Expression of Glucosylceramide Synthase, Converting Ceramide to Glucosylceramide, Confers Adriamycin Resistance in Human Breast Cancer Cells
1999; Elsevier BV; Volume: 274; Issue: 2 Linguagem: Inglês
10.1074/jbc.274.2.1140
ISSN1083-351X
AutoresYu Liu, Tie-Yan Han, Armando E. Giuliano, Myles C. Cabot,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoMultidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682–1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells. Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682–1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells. Glucosylceramide synthase (GCS) 1The abbreviations used are: GCS, glucosylceramide synthase (ceramide glucosyltransferase, UDP-glucose:N-acylsphingosined-glucosyltransferase (EC 2.4.1.80)); GC, glucosylceramide; FBS, fetal bovine serum; MCF-7/GCS, glucosylceramide synthase-transfected cell line; Tet, tetracycline; rtTA, reverse tetracycline transactivator; TNF-α, tumor necrosis factor-α; Bcl-2, B-cell leukemia oncoprotein, MDR, multidrug resistance; CMV, cytomegalovirus. transfers glucose from UDP-glucose to ceramide and produces GC. GC serves as the core structure for more than 300 glycolipids (1Basu S. Kaufman B. Roseman S. J. Biol. Chem. 1968; 243: 5802-5804Abstract Full Text PDF PubMed Google Scholar). Recently, it has been shown that human GCS is a glycoprotein containing 394 amino acids encoded from 1182 nucleotides, including a G+C-rich 5′ untranslated region of 290 nucleotides (2Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar). A large body of literature shows that ceramide, the substrate of GCS, exerts an important role mediating myriad biological activities. Ceramide is a pleiotropic cellular activator capable of inducing two mutually exclusive cellular functions, cell proliferation and cell death. Ceramide is now recognized as a messenger of signaling events that originate from different cell surface receptors, including interferon-γ, TNF-α, interferon-1β, CD95 (Fas/APO-1), nerve growth factor receptor, and CD28 (3Testi R. Trends Biochem. Sci. 1996; 21: 468-471Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 4Smyth M.J. Obeid L.M. Hannun Y.A. Adv. Pharmacol. 1997; 41: 133-154Crossref PubMed Scopus (62) Google Scholar, 5Haimovitz-Friedman A. Kolesnick R. Fuks Z. Br. Med. Bull. 1997; 53: 539-553Crossref PubMed Scopus (187) Google Scholar). Ceramide is also involved in the action of protein kinase C ζ, vavprotooncogene, 1α-25-dihydroxy vitamin D3, dexamethasone, ionizing radiation, and chemotherapeutic agents (3Testi R. Trends Biochem. Sci. 1996; 21: 468-471Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 4Smyth M.J. Obeid L.M. Hannun Y.A. Adv. Pharmacol. 1997; 41: 133-154Crossref PubMed Scopus (62) Google Scholar, 5Haimovitz-Friedman A. Kolesnick R. Fuks Z. Br. Med. Bull. 1997; 53: 539-553Crossref PubMed Scopus (187) Google Scholar). There have been several lines of evidence suggesting that loss of ceramide production is one cause of cellular resistance to apoptosis induced by either ionizing radiation, TNF-α, or adriamycin (6Chuma S.J. Nodzenski E. Beckett M.A. Kufe D.W. Quintans J. Weichselbaum R.R. Cancer Res. 1997; 57: 1270-1275PubMed Google Scholar, 7Cai Z. Bettaieb A. El Mahdani N. Legres L.G. Stancou R. Masliah J. Chouaib S. J. Biol. 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GC has recently been shown to be associated with resistance to chemotherapy (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 13Cabot M.C. Giuliano A.E. Breast Cancer Res. Treat. 