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• W2100076132 abstract "Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246–6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis. Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246–6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis. GlcCer 1The abbreviations used are: GlcCer, glucosyl ceramide; MDR1, multi-drug resistance protein 1 (P-glycoprotein); MRP1, multidrug resistance protein 1; CsA, cyclosporin A; GSL, glycosphingolipid; Cer, ceramide; LacCer, lactosyl ceramide (Galβ1–4Glc ceramide); GM3, monosialoganglioside (SAα2–3Galβ1–4Glc ceramide); Gb3, globotriaosyl ceramide (Galα1–4Galβ1–4Glc ceramide); Gb4, globotetraosyl ceramide (GalNacβ1–3Galα1–4Galβ1–4Glc ceramide); CTH, ceramide trihexoside; ER, endoplasmic reticulum; KOT, knock-out; VT1, verotoxin 1; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MDCK, Madin-Darby canine kidney cells. is the precursor of the majority of eukaryotic GSLs (1Stults C.L.M. Sweeley C.H. Macher B.A. Methods Enzymol. 1989; 179: 167-214Crossref PubMed Scopus (234) Google Scholar). The UDP galactose β1–4 glucosyl ceramide glycosyltransferase (LacCer synthase) within the Golgi lumen then provides the common precursor, LacCer for the synthesis of most complex GSL. Drug-resistant tumor cells have been shown to increase GlcCer synthesis (2Lavie 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 (310) Google Scholar, 3Lucci A. Cho W. Han T. Giuliano A. Morton D. Cabot M. Anticancer Res. 1998; 18: 475-480PubMed Google Scholar), and this has been postulated as a mechanism for drug resistance by providing an alternative biosynthetic destination for ceramide, which might otherwise induce apoptosis (4Liu 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 (240) Google Scholar). GlcCer is synthesized from ER-derived ceramide, on the cytosolic cis-Golgi surface (5Coste H. Martel M.B. Got R. Biochim. Biophys. Acta. 1986; 858: 6-12Crossref PubMed Scopus (114) Google Scholar, 6Jeckel D. Karrenbauer A. Burger K.N.J. van Meer G. Wieland F. J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (259) Google Scholar, 7Warnock D. Lutz M. Blackburn W. Young W. Baenziger J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2708-2712Crossref PubMed Scopus (107) Google Scholar), and may be transported to the plasma membrane from this site (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). However, the active sites of the glycosyltransferases responsible for further elongation of GSLs are located within the Golgi lumen (9Lannert H. Bunning C. Jeckel D. Wieland F. FEBS Lett. 1994; 342: 91-96Crossref PubMed Scopus (88) Google Scholar, 10Burger K. van der Bijl P. van Meer G. J. Cell Biol. 1996; 133: 15-28Crossref PubMed Scopus (90) Google Scholar). Thus, a mechanism for the translocation of GlcCer from the cytosolic surface of the Golgi to the lumen must exist. We have shown that transfection of cells with the mdr1 gene results in a marked elevation of GlcCer, LacCer, and Gb3 levels with a concomitant increase in cell sensitivity to the Gb3-binding verotoxin 1. Both effects were reversed in the presence of MDR1 inhibitors (11Lala P. Ito S. Lingwood C.A. J. Biol. Chem. 2000; 275: 6246-6251Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We proposed that MDR1 can function as a GlcCer translocase to flip GlcCer to the inner Golgi surface to enhance LacCer and subsequent Gb3 synthesis. MDR1 has been shown to function as a membrane translocase for chemically modified short chain fatty acid GlcCer (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). However such derivatized, indicator GSLs have been considered possible drug substrates for MDR1, and the ability of MDR1 to translocate natural, long-chain GSLs has been questioned on the basis of lack of overt phenotype in MDR1 knockout