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• W2018695514 abstract "Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 μg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OXR), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OXR showed no enhanced calcium uptake in response to 25-hydroxycholesterol. Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 μg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OXR), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OXR showed no enhanced calcium uptake in response to 25-hydroxycholesterol. oxidized low density lipoprotein low density lipoprotein sterol regulatory element sterol regulatory element-binding protein Chinese hamster ovary phosphate-buffered saline low density lipoprotein 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate terminal deoxyuridine nick end labeling 25-hydroxycholesterol 7-amido-4-methylcoumarin Oxidized low density lipoprotein (oxLDL)1 plays an important role in atherogenesis (1.Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9970) Google Scholar, 2.Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar). oxLDL can be generated in vivoby at least three classes of mechanisms: 1) autoxidation in the presence of transition metals (3.Lamb D.J. Michinson M.J. Leake D.S. FEBS Lett. 1995; 374: 12-16Crossref PubMed Scopus (81) Google Scholar, 4.Morel D.W. Hessler J.R. Chisolm G.M. J. Lipid Res. 1983; 24: 1070-1076Abstract Full Text PDF PubMed Google Scholar); 2) cell-mediated oxidation (5.Morel D.W. DiCorleto P.E. Chisolm G.M. Arteriosclerosis. 1984; 4: 357-364Crossref PubMed Google Scholar, 6.Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Scopus (1415) Google Scholar, 7.Hiramatsu K. Rosen H. Heinecke J.W. Wolfbauer G. Chait A. Arteriosclerosis. 1987; 7: 55-60Crossref PubMed Google Scholar); and 3) plasma enzyme-mediated oxidation (8.Yla-Herttuala S. Luoma J. Viita H. Hiltunen T. Sisto T. Nikkari T. J. Clin. Invest. 1995; 95: 2692-2698Crossref PubMed Scopus (128) Google Scholar, 9.Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1127) Google Scholar, 10.Parthasarathy S. Steinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar, 11.Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Scopus (239) Google Scholar). oxLDL plays a role in many early events of atherosclerosis; it induces the expression of adhesion molecules on endothelial cells (12.Kume N. Cybulsky M.I. Gimbrone Jr., M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (723) Google Scholar), the transformation of macrophages and smooth muscle cells to foam cells (13.Henriksen T. Mahoney E.M. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6499-6503Crossref PubMed Scopus (816) Google Scholar), the production of various proinflammatory cytokines and growth factors by almost all vascular cells (14.Kume N. Gimbrone Jr., M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (298) Google Scholar, 15.Nakano T. Raines E.W. Abraham J.A. Klagsbrun M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1069-1073Crossref PubMed Scopus (123) Google Scholar), the proliferation and migration of vascular cells (16.Quinn M.T. Parthasarathy S. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2805-2809Crossref PubMed Scopus (584) Google Scholar, 17.Auge N. Andrieu N. Negre-Salvayre A. Thiers J.-C. Levade T. Salvayre R. J. Biol. Chem. 1996; 271: 19251-19255Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 18.Yui S. Sasaki T. Miyazaki A. Horiuchi S. Yamazaki M. Arterioscler. Thromb. 1993; 13: 331-337Crossref PubMed Scopus (104) Google Scholar), and the retardation of endothelial regeneration (19.Murugesan G. Fox P.L. J. Clin. Invest. 