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• W2092111990 abstract "We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol. We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol. Vitamin A (retinol) and its natural metabolites and synthetic derivatives, known as retinoids, play important roles in regulating cell proliferation, differentiation, and embryonic development (1Means A.L. Gudas L.J. The roles of retinoids in vertebrate development..Annu. Rev. Biochem. 1995; 64: 210-233Crossref Scopus (268) Google Scholar, 2Hofmann C. Eichele G. Retinoids in development..in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 387-441Google Scholar, 3Gudas L.J. Sporn M.B. Roberts A. Cellular biology and biochemistry of the retinoids..in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 443-520Google Scholar). The physiological activities of retinoids are primarily carried out via nuclear receptors that transcriptionally activate certain retinoid-responsive genes. Two types of retinoid receptors have been identified, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with three subtypes, α, β, and γ (4Lefebvre P. Martin P.J. Flajollet S. Dedieu S. Billaut X. Lefebvre B. Transcriptional activities of retinoic acid receptors..Vitam. Horm. 2005; 70: 199-264Crossref PubMed Scopus (110) Google Scholar, 5Chambon P. A decade of molecular biology of retinoic acid receptors..FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 6Gudas L.J. Retinoids and vertebrate development..J. Biol. Chem. 1994; 269: 15399-15402Abstract Full Text PDF PubMed Google Scholar). RAR/RXR heterodimers bind to specific DNA sequences known as retinoic acid response elements (7de Thé H. Vivanco-Ruiz M. Tiollais P. Stunnenberg H. Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor b gene..Nature. 1990; 343: 177-180Crossref PubMed Scopus (842) Google Scholar). Ligand binding of RARs and RXRs regulates the transcription of retinoic acid-responsive genes. The actions of RARs and RXRs on gene transcription also require a highly coordinated interaction with a large number of coactivators and corepressors (8Privalsky M.L. The role of corepressors in transcriptional regulation by nuclear hormone receptors..Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (258) Google Scholar).The appropriate intracellular retinoid concentrations are maintained by the activities of several metabolic enzymes (9Blaner W.S. Olson J.A. Retinol and retinoic acid metabolism..in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 229-256Google Scholar, 10Napoli J.L. Biochemical pathways of retinoid transport, metabolism, and signal transduction..Clin. Immun. Immunopathol. 1996; 80: 52-62Crossref PubMed Scopus (175) Google Scholar, 11Duester G. Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis..Biochemistry. 1996; 35: 12221-12227Crossref PubMed Scopus (233) Google Scholar). All-trans retinol (ROH; vitamin A) is the precursor of other natural retinoids. Retinol dehydrogenases oxidize ROH to all-trans retinaldehyde, and then all-trans retinaldehyde is further metabolized to all-trans retinoic acid (ATRA) by retinaldehyde dehydrogenases (11Duester G. Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis..Biochemistry. 1996; 35: 12221-12227Crossref PubMed Scopus (233) Google Scholar). Currently, four members of the retinaldehyde dehydrogenase family have been identified, retinaldehyde dehydrogenases 1, 2, 3, and 4 (12Zhao D. McCaffery P. Ivins K.J. Neve R.L. Hogan P. Chin W.W. Dräger U.C. Molecular identification of a major retinoic-acid-synthesizing enzyme, a retinaldehyde-specific dehydrogenase..Eur. J. Biochem. 1996; 240: 15-22Crossref PubMed Scopus (254) Google Scholar). ROH is converted to all-trans retinyl esters primarily by lecithin:retinol acyltransferase (LRAT), and retinyl esters (REs) are hydrolyzed to retinol by ester hydrolases (9Blaner W.S. Olson J.A. Retinol and retinoic acid metabolism..in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 229-256Google Scholar, 13Haeseleer F. Huang J. Lebioda L. Saari J.C. Palczewski K. Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal..J. Biol. Chem. 1998; 273: 21790-21799Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 14Napoli J.L. Retinoic acid: its biosynthesis and metabolism..Prog. Nucleic Acid Res. Mol. Biol. 1999; 63: 139-188Crossref PubMed Google Scholar, 15Mondal M.S. Ruiz A. Hu J. Bok D. Rando R.R. Two histidine residues are essential for catalysis by lecithin retinol acyl transferase..FEBS Lett. 2001; 489: 14-18Crossref PubMed Scopus (15) Google Scholar). REs are regarded as the storage forms of retinol (9Blaner W.S. Olson J.A. Retinol and retinoic acid metabolism..in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 229-256Google Scholar). ATRA is oxidized to more polar metabolites, such as 4-oxo-retinoic acid, by a cytochrome P450 family member (CYP26) (16Abu-Abed S.S. Beckett B.R. Chiba H. Chithalen J.V. Jones G. Metzger D. Chambon P. Petkovich M. Mouse P450RAI (CYP26) expression and retinoic acid-inducible retinoic acid metabolism in F9 cells are regulated by retinoic acid receptor gamma and retinoid X receptor alpha..J. Biol. Chem. 1998; 273: 2409-2415Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Lampen A. Meyer S. Nau H. Effects of receptor-selective retinoids on CYP26 gene expression and metabolism of all-trans-retinoic acid in intestinal cells..Drug Metab. Dispos. 2001; 29: 742-747PubMed Google Scholar, 18Sonneveld E. van den Brink C.E. van der Leede B. J.M. Schulkes R. K. A.M. Petkovich M. van der Burg B. van der Saag P.T. Human retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells..Cell Growth Differ. 1998; 9: 629-637PubMed Google Scholar, 19White J.A. Beckett-Jones B. Guo Y-D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP450)..J. Biol. Chem. 1997; 272: 18538-18541Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar).Retinoids are required for the appropriate differentiation of normal rodent prostate epithelial cells (20Lasnitzki I. Goodman D.S. Hypovitaminosis A in the mouse prostate gland cultured in chemically defined medium..Exp. Cell Res. 1962; 28: 40-51Crossref Scopus (39) Google Scholar). Epidemiological data have implicated retinoids in the prevention of human prostate cancer (21Nanus D.M. Gudas L.J. Retinoids and prostate cancer..Prostate J. 2000; 2: 68-73Crossref Scopus (3) Google Scholar). An inverse correlation between serum levels of retinoids and prostate cancer incidence has been reported (22Hsing A.W. Comstock G.W. Abbey H. Polk B.F. Serologic precursors of cancer. Retinol, carotenoids, and tocopherol and risk of prostate cancer..J. Natl. Cancer Inst. 1990; 82: 941-946Crossref PubMed Scopus (198) Google Scholar, 23Reichman M.E. Hayes R.B. Ziegler R.G. Schatzkin A. Taylor P.R. Kahle L.L. Fraumeni Jr., J.F. Serum vitamin A and subsequent development of prostate cancer in the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study..Cancer Res. 1990; 50: 2311-2315PubMed Google Scholar). Moreover, human prostate cancer tissues contain five to eight times less ATRA than normal prostate or benign prostatic hyperplasia (24Pasquali D. Thaller C. Eichele G. Abnormal level of retinoic acid in prostate cancer tissues..J. Clin. Endocrinol. Metab. 1996; 81: 2186-2191Crossref PubMed Scopus (78) Google Scholar). We have shown that vitamin A (retinol) metabolism is altered in some types of human cancer, such as oral cavity cancer, skin cancer, and breast cancer (25Guo X. Gudas L.J. Metabolism of all-trans-retinol in normal human cell strains and squamous cell carcinoma lines from the oral cavity and skin: reduced esterification of retinol in SCC lines..Cancer Res. 1998; 58: 166-176PubMed Google Scholar, 26Guo X. Ruiz A. Rando R.R. Bok D. Gudas L.J. Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines..Carcinogenesis. 2000; 21: 1925-1933Crossref PubMed Scopus (57) Google Scholar, 27Guo X. Nanus D.M. Ruiz A. Rando R.R. Bok D. Gudas L.J. Reduced levels of retinyl esters and vitamin A in human renal cancers..Cancer Res. 2001; 61: 2774-2781PubMed Google Scholar). In addition, our laboratory showed that both the esterification of ROH to REs and the levels of LRAT, an enzyme involved in vitamin A esterification and storage, were greatly decreased in human prostate cancer cell lines and in patient tumor samples (28Guo X. Knudsen B.S. Peehl D.M. Ruiz A. Bok D. Rando R.R. Rhim J.S. Nanus D.M. Gudas L.J. Retinol metabolism and lecithin:retinol acyltransferase levels are reduced in cultured human prostate cancer cells and tissue specimens..Cancer Res. 2002; 62: 1654-1661PubMed Google Scholar).Huang et al. (29Huang J. Powell W.C. Khodavirdi A.C. Wu J. Makita T. Cardiff R.D. Cohen M.B. Sucov H.M. Roy-Burman P. Prostatic intraepithelial neoplasia in mice with conditional disruption of the retinoid X receptor alpha allele in the prostate epithelium..Cancer Res. 2002; 62: 4812-4819PubMed Google Scholar) have shown that mice with both RXRα alleles specifically disrupted in prostate epithelium develop prostate hyperplasia. Conversely, RXR selective retinoids demonstrated efficacy in the prevention of prostate cancer in a rodent model (30McCormick D.L. Rao K. V.N. Steele V.E. Lubet R.A. Kelloff G.J. Bosland M.C. Chemoprevention of rat prostate carcinogenesis by 9-cis retinoic acid..Cancer Res. 1999; 59: 521-524PubMed Google Scholar). RXR selective retinoids were shown to inhibit the clonal growth of prostate cancer cells (31de Vos S. Dawson M.I. Holden S. Le T. Wang A. Cho S.K. Chen D.L. Koeffler H.P. Effects of retinoid X receptor-selective ligands on proliferation of prostate cancer cells..Prostate. 