mice (12Borst P. Zelcer N. van Helvoort A. Biochim. Biophys. Acta. 2000; 1486: 128-144Crossref PubMed Scopus (263) Google Scholar). In the present study, we show that fibroblasts from such mice are indeed defective in GSL synthesis, in the manner predicted from the in vitro MDR1 inhibition studies now reported. In this study, we address the question as to whether MDR1, which is widely expressed in normal tissues (13Lum B.L. Gosland M.P. Hematol. Oncol. Clin. North Am. 1995; 9: 319-336Abstract Full Text PDF PubMed Google Scholar), is involved in GSL synthesis in cultured cells in general. These studies fill a major gap in understanding the mechanism of GSL biosynthesis and demonstrate a new, unsuspected distinction between acidic and neutral GSL synthesis. MDCK cells transfected with the human MDR1 cDNA were a gift from Dr. M. Gottesman (National Institutes of Health, Bethesda, MD) (14Pastan I. Gottesman M. Ueda K. Lovelace E. Rutherford A. Willingham M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4486-4490Crossref PubMed Scopus (476) Google Scholar). The human astrocytoma cell line SF-539, and IOMM Lee and CH157 MN meningioma cell lines were kindly provided by Dr. J. Rutka (Hospital for Sick Children, Toronto, Canada). Vero, HEp-2, SF-539 astrocytoma, ovarian carcinoma SK VLB (MDR variant of the parental SKOV3 cell line (15Bradley G. Naik M. Ling V. Canc. Res. 1989; 49: 2790-2796PubMed Google Scholar)), MDR1-MDCK cells were maintained in α-minimal essential medium supplemented with 5% or 10% FBS and 40 μg/ml gentamicin. MDR1-MDCK medium also contained 80 ng/ml colchicine, and SK VLB medium contained 1 μg/ml vinblastine. HeLa cells, NIH 3T3 cells, and meningioma IOMM Lee and CH157 MN cell lines were maintained in Dulbecco's modified Eagle's medium (with 4.5 gm/liter of glucose for HeLa and 3T3, and with 1 gm/liter of glucose for meningioma cell lines) with 10% FBS. Daudi Burkitt's lymphoma and Jurkat E6.1 cells were maintained in RPMI 1640 medium also supplemented with 10% FBS. ECV 304 bladder carcinoma cell line (16MacLeod R.A. Dirks W.G. Matsuo Y. Kaufmann M. Milch H. Drexler H.G. Int. J. Cancer. 1999; 83: 555-563Crossref PubMed Scopus (286) Google Scholar) was maintained in M199 medium with 10% FBS and 40 μg/ml gentamicin. KOT cells (KOT11 and KOT51 subclones) are epidermal fibroblasts derived from a triple knock-out (mdr1 a-/-/b-/-/mrp1-/-) mouse (17Wijnholds J. deLange E.C. Scheffer G.L. van den Berg D.J. Mol C.A. van der Valk M. Schinkel A.H. Scheper R.J. Breimer D.D. Borst P. J. Clin. Invest. 2000; 105: 279-285Crossref PubMed Scopus (346) Google Scholar, 18Johnson D.R. Finch R.A. Ping Lin Z. Zeiss C.J. Sartorelli A.C. Cancer Res. 2001; 61: 1469-1476PubMed Google Scholar) and were a generous gift from Dr. J. Wijnholds (Department of Ophthalmo-genetics, Netherlands Ophthalmic Research Institute). Subclones were taken after 25 passages, after which the cells were grown for another 25 passages prior to use in the present studies. For MDR1 inhibition, cells were grown in medium containing inhibitor at the highest concentration (±4 or 8 μm CsA for 4 days, or 5, 10, or 15 μm ketoconazole for 5 days), which our prior studies had shown to have no effect on cell growth. VT1 was purified as described previously (19Nutikka A. Binnington-Boyd B. Lingwood C.A. Methods Mol. Med. 2003; 73: 187-195PubMed Google Scholar). Target cells in logarithmic growth phase were cultured in microtiter plates and incubated in triplicate, with increasing dilutions of VT1. After 72 h, the cells remaining attached were stained with 0.1% crystal violet and quantitated using a microtiter plate reader as previously (20Kueng W. Silber E. Eppenberger U. Anal. Biochem. 