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• W2097146306 abstract "Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc nih mouse model of NPC disease. A library of 44,240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells.One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels. Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc nih mouse model of NPC disease. A library of 44,240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells. One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels. Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick Disease Type C: a lipid trafficking disorder.in: Valle D. 8th edition. The Metabolic and Molecular Bases of Inherited Disease. Vol. III. McGraw-Hill, New York2001: 3611-3633Google Scholar). Historically, NPC has been considered a disease of cholesterol metabolism, since a major lipid that is stored in visceral organs is cholesterol. However, in reality it is a complex lipid storage disorder, with increases in sphingomyelin, cholesterol, lysobisphosphatidic acid, neutral and acidic glycosphingolipids, and phospholipids seen in liver and spleen. The lipid storage pattern differs dramatically in the brain where gangliosides are the main storage material and cholesterol is stored to a lesser extent (2Zervas M. Dobrenis K. Walkley S.U. Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J. Neuropathol. Exp. Neurol. 2001; 60: 49-64Google Scholar). NPC patients exhibit progressive loss of motor skills, learning difficulties, dementia, and seizures (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick Disease Type C: a lipid trafficking disorder.in: Valle D. 8th edition. The Metabolic and Molecular Bases of Inherited Disease. Vol. III. McGraw-Hill, New York2001: 3611-3633Google Scholar), all of which signal central nervous system degeneration. Onset of symptoms in the majority of NPC children is school age, and the disease is generally fatal within a decade. NPC disease is caused by mutations in one of two genetic loci, NPC1 (3Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.-Y. Liscum L. Strauss J.F. Ohno K. Zigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. v. Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar) and NPC2 (4Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Google Scholar). NPC1 and NPC2 phenotypes are clinically and biochemically indistinguishable and the two genes may encode constituents of the same lipid transport pathway. The NPC1 gene product is a 1278 amino acid membrane protein found in late endosomes (5Watari H. Blanchette-Mackie E.J. Dwyer N.K. Glick J.M. Patel S. Neufeld E.B. Brady R.O. Pentchev P.G. Strauss 3rd, J.F. Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.Proc. Natl. Acad. Sci. USA. 1999; 96: 805-810Google Scholar, 6Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss 3rd J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.J. Biol. Chem. 1999; 274: 9627-9635Google Scholar, 7Higgins M.E. Davies J.P. Chen F.W. Ioannou Y.A. Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network.Mol. Genet. Metab. 1999; 68: 1-13Google Scholar). Mutations in NPC1 cause altered lipid transport kinetics and lysosomal accumulation of cholesterol and gangliosides (8Pentchev P.G. Blanchette-Mackie E.J. Liscum L. Biological implications of the Niemann-Pick C mutation.Subcellular Biochemistry. 1997; 28: 437-451Google Scholar, 9Liscum L. Niemann-Pick type C mutations cause lipid traffic jam.Traffic. 2000; 1: 218-225Google Scholar, 10Puri V. Watanabe R. Dominguez M. Sun X. Wheatley C.L. Marks D.L. Pagano R.E. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid storage diseases.Nat. Cell Biol. 1999; 1: 386-388Google Scholar). The biological function of NPC1 still is not clear; however, recent evidence suggests that NPC1 may have a permease activity (11Davies J.P. Chen F.W. Ioannou Y.A. Transmembrane molecular pump activity of Niemann-Pick C1 protein.Science. 