1997; 32: 115-121Crossref PubMed Scopus (48) Google Scholar). Recently, a selective RXR agonist was shown to prevent and overcome multidrug resistance in human prostate cancer PC-3 cells (32Yen W.C. Lamph W.W. A selective retinoid X receptor agonist bexarotene (LGD1069, Targretin) prevents and overcomes multidrug resistance in advanced prostate cancer..Prostate. 2006; 66: 305-316Crossref PubMed Scopus (35) Google Scholar). However, the mechanisms by which the inhibition of cell proliferation is achieved are still not clear. Therefore, it is important to explore the effects of RXR ligands on the metabolism of retinol, including the esterification of retinol.Some unsaturated fatty acids have been shown to be physiological RXR ligands (33Goldstein J.T. Dobrzyn A. Clagett-Dame M. Pike J.W. DeLuca H.F. Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor..Arch. Biochem. Biophys. 2003; 420: 185-193Crossref PubMed Scopus (64) Google Scholar, 34Steineger H.H. Arntsen B.M. Spydevold O. Sorensen H.N. Gene transcription of the retinoid X receptor alpha (RXRalpha) is regulated by fatty acids and hormones in rat hepatic cells..J. Lipid Res. 1998; 39: 744-754Abstract Full Text Full Text PDF PubMed Google Scholar). Phytanic acid (PA) and docosahexaenoic acid (DHA) are dietary ligands of RXRs (35de Urquiza A.M. Liu S. Sjoberg M. Zetterstrom R.H. Griffiths W. Sjovall J. Perlmann T. Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain..Science. 2000; 290: 2140-2144Crossref PubMed Scopus (642) Google Scholar, 36Kitareewan S. Burka L.T. Tomer K.B. Parker C.E. Deterding L.J. Stevens R.D. Forman B.M. Mais D.E. Heyman R.A. McMorris T. et al.Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR..Mol. Biol. Cell. 1996; 7: 1153-1166Crossref PubMed Scopus (164) Google Scholar, 37Lemotte P.K. Keidel S. Apfel C.M. Phytanic acid is a retinoid X receptor ligand..Eur. J. Biochem. 1996; 236: 328-333Crossref PubMed Scopus (141) Google Scholar, 38Radominska-Pandya A. Chen G. Photoaffinity labeling of human retinoid X receptor b (RXRb) with 9-cis-retinoic acid: identification of phytanic acid, docosahexaenoic acid, and lithocholic acid as ligands for RXRb..Biochemistry. 2002; 41: 4883-4890Crossref PubMed Scopus (37) Google Scholar). PA (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid generated by the oxidation of the phytol side chain of chlorophyll in mammals (39Jansen G.A. Mihalik S.J. Watkins P.A. Moser H.W. Jakobs C. Denis S. Wanders R.J. Phytanoyl-CoA hydroxylase is present in human liver, located in peroxisomes, and deficient in Zellweger syndrome: direct, unequivocal evidence for the new, revised pathway of phytanic acid alpha-oxidation in humans..Biochem. Biophys. Res. Commun. 1996; 229: 205-210Crossref PubMed Scopus (69) Google Scholar). Because humans cannot release phytol from chlorophyll, PA in the human body comes from dairy products and ruminant fats in the diet (40Baxter J.H. Absorption of chlorophyll phytol in normal man and in patients with Refsum's disease..J. Lipid Res. 1968; 9: 636-641Abstract Full Text PDF PubMed Google Scholar). DHA is a long-chain ω-3 polyunsaturated fatty acid that is present at high levels in fish oils. ω-3 fatty acids have various physiological functions (41Seo T. Blaner W.S. Deckelbaum R.J. Omega-3 fatty acids: molecular approaches to optimal biological outcomes..Curr. Opin. Lipidol. 2005; 16: 11-18Crossref PubMed Scopus (98) Google Scholar). DHA inhibits cell proliferation and induces apoptosis of breast cancer cells (42Schley P.D. Jijon H.B. Robinson L.E. Field C.J. Mechanisms of omega-3 fatty acid-induced growth inhibition in MDA-MB-231 human breast cancer cells..Breast Cancer Res. Treat. 