1996; 97: 2736-2744Crossref PubMed Scopus (98) Google Scholar), and it increases the procoagulant activity on the vascular cells (20.Aupeix K. Toti F. Satta N. Bischoff P. Freyssinet J.M. Biochem. J. 1996; 314: 1027-1033Crossref PubMed Scopus (48) Google Scholar). These changes eventually result in the formation of atheromatous lesions. Local oxidative degradation of trapped LDL may generate lipid-derived inflammatory mediators, such as oxysterols, lysophospholipids, and fatty acid peroxides. oxLDL and its lipid components have also been shown to be cytotoxic for cultured vascular smooth muscle cells, endothelial cells, macrophages, fibroblasts, and central nervous system cells (5.Morel D.W. DiCorleto P.E. Chisolm G.M. Arteriosclerosis. 1984; 4: 357-364Crossref PubMed Google Scholar, 21.Sugawa M. Ikeda S. Kushima Y. Takashima Y. Cynshi O. Brain Res. 1997; 761: 165-172Crossref PubMed Scopus (48) Google Scholar, 22.Papassotiropoulos A. Ludwig M. Naib-Majami W. Rao G.S. Neurosci. Lett. 1996; 209: 33-36Crossref PubMed Scopus (28) Google Scholar, 23.Draczynska-Lusiak B. Chen Y.M. Sun A.Y. Neuroreport. 1998; 9: 527-532Crossref PubMed Scopus (77) Google Scholar). Recently, this cytotoxicity has been partly attributed to induction of apoptosis. oxLDL induces both the morphological changes and DNA fragmentation characteristic of apoptosis in cultured smooth muscle cells (24.Bjorkerud S. Bjorkerud B. Am. J. Pathol. 1996; 149: 367-380PubMed Google Scholar), macrophages (24.Bjorkerud S. Bjorkerud B. Am. J. Pathol. 1996; 149: 367-380PubMed Google Scholar, 25.Bjorkerud B. Bjorkerud S. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 416-424Crossref PubMed Scopus (165) Google Scholar), endothelial cells (26.Harada-Shiba M. Kinoshita M. Kamido H. Shimokado K. J. Biol. Chem. 1998; 273: 9681-9687Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 27.Escargueil-Blanc I. Meilhac O. Pieraggi M.-T. Arnal J.-F. Salvayre R. Negre-Salvayre A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 331-339Crossref PubMed Scopus (135) Google Scholar), and lymphoid cells (29.Escargueil-Blanc I. Salvayre R. Negre-Salvayre A. FASEB J. 1994; 8: 1075-1080Crossref PubMed Scopus (118) Google Scholar). It has been shown that the apoptosis-inducing activity was recovered in the neutral lipid fraction of oxLDL, and various oxysterols in this fraction induce apoptosis in endothelial cells (26.Harada-Shiba M. Kinoshita M. Kamido H. Shimokado K. J. Biol. Chem. 1998; 273: 9681-9687Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), monocytic cell lines (30.Aupeix K. Weltin D. Mejia J.E. Christ M. Marchal J. Freyssinet J.M. Bischoff P. Immunobiology. 1995; 194: 415-428Crossref PubMed Scopus (102) Google Scholar), thymocytes (31.Christ M. Luu B. Mejia J.E. Moosbrugger I. Bischoff P. Immunology. 1993; 78: 455-460PubMed Google Scholar), and smooth muscle cells (32.Ares M.P. Porn-Ares M.I. Thyberg J. Juntti-Berggren L. Berggren P.O. Diczfalusy U. Kallin B. Bjorkhem I. Orrenius S. Nilsson J. J. Lipid Res. 1997; 38: 2049-2061Abstract Full Text PDF PubMed Google Scholar). However, it is not clear how oxLDL or its active components induce apoptosis in these and other vascular cells. Different agents, such as tumor necrosis factor, γ radiation, UV radiation, hydrogen peroxide (33.de Bono D.P. Yang W.D. Atherosclerosis. 1995; 114: 235-245Abstract Full Text PDF PubMed Scopus (68) Google Scholar), and growth factor removal (34.Araki S. Shimada Y. Kaji K. Hayashi H. Biochem. Biophys. Res. Commun. 1990; 168: 1194-1200Crossref PubMed Scopus (224) Google Scholar), induce apoptosis in many cell types by both unique and common mechanisms. For example, Fas and tumor necrosis factor receptor family members transduce the signal of apoptosis through death domain-containing molecules, such as FADD (35.Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (481) Google Scholar), whereas many other agents induce apoptosis by pathways that do not involve protein molecules with a death domain. On the other hand, almost all known agents that induce apoptosis share the activation of caspases and the execution phase of the death program (36.Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1610) Google Scholar). CHO cells are well suited for mutational analysis of complex cellular pathways (37.Leonard S. Sinensky M. Biochim. Biophys. Acta. 1988; 947: 101-112Crossref PubMed Scopus (23) Google Scholar). Because of this, it is possible to use transfected or mutant CHO cell lines for genetic analysis of the gene products and functions involved in the programmed cell death induced by oxLDL or its components. In this study, we have used CHO-K1 cells stably transfected with CD36 to study the cytotoxic effects of oxLDL and 25-hydroxycholesterol. We have also generated a CHO/CD36 mutant cell line resistant to killing by these two agents. CHO-K1 cells were purchased from the American Type Culture Collection (Manassas, VA). All cell culture reagents were obtained from Life Technologies, Inc. 25-Hydroxycholesterol was from Steraloids Inc. (Wilton, NH). 45Ca2+ was from Amersham Pharmacia Biotech. DiI and fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies were from Molecular Probes, Inc. (Eugene, OR). Anti-CD36 monoclonal antibody was from Pharmingen. Horseradish peroxidase-conjugated goat anti-mouse IgM and IgG and the micro-BCA protein assay kit were from Pierce. The plasmid pcDNA3 carrying human CD36FLAG cDNA was generously provided by Dr. D. M. Lublin (Department of Pathology, Washington University School of Medicine, St. Louis, MO). SREBP constructs were kindly provided by Dr. T. F. Osborne (Department of Molecular Biology and Biochemistry, University of California, Irvine, CA). pTK(K×3)CAT was provided by Dr. David Russell (University of Texas, Southwestern, Dallas, TX). THP-1 cells (TIB-202) were obtained from the American Type Culture Collection and were grown in RPMI 1640 medium supplemented with 10 mm HEPES, 2 mm glutamine, 10% fetal bovine serum, 50 μm 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI medium) under a humidified 5% CO2 atmosphere. For experiments, cells were plated on coverslips at 1 × 106 cells/cm2 in RPMI medium containing 100 nm phorbol 12-myristate-13-acetate (Alexis, San Diego, CA). The cells were allowed to attach and differentiate into macrophages for 72 h. For isolation of permanent transfectants expressing CD36, CHO-K1 cells were grown in Ham's F-12 medium containing 5% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (F12FC5) at 37 °C and 5% CO2and transfected with pcDNA3, carrying human CD36FLAG cDNA and neomycin resistance, by the lipofectam method using a mammalian transfection kit (Stratagene, La Jolla, CA). The neomycin-resistant cells were selected using 500 μg/ml G418 (Life Technologies, Inc.) in F12FC5. Resistant colonies were isolated and assayed for the presence of CD36 and DiI-oxLDL binding, and internalization activity as described below. Colonies expressing CD36 activity were expanded and maintained in F12FC5 containing 500 μg/ml G418. For 25-hydroxycholesterol cytotoxicity assays, CHO-K1 cells were seeded at a density of 500 or 1000 cells/35- or 60-mm dish in F12FC5 on day 0. On day 1, the cells were rinsed with phosphate-buffered saline (PBS) twice and then fed either Nutridoma-SP (1% in Ham's F-12) or F12FC5 containing oxysterols or oxLDL as described in the figure legends. Following incubation, cells were fed fresh F12FC5 and allowed to grow for 5 days. The surviving colonies were then fixed and stained with crystal violet as described (38.Sinensky M. Armagast S. Mueller G. Torget R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6621-6623Crossref PubMed Scopus (13) Google Scholar). Transient transfections of pTK(K×3)CAT (3 μg) were performed on 5 × 104 cells/60-mm plate using the Stratagene Transfection MBS Mammalian Transfection Kit according to the instruction manual with an internal standard expressing β-galactosidase (2 μg), pCMVβ-gal (Promega), to correct for transfection efficiency. Chloramphenicol acetyltransferase assays were as described previously (57.Thewke D.P. Panini S.R. Sinensky M. J. Biol. Chem. 1998; 273: 21402-21407Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Cells (50,000) were plated on glass coverslips and incubated for different periods of time in F12FC5 with or without 10–50 μg/ml oxLDL. After washing with PBS, the cells were fixed in 4% buffered paraformaldehyde for 30 min at room temperature. Coverslips were rinsed, and cells were permeabilized with 1% Triton X-100 in 100 mm sodium citrate buffer and incubated for 1 h at 37 °C with terminal deoxynucleotidyltransferase and fluorescein isothiocyanate-dUTP to label the fragmented DNA. After completion, coverslips were mounted in anti-fade mounting solution and observed under a Nikon Diaphot-200 microscope. LDL (1.019 <d < 1.063) was prepared from normal human serum by sequential ultracentrifugation as described previously (39.Havel R. Eder H. Braigon J. J. Clin. Invest. 1955; 39: 1345-1363Crossref Scopus (6479) Google Scholar). Extensively oxidized LDL was prepared by incubation with CuCl2 as described previously (40.Brown A.J. Leong S.L. Dean R.T. Jessup W. J. Lipid. Res. 1997; 38: 1730-1745Abstract Full Text PDF PubMed Google Scholar). Oxidation of LDL was evaluated by agarose gel electrophoresis. The oxLDL had 3–5-fold higher R F values on agarose gel electrophoresis compared with native LDL. DiI-labeled oxLDL was prepared as described elsewhere (41.Pitas R.E. Innerarity T.L. Weinstein J.N. Mahley R.W. Arteriosclerosis. 1981; 1: 177-185Crossref PubMed Google Scholar). Essentially, oxLDL was incubated with the DiI probe in human lipoprotein-deficient serum for 12 h at 37 °C, using the following relative amounts: 300 μl (300 μg) of DiI, 3 mg of lipoprotein lipid, 2 ml of lipoprotein-deficient serum. Subsequently, the labeled lipoproteins are reisolated by ultracentrifugation for 2.5 h at 99,000 rpm in a TL-100 centrifuge. Labeled lipoproteins were stored at 4 °C in the dark and used within 2 weeks after their preparation. Transfected cells were grown overnight on glass coverslips and then incubated with 10 μg/ml DiI-oxLDL for 1 h at 37 °C. After washing with PBS, cells were fixed in freshly prepared 4% formaldehyde in PBS and mounted. Cell association of DiI-labeled oxLDL was observed by fluorescence microscopy using a Texas Red filter cube. Alternatively, after fixation, standard immunofluorescence was performed with anti-CD36- or anti-FLAG-specific monoclonal antibodies. Detection of oligonucleosomal DNA fragments was done as described previously (42.Meßmer U.K. Reed J.C. Brüne B. J. Biol. Chem. 1996; 271: 20192-20197Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Basically, cells at an initial density of 5 × 105/100-mm dish were cultured for 24 h in F12FC5 plus oxLDL or oxysterols and then harvested, lysed, and centrifuged to separate DNA fragments from intact chromatin. Supernatants were precipitated overnight at −20 °C with 2 volumes of ethanol and centrifuged at 13,000 × g for 15 min. Then pellets were incubated for 30 min at 37 °C in 500 μl of Tris-EDTA (TE) buffer supplemented with 100 μg/ml RNase A. Samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and once again with chloroform/isoamylalcohol (24:1). DNA was precipitated, and pellets were recovered by centrifugation (13,000 × g, 15 min), air-dried, resuspended in 10 μl of TE buffer, supplemented with 2 μl of sample buffer (0.25% bromphenol blue, 30% glycerol), and electrophoretically separated on a 1.3% agarose gel. After electrophoresis, they were stained in ethidium bromide (1 μg/ml). Pictures were taken by UV transillumination. CHO-K1 cells were plated and treated as described above. After treatment, both treated and nontreated cells were harvested and collected