2000; 290: 2295-2298Google Scholar). Molecular cloning of NPC2 reveals that the protein is likely to be a soluble lysosomal protein with cholesterol binding activity (4Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Google Scholar). NPC disease does not affect lipoprotein levels or produce premature vascular disease. Thus, therapies that target hypercholesterolemia are not effective against NPC (12Erickson R.P. Garver W.S. Camargo F. Hossain G.S. Heidenreich R.A. Pharmacological and genetic modifications of somatic cholesterol do not substantially alter the course of CNS disease in Niemann-Pick C mice.J. Inherit. Metab. Dis. 2000; 23: 54-62Google Scholar). Bone marrow and liver transplantation in NPC patients and an NPC mouse model have not slowed the progression of neurological symptoms (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick Disease Type C: a lipid trafficking disorder.in: Valle D. 8th edition. The Metabolic and Molecular Bases of Inherited Disease. Vol. III. McGraw-Hill, New York2001: 3611-3633Google Scholar). Currently, there is no therapy that alleviates the progressive neurodegeneration that is seen in NPC patients. Without a defined biological function for NPC1, it is difficult to develop a cell-based, target-specific high throughput screen for therapeutic compounds that might reverse or prevent the neurodegeneration. Instead, we have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that alter key aspects of the NPC phenotype. Amphotericin B is a polyene antibiotic that forms aqueous pores in sterol rich membranes, resulting in lysis and cell death. We previously found that Chinese hamster ovary cells with an NPC phenotype are resistant to amphotericin B-mediated toxicity (13Dahl N.K. Reed K.L. Daunais M.A. Faust J.R. Liscum L. Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of LDL-derived cholesterol.J. Biol. Chem. 1992; 267: 4889-4896Google Scholar), presumably due to lysosomal cholesterol storage resulting in lowered plasma membrane cholesterol content. To identify compounds that induce cholesterol movement from NPC lysosomes, a 44,240 member compound library was screened for those that cause LDL-dependent amphotericin B-mediated killing in NPC cells. The screen has identified one compound that induces specific pathways of intracellular cholesterol transport in NPC cells. Sodium [3H]acetate (100 mCi/mmol), [9,10-3H]oleic acid (5 Ci/mmol), cholesteryl [1-14C]oleic acid (51 mCi/mmol), [1,2-3H]cholesterol (45 Ci/mmol), [4-14C]cholesterol (56 mCi/mmol), and [1,2,6,7-3H]cholesteryl linoleate (84 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). FCS was from Hyclone (Logan, UT). Mediatech cellgro tissue culture medium was from Fisher Scientific; other tissue culture reagents were from Life Technologies, Inc. or from Sigma Chemical. Ninety-six-well tissue culture plates were from Corning. Lovastatin was from Merck Research Laboratory (Rahway, NJ) whereas pravastatin was from Bristol-Myers Squibb (Princeton, NJ). Other chemicals were from Sigma Chemical unless otherwise indicated. LDL was prepared by ultracentrifugation (14Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). Lipoprotein-deficient FCS was prepared as described, omitting the thrombin incubation (14Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). The following media were prepared: HD-10% FCS (a 1:1 mixture of Ham's F-12 and DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 25 mM HEPES, pH 7.3), HD-15% fetal calf lipoprotein-deficient serum (FCLPDS) and HD-1% FCLPDS (HD-10% FCS in which the FCS was replaced with 15% or 1% fetal calf lipoprotein-deficient serum). HD-15% FCLPDS/sm is HD-15% FCLPDS supplemented with a statin (20 μM lovastatin or pravastatin to inhibit endogenous cholesterol synthesis) and 0.5 mM mevalonate (which enters the cholesterol biosynthetic pathway past the point of statin inhibition and provides essential nonsteroidal isoprenoids). Normal and NPC mouse ovarian granulosa cells were generously provided by Jerome F. Strauss III (University of Pennsylvania, Philadelphia, PA) and Peter G. Pentchev (National Institute of Neurological Disorders and Stroke, National Institutes of Health). NPC cells were from the npc nih mouse model (15Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: Mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar). Cells were grown in a monolayer in HD-10% FCS in a humidified incubator (5% CO2) at 37°C. Stock flasks were washed with HBSS, trypsinized and re-seeded three times per week. All test compounds identified in this study were dissolved in 100% Me2SO and added to the tissue culture wells at a final concentration of 1% Me2SO. 