Uncoupling Ceramide Glycosylation by Transfection of Glucosylceramide Synthase Antisense Reverses Adriamycin Resistance
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.7138
ISSN1083-351X
AutoresYu Liu, Tie-Yan Han, Armando E. Giuliano, Nora Hansen, Myles C. Cabot,
Tópico(s)Adenosine and Purinergic Signaling
ResumoPrevious work from our laboratory demonstrated that increased competence to glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cancer cells (Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140–1146). This was achieved by cellular transfection with glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide. With this, we hypothesized that a decrease in cellular ceramide glycosylation would result in heightened drug sensitivity and reverse adriamycin resistance. To down-regulate ceramide glycosylation potential, we transfected adriamycin-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new cell line, MCF-7-AdrR/asGCS. Reverse transcription-polymerase chain reaction assay and Western blot analysis revealed marked decreases in both GCS mRNA and protein in MCF-7-AdrR/asGCS cells compared with the MCF-7-AdrR parental cells. MCF-7-AdrR/asGCS cells exhibited 30% less GCS activity by in vitro enzyme assay (19.7 ± 1.1 versus 27.4 ± 2.3 pmol GC/h/μg protein,p < 0.001) and were 28-fold more sensitive to adriamycin (EC50, 0.44 ± 0.01 versus12.4 ± 0.7 μm, p < 0.0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C6-ceramide compared with parental cells (EC50= 4.0 ± 0.03 versus 9.6 ± 0.5 μm,p < 0.0005). Under adriamycin stress, GCS antisense transfected cells compared with parental cells displayed time- and dose-dependent increases in endogenous ceramide and dramatically higher levels of apoptotic effector, caspase-3. Western blotting showed that adriamycin sensitivity, introduced by asGCS gene transfection, was independent of P-glycoprotein and Bcl-2 expression. In summary, this work shows that transfection of GCS antisense tempers the expression of native GCS and restores cell sensitivity to adriamycin. Therefore, limiting the potential to glycosylate ceramide, which is an apoptotic signal in chemotherapy and radiotherapy, provides a promising approach to combat drug resistance. Previous work from our laboratory demonstrated that increased competence to glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cancer cells (Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140–1146). This was achieved by cellular transfection with glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide. With this, we hypothesized that a decrease in cellular ceramide glycosylation would result in heightened drug sensitivity and reverse adriamycin resistance. To down-regulate ceramide glycosylation potential, we transfected adriamycin-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new cell line, MCF-7-AdrR/asGCS. Reverse transcription-polymerase chain reaction assay and Western blot analysis revealed marked decreases in both GCS mRNA and protein in MCF-7-AdrR/asGCS cells compared with the MCF-7-AdrR parental cells. MCF-7-AdrR/asGCS cells exhibited 30% less GCS activity by in vitro enzyme assay (19.7 ± 1.1 versus 27.4 ± 2.3 pmol GC/h/μg protein,p < 0.001) and were 28-fold more sensitive to adriamycin (EC50, 0.44 ± 0.01 versus12.4 ± 0.7 μm, p < 0.0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C6-ceramide compared with parental cells (EC50= 4.0 ± 0.03 versus 9.6 ± 0.5 μm,p < 0.0005). Under adriamycin stress, GCS antisense transfected cells compared with parental cells displayed time- and dose-dependent increases in endogenous ceramide and dramatically higher levels of apoptotic effector, caspase-3. Western blotting showed that adriamycin sensitivity, introduced by asGCS gene transfection, was independent of P-glycoprotein and Bcl-2 expression. In summary, this work shows that transfection of GCS antisense tempers the expression of native GCS and restores cell sensitivity to adriamycin. Therefore, limiting the potential to glycosylate ceramide, which is an apoptotic signal in chemotherapy and radiotherapy, provides a promising approach to combat drug resistance. glucosylceramide glucosylceramide synthase (ceramide glucosyltransferase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) GCS antisense fetal bovine serum MCF-7 adriamycin-resistant cells MCF-7-AdrR GCS antisense-transfected cells reverse transcription polymerase chain reaction phosphate-buffered saline Ceramide, now recognized as a second messenger in cellular apoptotic signaling events, has been shown to play a role in chemotherapy and radiotherapy of cancer (1.Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (730) Google Scholar, 2.Hannun Y.A. Blood. 1997; 89: 1845-1853Crossref PubMed Google Scholar). Loss of ceramide production is one cause of cellular resistance to apoptosis induced by either ionizing radiation or tumor necrosis factor-α and adriamycin (2.Hannun Y.A. Blood. 1997; 89: 1845-1853Crossref PubMed Google Scholar, 3.Chuma S.J. Nodzenski E. Beckett M.A. Kufe D.W. Quintans J. Weichselbaum R.R. Cancer Res. 1997; 57: 1270-1275PubMed Google Scholar, 4.Bose R. Verheil M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar, 5.Cai Z. Bettaieb A. El Mahdani N. Legres L.G. Stancou R. Masliah J. Chouaib S. J. Biol. Chem. 1997; 272: 6918-6926Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 6.Santana 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 (727) Google Scholar, 7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Accumulation of glucosylceramide (GC),1 a simple glycosylated form of ceramide, is a characteristic of some multidrug-resistant cancer cells and tumors derived from patients who are less responsive to chemotherapy (8.Lavie Y. Cao H.T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 9.Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticarcer Res. 1998; 18: 475-480PubMed Google Scholar). The study of GC metabolism, as a molecular determinant of the drug-resistant phenotype, has been a subject of recent attention. Modification of ceramide metabolism by blocking the glycosylation pathway has been shown to increase cancer cell sensitivity to cytotoxics (10.Lucci A. Han T.Y. Liu Y.Y. Giuliano A.E. Cabot M.C. Int. J. Oncol. 1999; 15: 541-546PubMed Google Scholar, 11.Lavie Y. Cao H. Volner A. Lucci A. Han T.-H. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 12.Lucci A. Han T.Y. Liu Y.Y. Giuliano A.E. Cabot M.C. Cancer. 1999; 86: 299-310Crossref Scopus (83) Google Scholar). Further, drug combinations that enhance ceramide generation and limit glycosylation have been shown to enhance kill in cancer cell models (11.Lavie Y. Cao H. Volner A. Lucci A. Han T.-H. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 12.Lucci A. Han T.Y. Liu Y.Y. Giuliano A.E. Cabot M.C. Cancer. 1999; 86: 299-310Crossref Scopus (83) Google Scholar). Other work has shown that ceramide toxicity can be potentiated in experimental metastasis of murine Lewis lung carcinoma and human neuroepithelioma cells by inclusion of a glucosylceramide synthase inhibitor (13.Inokuchi J. Jimbo M. Momosaki K. Shimeno H. Nagamatsu A. Radin N. Cancer Res. 1990; 50: 6731-6737PubMed Google Scholar, 14.Spinedi A. Bartolomeo S.D. Piacentini M. Cell Death Differ. 1998; 5: 785-791Crossref PubMed Scopus (40) Google Scholar). These findings assign biological significance to ceramide metabolism as it relates to circumvention of resistance to antineoplastic agents. The increased capacity for ceramide glycosylation in GCS-transfected human breast cancer cells conferred resistance to adriamycin and to tumor necrosis factor-α (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15.Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Crossref PubMed Scopus (73) Google Scholar). Both agents are known to activate ceramide generation and potentiate apoptosis (1.Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (730) Google Scholar, 2.Hannun Y.A. Blood. 1997; 89: 1845-1853Crossref PubMed Google Scholar, 7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15.Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Crossref PubMed Scopus (73) Google Scholar). From this, we hypothesized that transfection of asGCS, to limit cellular ceramide glycosylation, would overcome adriamycin resistance. By introducing asGCS to modulate GCS activity in adriamycin-resistant human breast cancer cells, we successfully decreased native GCS expression and restored cellular sensitivity to adriamycin and to C6-ceramide. The present study shows further that ceramide generation is a major factor in the cytotoxicity of adriamycin and suggests that asGCS would be a novel force to overcome adriamycin resistance. [3H]UDP-glucose (40 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). 