1989; 182: 16-19Crossref PubMed Scopus (597) Google Scholar). Non-adherent Daudi and Jurkat cell survival data were calculated by 1% Trypan Blue dye exclusion. For MDR1 inhibition studies, CsA or ketoconazole was added to cells 4 or 5 days, respectively, prior to VT1 and maintained during the cytotoxicity assay. GSLs were extracted from 5 × 106 exponential growth phase cells. Adherent cells were scraped, and all cells were pelleted by centrifugation at 1000 × g for 10 min. The cell pellet was extracted with 20 volumes of CHCl3/CH3OH (2:1) as described (21Pudymaitis A. Armstrong G. Lingwood C.A. Arch. Biochem. Biophys. 1991; 286: 448-452Crossref PubMed Scopus (36) Google Scholar). The extract was partitioned against water, and the lower phase was applied to a silica column in CHCl3/CH3OH (98:2). The column was washed extensively with CHCl3 and the glycolipid fraction eluted with CH3COCH3/CH3OH (9:1) (22Nutikka A. Binnington-Boyd B. Lingwood C. Methods Mol. Med. 2003; 73: 197-208PubMed Google Scholar). Gangliosides were separated from pooled upper and lower phases by DEAE-Sephadex G-25 chromatography and eluted using 0.4 m ammonium acetate in methanol (23Yogeeswaran G. Hakomori S. Biochemistry. 1975; 14: 2151-2156Crossref PubMed Scopus (80) Google Scholar). Equivalent GSL aliquots, based on cell number, were separated by TLC, and the plates were dried and blocked with 1% gelatin in water at 37 °C overnight. After washing with 50 mm Tris-buffered saline, pH 7.4, plates were incubated with 0.1 μg/ml VT1 for 1 h at room temperature. After washing, the plates were incubated with monoclonal anti-VT1 antibody (24Boulanger J. Petric M. Lingwood C.A. Law H. Roscoe M. Karmali M. J. Clin. Microbiol. 1990; 28: 2830-2833Crossref PubMed Google Scholar) (2 μg/ml) followed, after washing, by peroxidase-conjugated goat anti-mouse antibody. VT1 bound was visualized with 4-chloro-1-naphthol (22Nutikka A. Binnington-Boyd B. Lingwood C. Methods Mol. Med. 2003; 73: 197-208PubMed Google Scholar). Eighty percent confluent MDR1-MDCK cells were washed, scraped into PBS, and centrifuged for 5 min at 300 × g. The pellet was resuspended in buffer (10 mm Tris-HCl, pH 7.4, 10 mm KCl, 1.5 mm MgCl2,10 mm dithiothreitol, 0.5 m sucrose, and 10 mm phenylmethylsulfonyl fluoride) and homogenized using a Dounce homogenizer. The homogenate was centrifuged at 800 × g for 10 min at 4 °C, and the supernatant was further centrifuged at 10,000 × g for 10 min at 4 °C. The resulting supernatant was centrifuged at 100,000 × g for 10 min, and the pellet was resuspended in 10% of the supernatant (to give a crude, cell free “microsomal” fraction). Protein content was measured by BCA assay (Pierce). GSLs in MDR1-MDCK microsomes were metabolically labeled with UDP-[14C]galactose. 100 μl of the microsomal fraction (100 μg of protein) was incubated in Tris-HCl buffer, pH 7.4 (10 mm), and MnCl2 (10 mm) ± 4 μm CsA at 37 °C for 2 h, with shaking. 0.2 μCi of UDP-[14C]galactose was then added to a 300-μl final volume and incubated for a further 3 h at 37 °C with shaking. For some assays, exogenous liposomal GlcCer (60 μg sonicated in 20 μl of H2O) was added during the latter incubation. MDR1-MDCK Cells—GSLs of exponential growth phase cells were metabolically labeled with [14C]serine (0.5 μCi/ml) for 72 h, ± 4 μm CsA. Cells were rinsed twice with PBS and harvested by gently scraping. Cellular lipids were extracted and applied on DEAE-Sephadex as above. Neutral GSLs were unbound, and gangliosides were eluted with ammonium acetate (23Yogeeswaran G. Hakomori S. Biochemistry. 