1997; 46 (abstr.): 293Google Scholar, 20Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 21Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticancer Res. 1998; 18: 475-480PubMed Google Scholar). Accumulation of GC is a characteristic of some MDR cancer cells and tumors derived from patients who are less responsive to chemotherapy (20Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 21Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticancer Res. 1998; 18: 475-480PubMed Google Scholar). Further studies have shown that MDR modulators of varying chemical structure inhibit the production of GC (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 21Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticancer Res. 1998; 18: 475-480PubMed Google Scholar). Although these studies document the accumulation of GC in multidrug-resistant cancer, little is known about the expression of GCS in MDR cells and its relationship to drug resistance. In particular, it is not known whether increased levels of GC are due to GCS gene expression or to other drug resistance factors that play a role in modulating GCS activation or GCS degradation. The retroviral Tet-off/Tet-on vector is a highly regulatory, versatile mammalian expression system (22Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2076) Google Scholar, 23Resnitzky D. Gossen M. Bujard H. Reed S.I. Mol. Cell. Biol. 1994; 14: 1669-1679Crossref PubMed Scopus (1006) Google Scholar, 24Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4324) Google Scholar, 25Yin D.X. Zhu L. Schimke R.T. Anal. Biochem. 1996; 235: 195-201Crossref PubMed Scopus (76) Google Scholar, 26Faris M. Kokot N. Lee L. Nel A. J. Biol. Chem. 1996; 271: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Expression of a target gene inserted in multiple cloning sites under CMV promoter control can be mediated simply by withdrawal of tetracycline or addition of doxycycline. Utilizing a retroviral Tet-on system, we established the MCF-7/GCS cell line from MCF-7 breast adenocarcinoma cells. The MCF-7/GCS-transfected cells express high levels of GCS activity, and they are resistant to cytotoxicity imparted by adriamycin and ceramide. [3H]UDP-glucose (40 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). EcoLume (liquid scintillation mixture) was from ICN (Costa Mesa, CA), and [α-32P]dCTP (6,000 Ci/mmol) was from Amersham Pharmacia Biotech. C6-Ceramide (N-hexanoylsphingosine) was purchased from LC Laboratories (Woburn, MA). Sulfatides (ceramide galactoside 3-sulfate) were from Matreya (Pleasant Gap, PA), and phosphatidylcholine (1, 2-dioleoyl-sn-glycero-3-phosphocholine) was from Avanti Polar Lipids (Alabaster, AL). C219, the monoclonal antibody against human P-glycoprotein, was from Signet Laboratories (Dedham, MA), and Bcl-2 (Ab-1) monoclonal antibody against human Bcl-2 was from Oncogene Research Products (Cambridge, MA). Hygromycin B was purchased from Boehringer Mannheim. Doxycycline hydrochloride, adriamycin (doxorubicin hydrochloride), and other chemicals were purchased from Sigma. FBS was purchased from HyClone (Logan, UT). RPMI 1640 medium and Dulbecco's modified Eagle's medium (high glucose) were from Life Technologies, Inc., and cultureware was from Corning-Costar (Cambridge, MA). Human breast adenocarcinoma cells, MCF-7 cells, and MCF-7 adriamycin-resistant cells (MDR clone) were kindly provided by Drs. Kenneth Cowan and Merrill Goldsmith (National Institutes of Health, NCI, Bethesda, MD). Cells were maintained in RPMI 1640 medium containing 10% (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 584 mg/literl-glutamine. Cells were cultured in a humidified, 5% CO2 atmosphere tissue culture incubator and subcultured weekly using a trypsin-EDTA (0.05%-0.53 mm) solution. Transfected cells, MCF-7/GCS, were cultured in RPMI 1640 medium containing 10% FBS and 200 μg/ml hygromycin in addition to the above components. pCG-2, a Bluescript II KS containing GlcT-1 (see Ref. 2Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar for terminology for GCS) in the EcoRI site was kindly provided by Drs. Shinichi Ichikawa and Yoshio Hirabayashi (Institute of Chemical and Physical Research, RIKEN, Saitama, Japan). The gene encoding human glucosylceramide synthase was immune-selected by monoclonal antibody M2590 from a human melanoma cell (SK-Mel-28) library (2Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar). The full-length cDNA of human GCS was subcloned into the EcoRI site in the pTRE, Tet-repressible expression plasmid. The Tet-on gene expression system was purchased fromCLONTECH (Palo Alto, CA). This system contains three vectors, pTet-on, pTRE, and pTK-Hyg. The pTet-on vector (pUHD17–1neo) expresses a doxycycline-controlled rtTA that is a fusion protein of a reverse Tet repressor and the C-terminal domain of protein 16 of herpes simplex virus, constitutively expressed under control of human CMV promoter (22Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2076) Google Scholar, 26Faris M. Kokot N. Lee L. Nel A. J. Biol. Chem. 1996; 271: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The pTRE vector (pUHD10–3) contains a multiple cloning site to accept any cDNA to be expressed followed by an SV40 polyadenylation sequence (24Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4324) Google Scholar). The promoter region upstream from the multiple cloning site contains a minimal human CMV promoter (P minCMV) with heptamerized tet operators. This promoter is silent in the absence of binding of rtTA to the tet operators. However, when the reverse Tet repressor of the rtTA binds to the tet operators, the virion protein 16 domain of the rtTA can activate P minCMV activity to a very high level and switch on expression of the target gene, GCS. Binding of doxycycline to the reverse Tet repressor domain of the rtTA can almost completely activate rtTA binding to the promoter (22Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2076) Google Scholar, 26Faris M. Kokot N. Lee L. Nel A. J. Biol. Chem. 1996; 271: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sense orientation of the GCS cDNA was analyzed with Vector NTI 4.0 and doubly checked by restriction enzyme digestion withHindIII and with XhoI plus NotI. The pTK-Hyg vector, which has a hygromycin-resistant gene under control of the mouse β-globin promoter, was used to select the stable transfectants. When MCF-7 cells reached 20% confluence, pTet-on DNA (10 μg/ml, 100-mm dish) was introduced by co-precipitation with calcium phosphate (Mammalian Transfection Kit, Stratagene, La Jolla, CA). The transfected cells were selected in RPMI 1640 medium containing 10% FBS and 400 μg/ml G418. Each G418-resistant clone was screened by luciferase assay, after transient transfection with pTRE-Luc vector containing the reporter gene, luciferase. pTK-Hyg (10 μg DNA) and pTRE-GCS (10 μg DNA) were introduced into the selected MCF-7 Tet-on cells by co-precipitation with calcium phosphate. The GCS-transfected cells were primarily selected in RPMI medium containing 10% FBS and 200 μg/ml hygromycin. As a control for transfection, MCF-7 Tet-on cells were co-precipitated with pTK-Hyg and pTRE plasmid without GCS cDNA. This procedure was performed as described previously (22Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2076) Google Scholar, 24Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4324) Google Scholar, 25Yin D.X. Zhu L. Schimke R.T. Anal. Biochem. 1996; 235: 195-201Crossref PubMed Scopus (76) Google Scholar). After MCF-7 cell transfection with pTet-on vector, each G418-resistant clone was grown for 16 h in 6-well plates (4000 cells/well) in 10% FBS RPMI medium, then shifted to 10% FBS Dulbecco's modified Eagle's medium. After a 6-h incubation, pTRE-Luc (1.5 μg of DNA) was introduced by co-precipitation with calcium phosphate. After culturing in 10% FBS Dulbecco's modified Eagle's medium for 18 h and in 10% FBS RPMI medium for 48 h, luciferase activity was measured using a commercial luciferase assay system according to the instruction manual (Promega, Madison, WI). Incubation for 48 h in 3.0 μg/ml doxycycline was used to induce expression of rtTA protein. Cellular extracts (100 μg of protein) from each clone were used. The activity of luciferase was measured by scintillation spectroscopy 2 min after the addition of substrate. MCF-7 cells transfected with pTRE-Luc were used as controls. To determine the expression of GCS in the hygromycin-resistant clones, a modified radioenzymatic assay was utilized (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 27Shukla G.S. Radin N.S. Arch. Biochem. Biophys. 