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). Adriamycin (doxorubicin hydrochloride) and other chemicals were purchased from Sigma. FBS was purchased from HyClone (Logan, UT). RPMI medium 1640 and Dulbecco's modified Eagle's medium (high glucose) were from Life Technologies, Inc., and cultureware was from Corning Costar (Cambridge, MA). GCS antiserum (from rabbit) was kindly provided by Drs. D. L. Marks and R. E. Pagano (Mayo Clinic and Foundation, Rochester, MN). Anti-Xpress tag antibody was from Invitrogen (Carlsbad, CA). C219, the monoclonal antibody against P-glycoprotein, was from Signet Laboratories (Dedham, MA), and Bcl-2 monoclonal antibody (Ab-1) against human Bcl-2 was from Oncogene Research Products (Cambridge, MA). The human breast adenocarcinoma cell line, MCF-7-AdrR, which is resistant to adriamycin (16.Cowan K.H. Bastist G. Tulpule A. Sinha B.K. Myers C.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9328-9332Crossref PubMed Scopus (282) Google Scholar), was kindly provided by Dr. Kenneth Cowan and Dr. Merrill Goldsmith (NCI, National Institutes of Health, 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 trypsin-EDTA (0.05%, 0.53 mm) solution. The stably transfected cells, MCF-7-AdrR/asGCS, were cultured in RPMI 1640 medium containing 400 μg/ml G418 (geneticin) in addition to the above components. Giemsa staining was performed as described (17.Freshney R.I. Culture of Animal Cells: A Manual of Basic Technique. 3rd Ed. Wiley-Liss, Inc., New York1994Google Scholar). Cells were seeded in 60-mm dishes (105 cells/dish) in 10% FBS RPMI 1640 medium and grown for 2 days at 37 °C. After rinsing with PBS, cells were fixed with 50% methanol/PBS, followed by methanol, and stained with KaryoMAX Giemsa stain stock solution (Life Technologies, Inc.). Following washing with deionized water, cells were photomicrographed. The population doubling time of each cell line was measured. Briefly, cells were seeded in 24-well plates (104 cells/well) in 10% FBS RPMI 1640 medium and grown for 24-, 48-, 72-, and 96-h periods. After rinsing with PBS, cells were dispersed with trypsin/EDTA, suspended in medium, and counted by hemocytometer. pCG-2, a Bluescript II KS containing GlcT-1(Ref. 18.Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (220) Google Scholar; terminology for GCS) in the EcoRI site, was kindly provided by Dr. Shinichi Ichikawa and Dr. Yoshio Hirabayashi (The Institute of Chemical and Physical Research, Saitama, Japan). The full-length cDNA of human GCS was subcloned into theEcoRI site in the pcDNA 3.1/His A with XpressTM tag peptide (Invitrogen) in the upstream region. Xpress tag fuses at the N terminus of the cloned gene; therefore, GCS will be expressed as Xpress-GCS. The antisense and sense orientation of GCS cDNA was analyzed with Vector NTI 4.0 and doubly checked by restriction digestion. When MCF-7-AdrR cells reached 20% confluence, pcDNA 3.1-asGCS or pcDNA 3.1-GCS (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, isolated utilizing cloning cylinders, was propagated and later screened by GCS enzyme assay. pcDNA 3.1/his A plasmid, without GCS DNA, was used in control transfection. To determine the levels of GCS in the G418-resistant clones, a modified radioenzymatic assay was utilized (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 19.Shukla G.S. Radin N.S. Arch. Biochem. Biophys. 1990; 283: 372-378Crossref PubMed Scopus (33) Google Scholar). 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, 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), and brain sulfatides (0.9 mm). Other reaction components included sodium phosphate buffer (0.1 m), pH 7.8, EDTA (2.0 mm), MgCl2 (10 mm), dithiothreitol (1.0 mm), β-NAD (2.0 mm), and [3H]UDP-glucose (0.5 mm). Radiolabeled and unlabeled UDP-glucose were diluted to achieve the desired radiospecific activity (4,700 dpm/nmol). To terminate the reaction, tubes were placed on ice, and 0.5 ml of isopropanol and 0.4 ml of Na2SO4 were added. After brief vortex mixing, 3 ml of t-butyl methyl ether was added, and the tubes were mixed for 30 s. After centrifugation, 0.5 ml of upper phase, which contained GC, was withdrawn and mixed with 4.5 ml of EcoLume for analysis of radioactivity by liquid scintillation spectroscopy. Cellular mRNA was purified using a mRNA isolation kit (Roche Molecular Biochemicals). Equal amounts of mRNA (5.0 ng) were used for RT-PCR. Under upstream primer (5′-CCTTTCCTCTCCCCACCTTCCTCT-3′) and downstream primer conditions (5′-GGTTTCAGAAGAGAGACACCTGGG-3′), a 302-base pair fragment in the 5′-terminal region of the GCS gene was produced using the ProSTAR HF single-tube RT-PCR system (High Fidelity, Stratagene) in a thermocycler (Mastercycler Gradient, Eppendorf). mRNAs were reverse transcribed using Moloney murine leukemia virus reverse transcriptase at 42 °C for 15 min. DNA was amplified with TaqPlus Precision DNA polymerase in a 40-cycle PCR reaction, using the following conditions: denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 68 °C for 120 s. RT-PCR products were analyzed by 1% agarose gel electrophoresis stained with ethidium bromide. β-Actin (Life Technologies, Inc.) was used as control for even loading. Assays were performed as described previously (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 11.Lavie Y. Cao H. Volner A. Lucci A. Han T.-H. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Briefly, cells were seeded in 96-well plates (2,000 cells/well) in 0.1 ml RPMI 1640 medium containing 10% FBS and cultured 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 (Promega, Madison, WI). Absorbance at 490 nm was recorded using a Microplate Fluorescent Reader, model FL600 (Bio-Tek, Winooski, VT). Analysis was performed as described previously (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 8.Lavie Y. Cao H.T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Cells were seeded in 6-well plates (60,000 cells/well) in 10% FBS RPMI 1640 medium. After 24 h, cells were shifted to 5% FBS medium with or without adriamycin and grown for the indicated times. Cellular lipids were radiolabeled by adding [3H]palmitic acid (2.5 μCi/ml culture medium) for 24 h. After removal of medium, cells were rinsed twice with PBS (pH 7.4), and total lipids were extracted as described (8.Lavie Y. Cao H.T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The resulting organic lower phase was withdrawn and evaporated under a stream of nitrogen. Lipids were resuspended in 100 μl of chloroform/methanol (1:1, v/v), and aliquots were applied to TLC plates. Ceramide was resolved using a solvent system containing chloroform/acetic acid (90:10, v/v). Commercial lipid standards were co-chromatographed. After development, lipids were visualized by iodine vapor staining, and the ceramide area was 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. Caspase-3 activity was assayed by DEVD-AFC cleavage, using the ApoAlert Caspase-3 assay kit (CLONTECH, Palo Alto, CA). The assay was performed as described previously (15.Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Crossref PubMed Scopus (73) Google Scholar). Cells were seeded in 100-mm dishes (500,000 cells/dish) in 10% FBS RPMI 1640 medium. After 24 h, cells were shifted to 5% FBS RPMI 1640 medium without or with adriamycin and grown for 24 and 48 h. Following harvest, cells (106/vial) were lysed on ice for 10 min with 50 μl of lysis buffer, and cell debris was removed by centrifugation at 4 °C at 10,000 × g for 5 min. The soluble fraction was incubated with 50 μm conjugated substrate DEVD-AFC in a 100-μl reaction volume at 37 °C for 60 min. The free AFC fluoresce was measured at λexcitation 400 nm and λemission 505 nm using a FL600 Microplate Fluorescence Reader. The caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde, was used to exclude nonspecific background in the enzymatic reaction. Western blots were performed using a modified procedure (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15.Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Crossref PubMed Scopus (73) Google Scholar, 20.Watanabe R. Wu K. Paul P. Marks D.L. Kobayashi T. Pittelknow M.R. Pagano R.E. J. Biol. Chem. 1998; 273: 9651-9655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Confluent cell monolayers were washed twice with PBS containing 1.0 mm phenylmethylsulfonyl fluoride and detached with trypsin-EDTA solution. Cells, pelleted by centrifugation, were solubilized in 1.0 ml of cold TNT buffer (20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1.0% Triton X-100, 1.0 mm phenylmethylsulfonyl fluoride, 1.0% 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 blot was blocked (3% fat-free milk powder in 10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween-20) and was immunoblotted with GCS antiserum (1:1000) in binding solution (0.5% bovine serum