1975; 14: 2151-2156Crossref PubMed Scopus (80) Google Scholar). Lipids were separated by TLC and labeled species quantitated by phosphorimaging. Mouse Wild Type and Knockout (KOT) Fibroblasts—Cells (wild type and KOT11 fibroblasts) were grown, in duplicate, in the presence of 2.5 μCi/ml [14C]acetate or 1 μCi/ml [14C]serine for 60 h in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Medium was removed and replaced with 3 ml of CHCl3:CH3OH:H2O (1:2.2:0.2, v/v), and extraction was allowed to proceed for 10 min. 3 ml was then transferred to an 11-ml glass tube, and 0.88 ml of CHCl3, 0.78 ml of Hanks' balance salt solution, and 0.78 ml of 10 mm acetic acid were added. Cells were then vortexed, and the lower phase was removed. The lower phase was dried and separated by two-dimensional TLC (first dimension, CHCl3:CH3OH:25% NH4OH:H2O (65:35:4:4, v/v); second dimension, CHCl3:CH3COCH3: CH3OH:CH3COOH:H2O (50:20:10:10:5, v/v)) and radioactive species quantitated by phosphorimaging and identified relative to standards. Wild type and KOT 51 fibroblasts were similarly labeled with [14C]serine ± 20 μm of indomethacin, a selective inhibitor of MRP1 (25Touhey S. O'Connor R. Plunkett S. Maguire A. Clynes M. Eur. J. Cancer. 2002; 38: 1661-1670Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In this case, the dried lipid extract was saponified in 600 μl of 0.2 m KOH in MeOH for 3 h at 37 °C, neutralized with 1.6 ml of 10 mm HAc, 2 ml of CHCl3, 1.8 ml of MeOH, and partitioned. Both phases were recombined, and polar products were removed on a SepPak C18 column. The methanol-eluted fraction was then separated by two-dimensional TLC as above. Western Blot Analysis of MDR1—Cell pellets were washed three times with cold 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and solubilized in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.1% NaN3, 5% Nonidet P-40, 5 μg/ml aprotinin, 2 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride) for 1 h with shaking at 4 °C. After spinning for 20 min at 12,000 × g at 4 °C, SDS-PAGE (26Laemmeli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) and Western blotting were performed on 60-μg protein samples by a standard procedure (27Hill B. Whelan R. Hurst H. McClean S. Cancer Res. 1994; 73: 2990-2999Google Scholar) using anti-MDR1 monoclonal antibody C219 (1 μg/ml) (DAKO, Glostrup, Denmark). Immunostaining of Cell Surface MDR1—HeLa, MDR1-MDCK, Vero, SK VLB, IOMM Lee, and CH157 MN cells were grown on glass cover-slips. Washed cells were incubated with MRK-16 monoclonal anti-MDR1 antibody (10 μg/ml; Kamiya) for 1 h at 4 °C, followed by fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody, also at 4 °C for 1 h. Following extensive washing with 50 mm PBS, the coverslips were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, mounted with DABCO, and examined and photographed using a Leica DMIRE 2 (Leica Canada, Ontario) fluorescence microscope under incident UV illumination. All images were recorded at the same exposure. Post-embedding Immunogold Cryo-electronmicroscopy—Logarithmic phase MDR1-MDCK cells were scraped and fixed in 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 m Sorenson's phosphate buffer, pH 7.4, for 2–4 h at room temperature. Cells were then pelleted by centrifugation at 1000 × g for 5 min, washed thoroughly in phosphate buffer, embedded in 15% gelatin, cut into mm3-pieces, and infused with 2.3 m sucrose for several hours. The blocks were then mounted on aluminum cryo-ultramicrotomy pins and frozen in liquid nitrogen. Ultrathin cryo-sections were cut with a diamond knife at -95 °C using a Leica Ultracut R cryo-ultramicrotome (Leica Canada, Ontario). Sections were transferred to Formvar-coated nickel grids in a loop of molten sucrose, and the grids were washed thoroughly in PBS containing 0.15% glycine and 0.5% BSA and PBS containing BSA alone. Sections were then incubated with a polyclonal rabbit antibody against Golgi marker rab6 (32Goud B. Zahraoui A. Tavitian A. Saraste J. Nature. 