by centrifugation (800 ×g for 10 min). Cells were washed twice with ice-cold PBS and lysed with cell lysis buffer (10 mm Tris (pH 7.5), 130 mm NaCl, 1% Triton X-100, 10 mm NaF, 10 mm NaPi, and 10 mmNaPPi). Samples were incubated on ice for 10 min, passed through a 21-gauge syringe 15 times, and then centrifuged at 15,000 × g for 20 min at 4 °C. Protein concentration in the supernatant (total cell lysate) was measured by the micro-BCA kit. Triplicates of 250 μg of total cell lysate protein were incubated with Ac-DEVD-AMC (20 μm) caspase-3 substrate or substrate plus the specific inhibitor Ac-DEVD-CHO (100 nm) for 1.5 h at 37 °C in protease assay buffer (20 mm HEPES, pH 7.5, 10% glycerol, 2 mmdithiothreitol). Liberated AMC from Ac-DEVD-AMC was measured on a spectrofluorometer with an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Cells (1.5 × 105/35-mm plate) were incubated with 3 μg/ml 25-[26,27-3H]hydroxycholesterol (20,000 dpm/μg) for 18 h. The dishes were extracted twice with 3 ml of hexane/isopropyl alcohol (60:40). The fixed cells were digested for 3 h in 1 ml of 0.1 n NaOH, and 0.1-ml aliquots were assayed for protein (BCA, Pierce). The lipid extract was concentrated to 0.1 ml by evaporation under nitrogen and spotted onto silicic acid TLC plates, and the components were separated with the solvent system hexane/diethyl ether/HAc (80:20:1). The 25-hydroxycholesterol ester was visualized by fluorography, and the bands were scraped and quantitated by liquid scintillation counting. Kinetic measurements of calcium were performed by a dipping technique as described previously (58.Yarom M. Zurgil N. Zisapel N. J. Biol. Chem. 1985; 260: 16286-16293Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were plated on glass coverslips at a density of 5 × 105 cells/35 mm dish. After an overnight incubation in F12FC5, coverslips were sequentially washed in a reference buffer. At time 0, the coverslips were dipped in a beaker containing Ca2+ and 45Ca2+ in reference buffer with or without 25-hydroxycholesterol. After the indicated period of time, cells were washed and solubilized in 1% SDS, and radioactivity was measured by liquid scintillation. Chemical modifications of LDL, such as oxidation, convert LDL into a high affinity ligand of scavenger receptors. oxLDL is cytotoxic for many cell types in the vessel wall, and its interaction with the macrophage scavenger receptors and subsequent toxic effects play a crucial role in the initiation of the atherosclerotic lesion. To test if uptake of oxLDL is sufficient to elicit cytotoxic effects in a fibroblastic cell line, we have transfected CHO-K1 with CD36, a scavenger receptor structurally related to SR-BI and its human counterpart CLA-I. This receptor has been reported to bind oxLDL and acetylated LDL. (43.Calvo D. Gomez-Coronado D. Suarez Y. Lasuncion M.A. Vega M.A. J. Lipid. Res. 1998; 39: 777-788Abstract Full Text Full Text PDF PubMed Google Scholar, 53.Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar). Cell association of oxLDL to CHO-K1 expressing CD36 was examined by fluorescence microscopy after incubating DiI-labeled oxLDL with CHO-K1 stably transfected with a vector carrying CD36FLAG (CHO/CD36). Untransfected CHO cells displayed a diffuse light staining, possibly due to traces of free DiI or uptake through other scavenger receptors (Fig.1 A). However, when CHO/CD36 cells were incubated with DiI-oxLDL, a subset of cells, corresponding to the number of cells expressing CD36, as determined by immunofluorescence with monoclonal antibodies specific against CD36 or FLAG (results not shown), showed both an internal punctuated staining pattern (probably endosomes and lysosomes) and an intense plasma membrane staining (Fig. 1 B). Specificity of the interaction was proven by the ability of unlabeled oxLDL (50-fold excess) to inhibit the DiI-oxLDL staining (Fig. 1 C). These findings confirm previous reports (44.Nicholson A.C. Frieda S. Pearce A. Silverstein R.L. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 269-275Crossref PubMed Scopus (222) Google Scholar) that CD36 when expressed on CHO cells binds and allows internalization of oxLDL. Using a single-cell plating assay to determine cytotoxicity, we found that expression of CD36 in CHO cells renders the cells susceptible to killing by oxLDL (Fig.2). In this assay, 500 cells are plated on 35-mm dishes, subjected to different treatments, and allowed to form colonies, which can be stained and counted. Fig. 2 shows that treatment of CHO/CD36 with 10 μg/ml of oxLDL for 5 days eliminates formation of colonies, indicating cytotoxicity, whereas untransfected CHO-K1 cells were not affected by this treatment. oxLDL induced apoptosis in a time- and dose-dependent manner (Fig.3). Approximately 10 or 20% of the CHO/CD36 cells became TUNEL-positive after a 48-h incubation with 10 or 50 μg of protein/ml of oxLDL, respectively (Fig. 3, A andB). Commensurate with the results derived from the TUNEL assay, DNA ladders were present in genomic DNA extracted from CHO/CD36 cells incubated with similar oxLDL concentrations (Fig. 3 C). The fragmented DNA showed the distinct pattern of oligonucleosomes found in apoptotic cells. oxLDL (10 μg/ml) induced significant apoptosis in CHO/CD36 cells as early as at 16 h of incubation (data not shown), and the number of apoptotic cells increased up until 48 h of incubation. Incubation of CHO/CD36 with native LDL or incubation of CHO-K1 with oxLDL did not induce apoptosis beyond the control level (less than 1% of total cells) (data not shown). These findings indicate that oxLDL is able to induce apoptosis in cultured fibroblasts when the cells are able to bind and/or internalize oxLDL. Several recent studies have shown that much of the cytotoxicity of oxLDL is associated with the neutral lipid components, in particular the oxysterols (45.Chisolm G.M. Ma G. Irwin K.C. Martin L.L. Gunderson K.G. Linberg L.F. Morel D.W. DiCorleto P.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11452-11456Crossref PubMed Scopus (191) Google Scholar, 46.Sevanian A. Hodis H.N. Hwang J. McLeod L.L. Peterson H. J. Lipid. Res. 1995; 36: 1971-1986Abstract Full Text PDF PubMed Google Scholar). The cytotoxicity of oxysterols may be evidenced by a number of mechanisms, from effects on cholesterol synthesis to effects on cell membranes to induction of apoptosis. (30.Aupeix K. Weltin D. Mejia J.E. Christ M. Marchal J. Freyssinet J.M. Bischoff P. Immunobiology. 1995; 194: 415-428Crossref PubMed Scopus (102) Google Scholar,47.Ayala-Torres S. Moller P.C. Johnson B.H. Thompson E.B. Exp. Cell. Res. 1997; 235: 35-47Crossref PubMed Scopus (38) Google Scholar). In the next set of experiments, we addressed the following two main questions. 1) Are oxysterol-resistant mutants cross-resistant to oxLDL? 2) Does transcriptional down-regulation of cholesterol synthesis by oxysterols play a role in their induction of apoptosis? Fig. 4 shows the results of a representative single-cell plating experiment as a function of time of exposure to 3 μg/ml 25-hydroxycholesterol in the presence of serum as the exogenous source of cholesterol. A significant reduction (∼50%) in the number of viable cells was seen after a 6-h incubation. Furthermore, after a 12-h incubation, virtually all treated cells were dead. The mode of cell death was confirmed to be through apoptosis by TUNEL assay (Fig. 5, A andB), oligonucleosomal DNA laddering assay (Fig.5 C), and caspase 3 activation assay (TableI).Figure 525-Hydroxycholesterol induces apoptosis in CHO/CD36 cells. A, apoptosis assay. Cells (30,000) were plated on glass coverslips and incubated for 24 h in F12FC5 containing 0, 1, 3, or 5 μg/ml 25-hydroxycholesterol. Apoptosis was measured