1990; 293: 372-378Crossref Scopus (34) Google Scholar). After incubation in the absence or presence of doxycycline (3 μg/ml for 48 h), cells were homogenized by sonication in lysis buffer (50 mm Tris-HCl, pH 7.4, 1.0 μg/ml leupeptin, 10 μg/ml aprotinin, 25 μm phenylmethylsulfonyl fluoride). Microsomes were isolated by centrifugation (129,000 × g for 60 min). The enzyme assay, containing 50 μg of microsomal protein, in a final volume of 0.2 ml, was performed in a shaking water bath at 37 °C for 60 min. The reaction contained liposomal substrate composed of C6-ceramide (1.0 mm), phosphatidylcholine (3.6 mm; molecular weight, 786.15), and brain sulfatides (0.9 mm; molecular weight, 563). The liposomal substrate was prepared by mixing the components, evaporating the solvents under a stream of nitrogen, and sonicating in water over ice for 1 min using a microtip at 50% output (Kontes, Micro Ultrasonic Cell Disrupter). Other reaction components included sodium phosphate buffer (0.1m), pH 7.8, EDTA (2.0 mm), MgCl2(10 mm), dithiothreitol (1.0 mm), β-nicotinamide adenine dinucleotide (2.0 mm), and [3H]UDP-glucose (0.5 mm). Radiolabeled and unlabeled UDP-glucose were diluted to achieve the desired radiospecific activity (4700 dpm/nmol). To terminate the reaction, tubes were placed on ice, and 0.5 ml isopropanol and 0.4 ml Na2SO4 were added. After brief vortex mixing, 3 ml t-butyl methyl ether was added, and the tubes were mixed for 30 s. After centrifugation, 0.5 ml of the upper phase, which contained GC, was withdrawn and mixed with 4.5 ml of EcoLume for analysis of radioactivity by liquid scintillation spectroscopy. Analyses were performed as described previously (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Cellular lipids were radiolabeled by incubating cells with [3H]palmitic acid (2.5 μCi/ml culture medium) for 24 h. After removal of medium, cells were rinsed twice with phosphate-buffered saline (pH 7.4), and lipids were extracted (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). After nitrogen evaporation of solvents, total lipids were resuspended in 100 μl of chloroform/methanol (1:1, v/v), and aliquots were applied to TLC plates. Ceramide was resolved using solvent system I, which contained chloroform/acetic acid (90:10, v/v). GC was resolved using solvent system II, which contained chloroform/methanol/ammonium hydroxide (70:20:4, v/v). Commercial lipid standards were co-chromatographed. After development, lipids were visualized by iodine vapor staining, and areas of interest were scraped into 0.5 ml of water. EcoLume counting fluid (4.5 ml) was added, the samples were mixed, and radioactivity was quantitated by liquid scintillation spectrometry. RNA was extracted from cells using the single-step method described by Chomczynski and Sacchi (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65136) Google Scholar). Equal amounts of total RNA (15 μg) were denatured in 59% formamide/2.2m formaldehyde, size-separated by electrophoresis on 1% agarose-formaldehyde, and then blotted onto NitroPure nitrocellulose transfer membrane (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.43-7.48Google Scholar). GCS cDNA was prepared from pCG-2 plasmid, digested with EcoRI, and HindIII (Stratagene). The 1.1-kilobase pair fragment was then isolated by 1% low melt agarose electrophoresis using a commercial agarose gel DNA extraction kit (Boehringer Mannheim). Probing of 32P-GCS cDNA was performed by nick translation according to the instruction manual (Boehringer Mannheim). Nitrocellulose-plus membranes were hybridized with the 32P-GCS probe at 68 °C