021 @default.
• W1980260023 crossrefType "journal-article" @default.
• W1980260023 hasAuthorship W1980260023A5042474689 @default.
• W1980260023 hasAuthorship W1980260023A5042664406 @default.
• W1980260023 hasBestOaLocation W19802600231 @default.
• W1980260023 hasConcept C109316439 @default.
• W1980260023 hasConcept C153911025 @default.
• W1980260023 hasConcept C178790620 @default.
• W1980260023 hasConcept C185592680 @default.
• W1980260023 hasConcept C202751555 @default.
• W1980260023 hasConcept C2780263894 @default.
• W1980260023 hasConcept C2780381497 @default.
• W1980260023 hasConcept C2780639532 @default.
• W1980260023 hasConcept C502942594 @default.
• W1980260023 hasConcept C55493867 @default.
• W1980260023 hasConcept C61705674 @default.
• W1980260023 hasConcept C73588182 @default.
• W1980260023 hasConcept C86803240 @default.
• W1980260023 hasConceptScore W1980260023C109316439 @default.
• W1980260023 hasConceptScore W1980260023C153911025 @default.
• W1980260023 hasConceptScore W1980260023C178790620 @default.
• W1980260023 hasConceptScore W1980260023C185592680 @default.
• W1980260023 hasConceptScore W1980260023C202751555 @default.
• W1980260023 hasConceptScore W1980260023C2780263894 @default.
• W1980260023 hasConceptScore W1980260023C2780381497 @default.

• next