1990; 345: 553-556Crossref PubMed Scopus (254) Google Scholar) for 1 h. Following a thorough rinse in PBS/BSA, samples were incubated in a goat anti-rabbit IgG 5-nm gold complex (Amersham Biosciences, Quebec, Canada) for 1 h. This procedure was repeated on the same specimens except MRK-16 anti-MDR1 antibody was used as the primary antibody and goat anti-mouse IgG 10-nm gold complex as the secondary antibody. Sections were then rinsed thoroughly with PBS followed by distilled water and stabilized in a thin film of methylcellulose containing 0.2% uranyl acetate. Controls included the omission of primary and secondary antisera and the use of an irrelevant antibody (either poly- or monoclonal anti-glial fibrillary acidic protein). Samples were then examined in a JEM 1230 transmission electron microscope (JEOL, Peabody, MA), and images were recorded using a charge-coupled device camera (AMT Corp.). Controls were uniformly negative for gold particles. Image analysis was performed on a minimum of 100 images from each group at a nominal magnification of 100,000× using an image analysis program (Image Pro Plus, Media Cybernetics) to determine cell particle density and percentage particle colocalization (where colocalization was defined as 5- and 10-nm particles being within 20 nm of each other). Data Analysis and Statistics—Differences in CD50 values for each of the cell lines, obtained before and after treatment with CsA and ketoconazole, were compared by 2-tailed non-parametric Student's t test. Means and standard deviations for each experimental point, repeated at least three times, as well as the statistical analysis were performed by GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA). A value of p < 0.05 was considered significant. MDR1 Inhibitors Inhibit Gb3Synthesis and Protect Cells against VT1 Cytotoxicity—The Gb3-containing cell lines, MDR1-MDCK (canine epithelial), Vero (monkey epithelial), Hep-2 (human epithelial), HeLa (human cervical carcinoma), Daudi (human B lymphoid), SF-539 (human astrocytoma), SK VLB (human ovarian carcinoma), Iomm Lee (human meningioma), CH157 MN (human meningioma), and ECV 304 (human bladder carcinoma) cell lines were analyzed for neutral GSL and Gb3 content and VT1 sensitivity, before and after ketoconazole or CsA treatment (Fig. 1 and Table I). For each cell line, these drugs were used at the maximum concentration, which alone had no effect on cell viability. Inhibition of MDR1 using either drug protected all cell lines (except HeLa cells) against VT1 cytotoxicity (Table I). The lower phase GSL fraction from control, CsA-treated, or ketoconazole-treated cells was separated by TLC and Gb3 identified by VT1/TLC overlay. In each case, (except HeLa cells), the Gb3 (and LacCer and GlcCer) content was significantly reduced in treated cells, correlating with the protection afforded against VT1 cytotoxicity. In HeLa cells, VT1 sensitivity and their Gb3 content was largely unaffected by either MDR1 inhibitor. The Gb3 content and VT1 sensitivity of SK VLB and ECV 304 cells was less effectively reduced after CsA, as opposed to ketoconazole treatment, consistent with our finding that the vinblastine sensitivity of these cell lines was affected only by ketoconazole (not shown). Despite the fact that Gb3 synthesis in ECV 304 and SF-539 cells was significantly inhibited by CsA, a reduced effect on VT1 sensitivity was observed. NIH 3T3 cells are VT1-insensitive and contain only trace levels, if any, Gb3 (28Meivar-Levy I. Futerman A. J. Biol. Chem. 1999; 274: 4607-4612Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The neutral GSLs of mouse 3T3 cells were eliminated by CsA treatment. Similarly, human T cells do not express Gb3 (29Cohen A. Madrid-Marina V. Estrov Z. Freedman M. Lingwood C.A. Dosch H.-M. Int. Immunol. 