for 18 h. The filters were exposed at –70 °C for autoradiography. For even gel loading, 28 S RNA was used. The assay was performed as described previously (12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Briefly, after culture in the absence or presence of 3.0 μg/ml doxycycline for 48 h, cells were harvested and seeded in 96-well plates (2,000 cells/well), in 0.1 ml of RPMI 1640 medium containing 10% FBS in the absence or presence of 3.0 μg/ml doxycycline. Cultures were incubated at 37 °C for 24 h before addition of drug. Drugs were added in FBS-free medium (0.1 ml), and cells were cultured at 37 °C for the indicated periods. Drug cytotoxicity was determined using the Promega 96 Aqueous cell proliferation assay kit. Absorbance at 490 nm was recorded using an enzyme-linked immunosorbent assay reader (Molecular Devices, San Diego, CA). Western blots were performed using a modified procedure (30Yang J.M. Chin K.V. Hait W.N. Cancer Res. 1996; 56: 3490-3494PubMed Google Scholar, 31Blagosklonny M.V. Schulte T. Nguyen P. Trepel J. Neckers L.M. Cancer Res. 1996; 56: 1851-1854PubMed Google Scholar). Confluent cells were washed twice with phosphate-buffered saline containing 1.0 mmphenylmethylsulfonyl fluoride, and detached with trypsin-EDTA solution. Cells were pelleted by centrifuging at 500 × g for 5 min. Cell pellets were solubilized in 1.0 ml of cold TNT buffer (20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1% aprotinin) for 60 min with shaking. The insoluble debris was excluded by centrifugation at 12,000 × g for 45 min at 4 °C. The detergent soluble fraction was loaded in equal aliquots by protein and resolved using 4–20% gradient SDS-polyacrylamide gel electrophoresis. The transferred nitrocellulose blot was blocked with 3% fat-free milk powder in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween-20) at room temperature for 1 h. The membrane was then immunoblotted with monoclonal antibodies C219 (5 μg/ml) or Bcl-2 (Ab-1) (2.5 μg/ml) in Tris-buffered saline containing 0.5% bovine serum albumin (10 mm Tris-HCl, pH 8.0, and 150 mm NaCl) at 4 °C for 18 h. Detection was performed using ECL (Amersham Pharmacia Biotech). All data represent the mean ± S.D. Experiments were repeated two or three times. Student's ttest was used to compare mean values, and linear correlation between variables was tested using Pearson's correlation coefficient. MCF-7 cells were transfected with pTet vector and co-transfected with pTRE-GCS and pTK-Hyg. The stable, high expression clones were selected by screening GCS activity using the cell-free enzyme assay and by Northern blot. After transfection of pTet-on in MCF-7 cells, more than 30 G418-resistant clones were collected. Luciferase activity, which is a measure of expression of rtTA in the G418-resistant clones, was analyzed after 3 days of transient transfection with pTRE-luciferase vector. After stimulation with doxycycline, maximal expression of luciferase, 16,000-fold above that of MCF-7 cells, was found in clone 16. Luciferase activity in clone 16 in the absence of doxycycline was 15,000-fold higher than that of MCF-7 cells. Clone 1 demonstrated low basal rtTA expression; however, clone 1 was highly responsive to doxycycline, with induced luciferase activity that was 100-fold over MCF-7 cells. Clones 1 and 16 were selected as the optimal MCF-7 Tet-on clones for expression of rtTA. After co-transfection of pTRE-GCS and pTK-Hyg into clone 1 and clone 16 of MCF-7 Tet-on cells, 65 hygromycin-resistant clones were selected. Utilizing the [3H]UDP-glucose enzyme assay, we analyzed GCS activity and identified three clones that exhibited 5–11-fold increases in enzyme activity (Fig.1 A). Compared with a basal level of 17.2 ± 0.1 pmol of GC in MCF-7 wt cells, doxycycline-induced GCS activity in MCF-7/GCS12, MCF-7/GCS13, and MCF-7/GCS14 was 167.4 ± 17.2, 183.3 ± 12.4, and 90.2 ± 2.76 pmol of GC, respectively (Fig. 1 A). There were no differences