albumin in 10 mmTris-HCl, pH 8.0, 150 mm NaCl) at 4 °C for 18 h. To detect Xpress tag, P-glycoprotein, and Bcl-2, the antibodies of anti-Xpress tag (1:500), C219 (5 μg/ml), and Ab-1 (2.5 μg/ml), respectively, were used in place of GCS antiserum. Detection employing enzyme-linked chemiluminescence was performed using ECL (Amersham Pharmacia Biotech). All data represent the means ± S.D. Experiments were repeated two or three times. Student's ttest was used to compare mean values. The structure of pcDNA 3.1/his A-asGCS is shown in Fig.1 A. The GCS antisense was cloned into the EcoRI site, just downstream from the anti-Xpress tag sequence in pcDNA 3.1/his A. This plasmid was introduced into MCF-7-AdrR cells by calcium phosphate coprecipitation. G418 was used to select transfectants. We found that the number of G418-resistant clones in MCF-7-AdrR asGCS transfected cells was much lower than in MCF-7-AdrR cells transfected with pcDNA3.1/his A vector (54/106 versus 251/106). G418-resistant clones were further selected by measuring GCS activity using the cell-free radioenzymatic assay. In all, fifty-four G418-resistant clones of MCF-7-AdrR asGCS-transfected cells were obtained, and we identified one clone that exhibited a stable 30% decrease in GCS activity (Fig. 1 B). Compared with 27.4 ± 2.3 pmol of GC synthesized by MCF-7-AdrR parental cells, GCS activity in MCF-7-AdrR/asGCS was decreased to 19.7 ± 1.1 pmol of GC (Fig. 1 B, p < 0.001). There were no differences in GCS activities between the pcDNA 3.1/his A vector-transfected cells and parental MCF-7-AdrR cells (Fig.1 B). The asGCS-transfected and parental MCF-7-AdrR cells were stained with Giemsa. Representative photomicrographs are shown in Fig.1 C. MCF-7-AdrR/asGCS cells, including nuclei, are flatter and larger than the dome-shaped, more stellate MCF-7-AdrR cells. The asGCS cell line is also more cuboidal with less dense cytoplasm. The population doubling times for both cell lines were similar, 32 and 30 h for MCF-7-AdrR/asGCS and MCF-7-AdrR cells, respectively. Consistent with diminished GCS activity, GCS mRNA and GCS protein were reduced in MCF-7-AdrR/asGCS cells, compared with MCF-7-AdrR cells. Total mRNA was isolated from both cell lines and reverse transcribed and amplified through RT-PCR. A representative RT-PCR gel electropherograph is shown in Fig.2 A. As with that revealed by densitometric scanning, the mRNA in MCF-7-AdrR/asGCS cells was reduced 3-fold compared with that in MCF-7-AdrR cells (25.4%versus 77.5% of β-actin). GCS protein in cell lysates was resolved by SDS-polyacrylamide gel electrophoresis and identified using GCS antiserum. Western blotting showed that the total amount of GCS protein in MCF-7-AdrR/asGCS cells decreased by 32% compared with MCF-7-AdrR parental cells (77,520 and 112,860 optical density units, respectively) (Fig. 2 B, right and center bands). However, MCF-7-AdrR cells that were transfected with pcDNA 3.1/his A-GCS expressed greater amounts of GCS (Fig.2 B, left band, AdrR/GCS). MCF-7-AdrR/GCS cells were developed by stable transfection of sense orientation pcDNA 3.1/his A-GCS vector in MCF-7-AdrR cells. This GCS-transfected cell line displays 80% higher GCS activity than MCF-7-AdrR cells as measured by radioenzymatic assay. After transfection with pcDNA 3.1/his A-GCS vector, although the expressed GCS was fused with Xpress tag (-Asp-Leu-Tyr-Asp-Asp-Asp-Lys-), the upward shift in molecular mass (about 800 daltons) was undetectable by Western blot (Fig.2 B). To evaluate the expression of transfected GCS antisense gene, we employed a Xpress antibody to detect the production of Xpress-GCS fused protein (Fig. 1 A). We did not find the GCS-Xpress tag in either MCF-7-AdrR or MCF-7-AdrR/asGCS cells (Fig.2 C). However, the tag protein was highly expressed in MCF-7-AdrR GCS transfected cells (Fig. 2 C, center band). In MCF-7-AdrR/asGCS cells, what appears to be the Xpress-asGCS protein (Fig. 2 C, faint band) had a higher molecular mass compared with Xpress-GCS protein of MCF-7-AdrR/GCS and was present at only 15% the level of the latter (Fig. 2C, center band). Previous work from our laboratory revealed that overexpression of GCS elicits adriamycin resistance (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). After transfection of GCS antisense, we used adriamycin to assess the influence of antisense on cellular response to anthracyclines. Parental