by TUNEL as described in the legend to Fig. 3. B, gel electrophoresis of DNA. Cells at an initial density 5 × 105 were cultured for 24 h in F12FC5, with or without 3 μg/ml 25-hydroxycholesterol, and incubated for 24 h and then harvested and lysed, and DNA fragments were detected as described in the legend to Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ISpectrofluorometric analysis of caspase-3 activity for CHO cells treated with and without 25-hydroxycholesterolCHO-K1 Cell lysateAc-DEVD-AMCAc-DEVD-CHORelative AMC fluorescenceNet Relative AMC fluorescenceUntreated+−0.267 ± 0.0150.063 ± 0.021++0.203 ± 0.01225-OHC (48 h)+−0.560 ± 0.0200.457 ± 0.025++0.103 ± 0.005CHO-K1 cells were treated with 0 or 3 μg/ml 25-hydroxycholesterol for 48 h, and 250 μg of total cell lysate protein were assayed for caspase-3 as described under “Experimental Procedures.” Data are the average of triplicates ± S.D. Open table in a new tab CHO-K1 cells were treated with 0 or 3 μg/ml 25-hydroxycholesterol for 48 h, and 250 μg of total cell lysate protein were assayed for caspase-3 as described under “Experimental Procedures.” Data are the average of triplicates ± S.D. If oxysterols are the mediators of the cytotoxic effects of oxLDL, an oxysterol-resistant mutant should be resistant to killing by oxLDL. We mutagenized CHO/CD36 with methanesulfonic acid ethyl ester and selected colonies that grew in medium containing 3 μg/ml 25-hydroxycholesterol (25-OHC). As shown in Fig. 6 A, OXRcells are resistant to killing by 3 μg/ml 25-hydroxycholesterol. These cells were also resistant to killing by 35 μg/ml oxLDL (Fig.6 B). Fig. 6 C shows that the staining pattern of OXR cells with DiI-oxLDL is similar to that in CHO/CD36, suggesting that binding and internalization of oxLDL in the OXR cell line is unaltered from its parental cell line. A general defect in one or more steps in the apoptosis pathway per se (e.g. defective caspase 3, overexpression of Bcl-2, etc.) would produce a similar resistance phenotype. Therefore, we also confirmed that OXR cells were capable of undergoing apoptosis in response to another known apoptosis inducer. Fig.6 A shows that OXR cells are susceptible to staurosporine, a reagent that induces apoptosis through inhibition of protein kinase C, and therefore seemed to have a functioning apoptosis pathway at least in the execution phase of the death program. The best known biological activity of oxysterols is transcriptional repression of cholesterol biosynthesis, through inhibition of processing of the SREBPs, although other activities for these molecules have recently come to be appreciated (62.Edwards P.A. Ericsson J. Annu. Rev. Biochem. 1999; 68: 157-185Crossref PubMed Scopus (389) Google Scholar). Oxysterols have been reported to initiate apoptosis in CEM leukemic cells by interfering with the synthesis of cholesterol in the absence of an exogenous cholesterol source (47.Ayala-Torres S. Moller P.C. Johnson B.H. Thompson E.B. Exp. Cell. Res. 1997; 235: 35-47Crossref PubMed Scopus (38) Google Scholar). Since we carried out our incubations in the presence of serum, as an exogenous cholesterol source, we would expect that the apoptotic pathway would not be mediated by inhibition of cholesterol synthesis. However, we dir" @default.
• W2018695514 created "2016-06-24" @default.
• W2018695514 creator A5015055428 @default.
• W2018695514 creator A5017242409 @default.
• W2018695514 creator A5042017214 @default.
• W2018695514 creator A5050831698 @default.
• W2018695514 creator A5053033763 @default.
• W2018695514 creator A5078666853 @default.
• W2018695514 date "2000-03-01" @default.
• W2018695514 modified "2023-10-17" @default.
• W2018695514 title "Isolation of a Somatic Cell Mutant Resistant to the Induction of Apoptosis by Oxidized Low Density Lipoprotein" @default.
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