in either basal or doxycycline-induced GCS activity in transfection control cells (TC) or in the basal level of GCS in MCF-7 wt cells (Fig.1 A). In MCF-7/GCS13 and MCF-7/GCS14, the doxycycline-inducible GCS activities were 1.6- and 4.1-fold, respectively, above untreated cells. The MCF-7/GCS14 clone was designated MCF-7/GCS, and this clone was used in further experiments. Doxycycline-induced GCS mRNA was highly elevated in MCF-7/GCS cells compared with doxycycline-naive MCF-7/GCS cells. A representative Northern blot is shown in Fig. 1 B. Only traces of GCS mRNA were observed in MCF-7 cells, TC, and MCF-7/GCS cells without doxycycline (Fig. 1 B). The levels of ceramide and GC in MCF-7 and in MCF-7/GCS cells were assessed by steady-state radiolabeling of cultured cells using [3H]palmitic acid. As shown in Fig. 1 C, transfection with GCS elicited only a moderate decrease in ceramide, compared with MCF-7 cells. The decrease was not statistically significant. GC in MCF-7/GCS compared with MCF-7 cells increased slightly and accounted for 1.8 and 1.5%, respectively, of total cellular radiolabeled lipid. Recent work has revealed that effects of therapeutic doses of anthracyclines are closely related to the generation of ceramide, and elevated GC has been shown to be associated with adriamycin resistance in MDR cells (9Bose R. Verheil M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (794) Google Scholar, 11Zyad A. Benard J. Tursz T. Clarke R. Chouaib S. Cancer Res. 1994; 54: 825-831PubMed Google Scholar, 12Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 13Cabot M.C. Giuliano A.E. Breast Cancer Res. Treat. 1997; 46 (abstr.): 293Google Scholar). Adriamycin was used to assess the influence of GCS transfection on cellular response to anthracyclines. After pretreatment with doxycycline for 2 days, MCF-7/GCS cells were incubated with increasing concentrations of adriamycin for 4 days. Fig.2 A shows that MCF-7/GCS cells, compared with MCF-7 cells, are resistant to adriamycin toxicity. At 0.5, 1.0, 2.0, and 3.0 μm adriamycin, survival of transfected MCF-7/GCS cells was significantly greater than that of MCF-7 cells (p < 0.0005, Fig. 2 A). As presupposed, it was observed that MCF-7/GCS cells were also resistant to ceramide toxicity. At 2.5 and 5.0 μmC6-ceramide, MCF-7/GCS cell survival was significantly higher than that of MCF-7 cells (p < 0.0005, Fig.2 B). The EC50 of adriamycin in MCF-7/GCS cells was approximately 11 times greater than the EC50 observed in MCF-7 cells (4.01 ± 0.12 versus 0.37 ± 0.01 μm, p < 0.0005, Fig. 2 C). However, the EC50 in the TC group was nearly identical with that of MCF-7 cells, and there was no statistical difference between the two groups (Fig. 2 C). The EC50 of C6-ceramide in MCF-7/GCS cells was 4-fold greater than that observed in MCF-7 cells (12.07 ± 1.50 versus 3.10 ± 0.50 μm, p < 0.0005, Fig.2 C), and survival of TC cells was not statistically different from that of the parent cell line, MCF-7 (Fig.2 C). If ceramide resistance is induced by GCS expression in MCF-7/GCS cells, the resistance response should be tightly correlated with the level of the inducer, doxycycline. We found that increasing doxycycline concentrations correlated closely with increased expression of GCS, which in turn correlated well with increased resistance of the cells to C6-ceramide. After cells were exposed to increasing concentrations of doxycycline, higher expression of GCS mRNA was observed in MCF-7/GCS cells with 1.0 and 3.0 μg/ml doxycycline (Fig.3 A); the densities were 97 and 256, respectively (GCS band/28 S RNA × 100). In contrast, the mRNA was scarcely detectable at 0 and 0.1 μg/ml doxycycline, with densities measuring 16 and 18, respectively. Only traces of GCS mRNA were found in MCF-7 cells treated with doxycycline (Fig.3 A). GCS activity in MCF-7/GCS cells exposed to 0.1, 1.0, and 3.0 μg/ml doxycycline was signifi
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