1990; 2: 1-8Crossref PubMed Scopus (75) Google Scholar) and the neutral GSLs of the Jurkat T cell line (primarily GlcCer) were lost following culture with CsA (see Figs. 1 and 3a).Table IEffect of CsA or ketoconazole on cell line VT1 sensitivity The effect of MDR1 inhibition on the sensitivity of exponentially growing cell cultures to VT1 was determined. The VT1 dose required to kill 50% of cells (CD50) calculated from the dose response was increased for all cells (except HeLa), after culture of cells with CsA or ketoconazole as described in Fig. 1. For each experiment, the effect on cell sensitivity to vinblastine was simultaneously assayed to confirm efficacy to prevent MDR1-mediated drug efflux.Cell typeaDose-dependent VT1 cytotoxicity assayed in triplicate, repeated at least twicePretreatmentNone,CD50CsAKetoconazoleCD50Approximate increasep valueCD50Approximate increasep valueng/mlng/ml-foldng/ml-foldMDCK-MDR13.00 × 10-60.42105<0.00014.50 × 10-21050.0001Vero3.95 × 10-60.195 × 104<0.00012.60 × 10-36 × 102<0.0001SF 5390.0431.69390.00013.16 × 10-17.3<0.0001HEp-24.58 × 10-60.0143 × 1030.00112.40 × 10-45 × 1020.0011HeLa2.54 × 10-62.8 × 10-610.22 NSbNS, not significant3.32 × 10-610.08 NSIOMM Lee4.00 × 10-476.802 × 108<0.0001NDcND, not determinedCH157 MN0.3826.47700.0005NDDaudi4.391.78 × 1034 × 1020.0449.94 × 1032 × 103<0.0001SK VLB7.86 × 10-97.0 × 10-910.35 NS7.93 × 10-7100<0.0001ECV 3047.85 × 10-97.7 × 10-910.49 NS4.80 × 10-26 × 1050.0001a Dose-dependent VT1 cytotoxicity assayed in triplicate, repeated at least twiceb NS, not significantc ND, not determined Open table in a new tab Fig. 3Comparison of the effect of MDR1 inhibition on GM3/ganglioside and Gb3/neutral GSL synthesis.a, cellular GSL content. Panels A, C, and E: neutral GSL fractions; panels B, D, and F: ganglioside fractions; panels A and B correspond to Vero cells, panels C and D to MDR1-MDCK cells, and panels E and F to Jurkat cells. Lane 1, standard GSLs: panels A, C, and E: GlcCer, LacCer, Gb3, and Gb4; panels B and D: GM3; panel F: GM3 (upper) and GM1 (lower); lane 2: GSLs from untreated cells; and lane 3: GSLs from ketoconazole (Vero)- or CsA (MDR1-MDCK and Jurkat)-treated cells. Neutral GSLs were visualized by orcinol and VT1 overlay for Gb3. Gangliosides were visualized using resorcinol staining. Similar results were obtained in three experiments. b, metabolic labeling MDR1-MDCK cells were metabolically labeled with [14C]serine ± 4 μm CsA for 4 days, and the neutral and acidic GSLs were extracted, separated, and resolved by TLC and visualized by autoradiography. Panel A, neutral GSLs: lanes 1 and 4, stds; lane 2: control cells; and lane 3: CsA-treated cells. Panel B, gangliosides, lane 1: control cells; lane 2: CsA-treated cells, and lane 3: stds. The [14C]serine-labeled doublet in the neutral GSL fraction comigrates with sphingomyelin and is reactive with the molybdenum stain for phosphate (not shown). This experiment was repeated three times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of CsA on Cell-free Glycolipid Synthesis—GSL synthesis (LacCer and some Gb3) in a crude MDR1-MDCK microsomal fraction, monitored by [14C]UDP-galactose incorporation, was inhibited by CsA (Fig. 2). Addition of exogenous GlcCer increased LacCer labeling, but CsA inhibition was maintained. Selective CsA Inhibition of Neutral Glycolipid as Opposed to Ganglioside Synthesis—GlcCer is the precursor of both gangliosides and neutral GSLs. We therefore compared the effect of ketoconazole or CsA on the synthesis of Gb3 and GM3, the simplest ganglioside, in MDR1-MDCK and Vero cells. The effect of CsA on Jurkat cell GlcCer, the only neutral GSL made, and ganglioside content, was compared. In Vero cells cultured with ketoconazole and MDR1-MDCK cells cultured with CsA, Gb3 levels were inhibited by >95%, whereas the level of GM3, the only ganglioside expressed, was unaffected. In Jurkat cells, GlcCer was reduced by >90%, whereas GM3, GM1, and one unidentified, more polar, ganglioside were unaltered after CsA treatment (Fig. 3a). Autoradiography of the metabolically labeled neutral and acidic GSL