and antisense transfected cell lines were treated with increasing concentrations of adriamycin for a three day period. Fig.3 A shows that MCF-7-AdrR/asGCS cells, compared with MCF-7-AdrR cells, were markedly more sensitive to adriamycin. At concentrations of 0.5 μm and higher, survival of MCF-7-AdrR/asGCS cells was significantly lower than MCF-7-AdrR cells (p < 0.0001, Fig. 3 A). The amount of drug provoking 50% cell death (EC50) was determined. The EC50 of adriamycin decreased 28-fold in MCF-7-AdrR/asGCS cells (0.44 ± 0.01 versus 12.4 ± 0.7 μm, p < 0.0001, Fig.3 B). As expected, we observed that MCF-7-AdrR/asGCS cells were also sensitive to ceramide. At higher concentrations of C6-ceramide (5–10 μm), MCF-7-AdrR/asGCS cell survival was significantly lower than MCF-7-AdrR cells (p < 0.0001). The EC50 of C6-ceramide in MCF-7-AdrR/asGCS cells was 2.4-fold less than that observed in MCF-7 AdrR cells (4.0 ± 0.03versus 9.6 ± 0.5 μm, p< 0.0005, Fig. 3 B). To further elucidate the dynamics of ceramide metabolism in drug sensitivity, we measured ceramide generation in the two cell lines. We found that adriamycin exposure dramatically elevated ceramide levels in GCS antisense-transfected cells. As shown in Fig.4, adriamycin treatment increased the levels of ceramide in MCF-7-AdrR/asGCS cells in a time- and dose-dependent manner. At 24 and 48 h post-treatment, ceramide levels in MCF-7-AdrR/asGCS cells increased 200 and 250%, respectively (Fig. 4 A). In sharp contrast, adriamycin treatment did not greatly modify ceramide levels in MCF-7-AdrR cells, which at 48 h increased only 16% above control. The result of increasing adriamycin dose on ceramide metabolism in the cell lines is shown in Fig. 4 B. Adriamycin at 0.5, 1.0, and 2.5 μm enhanced ceramide levels by 181, 188, and 246%, respectively, in MCF-7-AdrR/asGCS cells (Fig. 1 B), whereas MCF-7-AdrR cells displayed minimal response over the same dose range. In mammalian cells, ceramide induces apoptosis directly through effector caspases, such as caspase-3 (21.Yoshimura S. Banno Y. Nakashima S. Takenaka K. Sakai H. Nishimura Y. Sakai N. Shimizu S. Eguchi Y. Tsujimoto Y. Nozawa Y. J. Biol. Chem. 1998; 273: 6921-6927Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22.Monney L. Olivier R. Otter I. Jansen B. Poirier G.G. Borner C. Eur. J. Biochem. 1998; 251: 295-303Crossref PubMed Scopus (100) Google Scholar). To identify whether an alteration in ceramide metabolism in asGCS cells is related to adriamycin sensitivity via signal cascades, we analyzed caspase-3 activity in the parental and transfected cell lines. The data demonstrate that increased effector caspase-3 activity is consistent with changes in ceramide metabolism. At 10 μm adriamycin, the EC50 in MCF-7-AdrR cells, caspase-3 activity in MCF-7-AdrR/asGCS increased 290 and 980% over control, at 24 and 48 h, respectively (Fig. 5). In contrast, adriamycin treatment increased caspase-3 by 160% in MCF-7-AdrR cells, albeit only at 48 h (Fig. 5). In summary, caspase-3 activity in the GCS antisense-transfected cells was 3- and 6-fold greater in response to adriamycin treatment than observed in parental cells (p < 0.0001). This suggests that impaired GCS activity permits cells to maintain high levels of ceramide under adriamycin stress, activating caspase-3 for progression of programmed cell death. Because GCS antisense transfection resulted in enhanced drug sensitivity, we evaluated the expression of P-glycoprotein and Bcl-2. A representative Western blot of P-glycoprotein is shown in Fig.6 A. P-glycoprotein was found only in trace amounts in MCF-7 cells (adriamycin sensitive). Decreased expression of P-glycoprotein was not evident in MCF-7-AdrR/asGCS cells, when compared with the parent MCF-7-AdrR cell line (Fig.6 A). Bcl-2 was found only in trace amounts in MCF-7-AdrR and in MCF-7-AdrR/asGCS cells (Fig. 6 B), although Bcl-2 was highly expressed in MCF-7 cells, consistent with our prior finding (7.Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). We have introduced GCS antisense DNA into chemotherapy-resistant cancer cells and revealed that this transfection reverses cellular resistance to adriamycin and to C6-ceramide in the resulting MCF-7-AdrR/asGCS cell line. The parent line, MCF-7-AdrR was selected from MCF-7 cells by culturing in the presence of adriamycin (16.Cowan K.H. Bastist
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