'No Compound’ controls received vehicle alone. Cells were grown and treated as described in the figure legends. Cell viability was assessed using a colorimetric 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (16Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.J. Immunol. Methods. 1983; 65: 55-63Google Scholar, 17Korting H.C. Schindler S. Hartinger A. Kerscher M. Angerpointner T. Maibach H.I. MTT assay and neutral red release assay: Relative role in the prediction of the irritancy potential of surfactants.Life Sci. 1994; 55: 533-540Google Scholar) exactly as described (18Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). On day 0, NPC cells were seeded into 96-well plates (10,000/well) in HD-15% FCLPDS. On day 1, cells were refed HD-15% FCLPDS/sm with 25 μg/ml LDL. During the primary screening, eighty test compounds were added per plate to columns 1–10 in a final concentration of 15 μM in 1% Me2SO. Controls were added in the remaining two columns of the plate. After 4 h, cells were washed with HBSS and refed HD-1% FCLPDS with or without 25 μg/ml amphotericin B. After 2 h, cells were washed twice with HBSS and refed HD-10% FCS. On day 2, cell viability was assessed using a colorimetric MTT assay. Cells were refed HD-10% FCS with 2.5 mg/ml MTT. After 2 h, the medium was aspirated and 100% Me2SO added for 15 min. MTT cleavage was determined by reading the absorbence at 564 nm. Cells were cultured and incubated with 100 μM [3H]oleate (20,000 dpm/nmol) as described in the legends to Fig. 5, 6, and Table 2. Cellular cholesteryl [3H]oleate and [3H]triglycerides were isolated as described (19Liscum L. Faust J.R. Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts.J. Biol. Chem. 1987; 262: 17002-17008Google Scholar). After lipids were extracted, the monolayers were dissolved in 0.1 N NaOH and aliquots removed for protein determination (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). Correction for procedural losses was made by adding [14C]cholesteryl oleate (2,000 dpm, 20 μg) as an internal standard during the extraction.Fig. 6Esterification of cellular [3H]cholesterol. On day 0, normal and NPC cells were seeded in 12-well plates (20,000/well) in HD-10% FCS. On day 2, cells were refed HD-15% FCLPDS. On day 3, cells were refed HD-15% FCLPDS with or without NP-27 at 1.47 μM or NP-31 at 2.1 μM. [3H]cholesterol (1 μCi/ml) was added and the cells were incubated at 37°C for 6 h. Cells were then washed and the cellular content of [3H]cholesterol and [3H]cholesteryl ester was determined as described under Experimental Procedures. Data are expressed as the percentage of cellular [3H]cholesterol metabolized to [3H]cholesteryl esters and are the means ± SD of four experiments. *P < 0.05 compared with “no compound” control.View Large Image Figure ViewerDownload (PPT)TABLE 2[3H]oleate incorporation into [3H]triglycerides[3H]Triglyceride FormationExperimentTest CompoundNormal CellsNPC Cellsnmol/h/mg1None10.5 ± 1.518.6 ± 5.2NP-2718.0 ± 1.728.3 ± 5.82None11.1 ± 0.723.4 ± 1.7NP-3118.8 ± 3.534.4 ± 6.4On day 0, cells were seeded in 6-well plates (25,000/well) in HD-10% FCS. The next day, cells were washed with HBSS and refed HD-15% FCLPDS. On day 2, cells were refed HD-15% FCLPDS with or without 50 μg/ml LDL and containing compounds at their EC50 (1.47 μM and 2.10 μM for NP-27 and NP-31, respectively). After 18 h (day 3), cells were pulse-labeled with [3H]oleate for 1 h. The cellular content of [3H]triglycerides was determined as described under Experimental Procedures. The data are expressed as nmol [3H]triglyceride formed per h/mg protein and present mean ± SD of triplicate cultures. Open table in a new tab On day 0, cells were seeded in 6-well plates (25,000/well) in HD-10% FCS. The next day, cells were washed with HBSS and refed HD-15% FCLPDS. On day 2, cells were refed HD-15% FCLPDS with or without 50 μg/ml LDL and containing compounds at their EC50 (1.47 μM and 2.10 μM for NP-27 and NP-31, respectively). After 18 h (day 3), cells were pulse-labeled with [3H]oleate for 1 h. The cellular content of [3H]triglycerides was determined as described under Experimental Procedures. The data are expressed as nmol [3H]triglyceride formed per h/mg protein and present mean ± SD of triplicate cultures. Cells were cultured and incubated with [3H]acetate as described in the legend to Table 1. Cell monolayers were washed once quickly, once for 10 min, once quickly with Tris-buffered saline (50 mM Tris-Cl and 155 mM NaCl, pH 7.4), then extracted with hexane-isopropanol (3:2, v/v). Samples were saponified by the addition of 1 ml of 1 M KOH in ethanol and incubation at 80°C for 1 h. Samples were extracted with petroleum ether after which the water phase was adjusted to pH 3 by addition of 10 N HCl. The water phase was then extracted with petroleum ether and the organic extract was washed with water and dried. [3H]fatty acids were analyzed by thin layer chromatography using silica gel 60 plates resolved with heptane-ethyl ether-etic acid (90:30:1, v/v/v). Correction for procedural losses was made by adding cholesteryl linoleate (20 μg), cholesteryl oleate (20 μg), and [14C]oleic acid (2,000 dpm) as an internal standard during the hexane/isopropanol extraction.TABLE 1Fatty acid synthesis in normal and NPC cells[3H]Fatty Acid SynthesisTest CompoundNormal CellsNPC Cells% of 'no compound’ controlNP-27137.8 ± 23.3120.2 ± 21.0NP-31137.7 ± 32.7135.1 ± 30.0On day 0, cells were seeded in 12-well plates (20,000/well) in HD-10% FCS. On day 2, cells were refed HD-15% FCLPDS. On day 3, cells were refed 0.5 ml HD-15% FCLPDS and compounds were added at their EC50 (1.47 μM and 2.10 μM for NP-27 and NP-31, respectively). After 1 h, 30 μCi of [3H]acetate was added. After 2 h, cells were washed and the cellular content of [3H]fatty acids was determined as described in Experimental Procedures. The data are expressed as a percentage of the “no compound” controls, which were 416 dpm/μg and 418 dpm/μg for normal and NPC cells, respectively. The data represent the mean ± SD of three experiments. Open table in a new tab On day 0, cells were seeded in 12-well plates (20,000/well) in HD-10% FCS. On day 2, cells were refed HD-15% FCLPDS. On day 3, cells were refed 0.5 ml HD-15% FCLPDS and compounds were added at their EC50 (1.47 μM and 2.10 μM for NP-27 and NP-31, respectively). After 1 h, 30 μCi of [3H]acetate was added. After 2 h, cells were washed and the cellular content of [3H]fatty acids was determined as described in Experimental Procedures. The data are expressed as a percentage of the “no compound” controls, which were 416 dpm/μg and 418 dpm/μg for normal and NPC cells, respectively. The data represent the mean ± SD of three experiments. Cells were cultured and incubated with [3H]acetate as described in the legend to Table 3. Cellular [3H]sterol was quantified as described (21Liscum L. Ruggiero R.M. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick Type C fibroblasts.J. Cell Biol. 1989; 108: 1625-1636Google Scholar), except that thin-layer chromatography separation was performed using heptane-ethyl ether (90:60, v/v). Correction for procedural losses was made by adding [14C]cholesterol (2,000 dpm, 20 μg) as an internal standard during the extraction. The endogenously labeled [3H]sterols co-chromatograph with authentic cholesterol; however, they can be resolved by HPLC into [3H]cholesterol (70%) and [3H]desmosterol (30%). They are referred to as [3H]cholesterol.TABLE 3Cholesterol synthesis in normal and NPC cells[3H]Cholesterol SynthesisTest CompoundNormal CellsNPC Cells% of 'no compound’ controlNP-25101.1 ± 18.0111.8 ± 18.6NP-2796.9 ± 13.3102.6 ± 24.3NP-31109.9 ± 14.7111.3 ± 15.1On day 0, cells were seeded in 12-well plates (20,000/well) in HD-10% FCS. On day 1, cells were refed HD-15% FCLPDS. On day 3, cells were refed 0.5 ml HD-15% FCLPDS and compounds were added at their EC50 (1.30 μM, 1.47 μM, and 2.10 μM for NP-25, NP-27, and NP-31, respectively). After 1 h, 30 μCi of [3H]acetate was added. After 2 h, cells were washed, cellular lipids were extracted and subjected to saponification, and the cellular content of [3H]sterol was determined as described in Experimental Procedures. The data are expressed as a percentage of the 'no compound’ controls, which were 83.9 dpm/μg and 50.5 dpm/μg for normal and NPC cells, respectively. The data represent the mean ± SD of four experiments. Open table in a new tab On day 0, cells were seeded in 12-well plates (20,000/well) in HD-10% FCS. On day 1, cells were refed HD-15% FCLPDS. On day 3, cells were refed 0.5 ml HD-15% FCLPDS and compounds were added at their EC50 (1.30 μM, 1.47 μM, and 2.10 μM for NP-25, NP-27, and NP-31, respectively). After 1 h, 30 μCi of [3H]acetate was added. After 2 h, cells were washed, cellular lipids were extracted and subjected to saponification, and the cellular content of [3H]sterol was determined as described in Experimental Procedures. The data are expressed as a percentage of the 'no compound’ controls, which were 83.9 dpm/μg and 50.5 dpm/μg for normal and NPC cells, respectively. The data represent the mean ± SD of four experiments. Cells were cultured and incubated with [3H]cholesterol as described in the legend to Fig. 6. Cells were washed and [3H]cholesterol and [3H]cholesteryl esters were isolated and quantified as described (18Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). Cells were cultured and incubated with LDL that is labeled with [3H]cholesteryl linoleate ([3H]CL-LDL) as described in the legend to Fig. 7. Cells were treated with cholesterol oxidase using the method of Slotte et al. (22Slotte J.P. Hedstrom G. Rannstrom S. Ekman S. Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior.Biochim. Biophys. Acta. 1989; 985: 90-96Google Scholar). Wells were then washed, lipids were extracted, and [3H]cholesterol and [3H]cholestenone quantified as described (23Underwood K.W. Andemariam B. McWilliams G.L. Liscum L. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport.J. Lipid Res. 1996; 37: 1556-1568Google Scholar). Statistical comparisons were made using a Student's t-test or Mann-Whitney test (VassarStats; http://faculty.vassar.edu/~lowry/VassarStats.html). The goal of this study was to identify compounds that alter the disease phenotype of cultured NPC cells. Since the biological function of NPC1 is unknown, we were unable to design a screen for compounds that would directly act on NPC1. Instead, we focused on a likely downstream effect of NPC1 activity, i.e., cholesterol transport from lysosomes to other cellular membranes. The screen for these compounds required an immortalized cell culture model for NPC disease and a biochemical assay that could be adapted for high throughput screening. The cell culture models that we used were simian virus 40-transformed mouse ovarian granulosa cell lines obtained from wild-type and npc nih BALB/c mice (24Amsterdam A. Hanukoglu I. Suh B.S. Keren-Tal D. Plehn-Dujowich D. Sprengel R. Rennert H. Strauss J.F. J. Steroid Biochem. Mol. Biol. 1992; 43: 875-884Google Scholar, 25Gu J.Z. Carstea E.D. Cummings C. Morris J.A. Loftus S.K. Zhang D. Coleman K.G. Cooney A.M. Comly M.E. Fandino L. Roff C. Tagle D.A. Pavan W.J. Pentchev P.G. Rosenfeld M.A. Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.Proc. Natl. Acad. Sci. USA. 1997; 94: 7378-7383Google Scholar). The NPC1 gene in npc nih BALB/c mice has a retrotransposon-like insertion that leads to prematurely truncated NPC1 protein of approximately 500 amino acids (15Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: Mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar). The murine model exhibits a pathological and clinical phenotype resembling the classical form of the human disease and has a shortened life span (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick Disease Type C: a lipid trafficking disorder.in: Valle D. 8th edition. The Metabolic and Molecular Bases of Inherited Disease. Vol. III. McGraw-Hill, New York2001: 3611-3633Google Scholar, 15Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: Mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar, 26Liu Y. Wu Y.P. Wada R. Neufeld E.B. Mullin K.A. Howard A.C. Pentchev P.G. Vanier M.T. Suzuki K. Proia R.L. Alleviation of neuronal ganglioside storage does not improve the clinical course of the Niemann-Pick C disease mouse.Hum. Mol. Genet. 2000; 9: 1087-1092Google Scholar). Cells cultured from the npc nih mouse have the characteristic cholesterol storage and delayed movement of exogenous cholesterol to acyl-CoA cholesterol acyltransferase (ACAT) in the endoplasmic reticulum that is seen in human NPC fibroblasts (27Pentchev P.G. Comly M.E. Kruth H.S. Patel S. Proestel M. Weintroub H. The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease.J. Biol. Chem. 1986; 261: 2772-2777Google Scholar). An amphotericin B cytotoxicity assay was used to distinguish between NPC cells that express the disease phenotype and those that have had their cholesterol homeostasis restored by the action of a test compound. Polyene antibiotics, such as amphotericin B and filipin, complex with sterols and form pores, lysing cells that have a threshold amount of plasma membrane sterol (28Saito Y. Chou S.M. Silbert D.F. Animal cell mutants defective in sterol metabolism: A specific selection procedure and partial characterization of defects.Proc. Natl. Acad. Sci. USA. 1977; 74: 3730-3734Google Scholar, 29Hidaka K. Endo H. Akiyama S. Kuwano M. Isolation and characterization of amphotericin B-resistant cell lines in Chinese hamster cells.Cell. 1978; 14: 415-421Google Scholar). The rationale behind the cytotoxicity assay is depicted in Fig. 1. Normal cells grown in lipoprotein-deficient serum plus a statin (to inhibit endogenous cholesterol synthesis) for 24 h are relatively resistant to amphotericin B because the cholesterol content of the plasma membrane is reduced. When LDL is added to normal cells, LDL's cholesteryl esters are hydrolyzed in lysosomes. The cholesterol released is transported to the plasma membrane and renders the cells amphotericin B sensitive (30Krieger M. Martin J. Segal M. Kingsley D. Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis.Proc. Natl. Acad. Sci. USA. 1983; 80: 5607-5611Google Scholar). However, NPC cells survive amphotericin B treatment because LDL-cholesterol (LDL-C) transport to the plasma membrane is delayed (13Dahl N.K. Reed K.L. Daunais M.A. Faust J.R. Liscum L. Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of LDL-derived cholesterol.J. Biol. Chem. 1992; 267: 4889-4896Google Scholar). Our goal was to identify compounds that would promote the movement of LDL-C to the plasma membrane, rendering NPC cells amphotericin B sensitive. We first adapted our standard amphotericin B protocol to be conducted on a smaller scale with shorter time incubations, which would permit high throughput screening. An experiment designed to illustrate amphotericin B killing of normal and NPC cells is shown in Fig. 2. In this experiment, normal and NPC cells were cultured for 24 h in HD-15% FCLPDS/sm. Growth in the absence of both endogenous cholesterol synthesis and an exogenous cholesterol supply lowers the plasma membrane cholesterol content. (Forty-eight hours of growth in this medium is needed for full amphotericin B survival of cultured cells (13Dahl N.K. Reed K.L. Daun" @default.
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