fraction of MDR1-MDCK cells cultured with or without CsA, shows that, although Gb3 synthesis was significantly reduced, the synthesis of GM3 and the more complex gangliosides were not inhibited at all by CsA (Fig. 3b). Sphingomyelin was identified in the [14C]serine-labeled neutral GSL fraction (Fig. 3a), and labeling was not inhibited, even slightly enhanced, in CsA-treated cells. Glycolipid/Phospholipid Profile of Mouse mdr1a, 1b mrp1 Knockout Fibroblasts—Mice contain two MDR1 homologous genes, which have been knocked out in combination with MRP1 (17Wijnholds J. deLange E.C. Scheffer G.L. van den Berg D.J. Mol C.A. van der Valk M. Schinkel A.H. Scheper R.J. Breimer D.D. Borst P. J. Clin. Invest. 2000; 105: 279-285Crossref PubMed Scopus (346) Google Scholar, 18Johnson D.R. Finch R.A. Ping Lin Z. Zeiss C.J. Sartorelli A.C. Cancer Res. 2001; 61: 1469-1476PubMed Google Scholar). Comparison of the metabolic labeling of GSLs of fibroblasts from knockout mice (mdr 1a-/-, 1b-/-mrp 1-/-), and wild type fibroblasts (Fig. 4 and Table II) showed the same effect we observed with CsA and ketoconazole treatment of cell lines. [14C]Acetate and [14C]serine labeling of GlcCer and LacCer in the KOT cells was markedly reduced (by >80 and 90%, respectively, Table II) as compared with wild type fibroblasts. Synthesis of an uncharacterized CTH Table II species was also reduced, but less severely. In contrast, labeling of GM3 ganglioside was not significantly affected. Phospholipid labeling was also altered in the KOT cells: sphingomyelin labeling was increased, and phosphatidyl choline labeling with [14C]serine was reduced. Neutral lipid labeling from either [14C]acetate or [14C]serine was also significantly reduced in KOT cells.Table IIQuantitation of metabolic labeling of GSL of MDR1 knockout fibroblasts with [14C]acetate or [14C]serine (shown inFig. 4) The metabolically radiolabeled lipids extracted from cells (in duplicate) and separated by two-dimensional TLC (Fig. 4) were quantitated by PhosphorImager analysis. By both [14C]acetate (A) and [14C]serine (B) labeling, the synthesis of neutral GSLs GlcCer and LacCer was severely reduced, whereas GM3 ganglioside synthesis was not inhibited in the KOT cells, as compared to the wild type control. Phospholipid synthesis was relatively unaffected except for increased SM synthesis and a marked decrease in PC labeling from [14C]serine. Neutral lipid labeling was reduced in KOT cells.Total phospholipidTotal lipidControlaAverage KOT value of duplicate experiments relative to average value for wild type cellsWild typeKOT 11Wild typeKOT 11%A) [14C]Acetate labelingGlcCer a0.490.060.390.0510GlcCer b0.620.110.490.1020LacCer0.710.070.380.0410GM31.231.260.961.15110CTHbCTH, ceramide trihexoside, not further characterized a0.400.230.320.2165CTHbCTH, ceramide trihexoside, not further characterized b0.230.190.180.1790Cer0.830.810.640.75110NL23.746.6518.436.0630xcUnidentified species indicated in Fig. 40.400.410.310.37110PCdPE, phosphatidyl ethanolamine; PS, phosphatidyl serine; PI, phosphatidyl inositol; SM, sphingomyelin56.7857.5344.2752.42110PE21.0018.2616.3416.6495PS/PI12.249.199.548.3780SM9.9915.047.7913.7165Total PL10010077.9491.13Total Lipid128.42109.74100.00100.00B) [14C]Serine labelingGlcCer a0.470.120.430.1125GlcCer b0.680.130.630.1220LacCer0.760.100.700.0915GM32.321.972.121.8985Cer0.741.020.670.98145" @default.
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• W2100076132 date "2004-02-01" @default.
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• W2100076132 title "Role of Multiple Drug Resistance Protein 1 in Neutral but Not Acidic Glycosphingolipid Biosynthesis" @default.
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• W2100076132 doi "https://doi.org/10.1074/jbc.m305645200" @default.
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