2005; 92: 187-195Crossref PubMed Scopus (158) Google Scholar) and human colon cancer cells (43Danbara N. Yuri T. Tsujita-Kyutoku M. Sato M. Senzaki H. Takada H. Hada T. Miyazawa T. Okazaki K. Tsubura A. Conjugated docosahexaenoic acid is a potent inducer of cell cycle arrest and apoptosis and inhibits growth of colo 201 human colon cancer cells..Nutr. Cancer. 2004; 50: 71-79Crossref PubMed Scopus (40) Google Scholar). Merendino et al. (44Merendino N. Molinari R. Loppi B. Pessina G. D'Aquino M. Tomassi G. Velottia F. Induction of apoptosis in human pancreatic cancer cells by docosahexaenoic acid..Ann. N. Y. Acad. Sci. 2003; 1010: 361-364Crossref PubMed Scopus (27) Google Scholar) found that DHA also induced apoptosis of human pancreatic cancer cells.With respect to prostate cancer, dietary studies in humans have shown that both fish oils and DHA have protective properties (45Norrish A.E. Skeaff C.M. Arribas G.L. Sharpe S.J. Jackson R.T. Prostate cancer risk and consumption of fish oils: a dietary biomarker-based case-control study..Br. J. Cancer. 1999; 81: 1238-1242Crossref PubMed Scopus (170) Google Scholar). DHA can inhibit the growth of androgen-responsive LNCaP cells (46Chung B.H. Mitchell S.H. Zhang J.S. Young C.Y. Effects of docosahexaenoic acid and eicosapentaenoic acid on androgen-mediated cell growth and gene expression in LNCaP prostate cancer cells..Carcinogenesis. 2001; 22: 1201-1206Crossref PubMed Scopus (59) Google Scholar) and androgen-unresponsive PC-3 cells and DU-145 cells (47Rose D.P. Connolly J.M. Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines..Prostate. 1991; 18: 243-254Crossref PubMed Scopus (180) Google Scholar). Lampen, Meyer, and Nau (48Lampen A. Meyer S. Nau H. Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells..Biochim. Biophys. Acta. 2001; 1521: 97-106Crossref PubMed Scopus (26) Google Scholar) reported that PA and DHA enhanced the induction of CYP26A1 expression and retinoic acid oxidation by RAR ligands in cells. However, the effects of RXR ligands on the metabolism of retinol have not been examined.Therefore, we examined the effects of a synthetic RXR ligand (BMS 188649) (49Chen J.Y. Clifford J. Zusi C. Starrett J. Tortolani D. Ostrowski J. Reczek P.R. Chambon P. Gronemeyer H. Two distinct actions of retinoid-receptor ligands..Nature. 1996; 382: 819-822Crossref PubMed Scopus (181) Google Scholar) and these RXR natural ligands, PA and DHA, on the proliferation of human PC-3 prostate cancer cells (androgen-unresponsive prostate cancer cells). We also investigated the effects of these drugs on retinol metabolism in normal human prostate epithelial cells (PrECs), PC-3 cells, and PC-3 cells engineered to overexpress stably human LRAT. We found that both PA and DHA inhibited the proliferation of PC-3 cancer cells and PC-3 cells engineered to overexpress stably human LRAT. Furthermore, both drugs, combined with ROH, generated novel REs through different mechanisms in cultured PrECs and in PC-3 cells. Our results indicate that LRAT catalyzes the esterification of ROH and PA to retinyl phytanate, whereas retinyl docosahexaenoate generated in the presence of DHA and ROH is produced by an enzyme other than LRAT.MATERIALS AND METHODSMaterialsRadiolabeled retinol (all-trans 11,12-[3H]) was purchased from New England Nuclear/Dupont (Boston, MA). The specific RXR agonist BMS 188649 was from the Bristol-Myers Squibb Pharmaceutical Research Institute (Buffalo, NY), as described previously (50Egea P.F. Mitschler A. Moras D. Molecular recognition of agonist ligands by RXRs..Mol Endocrinol. 2002; 16: 987-997Crossref PubMed Scopus (0) Google Scholar, 51Lehmann J.M. Jong L. Fanjul A. Cameron J.F. Lu X.P. Haefner P. Dawson M.I. Pfahl M. Retinoids selective for retinoid X receptor response pathways..Science. 1992; 258: 1944-1946Crossref PubMed Scopus (326) Google Scholar). DHA was purchased from Sigma Chemical Co. (St. Louis, MO) and Cayman Chemical Co. (Ann Arbor, MI). Beta-flow and mono-flow scintillation fluids were purchased from National Diagnostics (Atlanta, GA). All primers for PCR were from MWG-Biotech, Inc. (High Point, NC). TRIzol reagent and Superscript III reverse transcriptase were from Invitrogen (Carlsbad, CA). The SYBR Green I Quantitect kit was purchased from Qiagen (Valencia, CA). All other chemicals used in this research, unless specified, were purchased from Sigma Chemical Co.Cell culturePrECs were purchased from Cambrex (Walkersville, MD) and were cultured according to the instructions of the manufacturer. PC-3 human prostate carcinoma cells were from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 supplemented with 10% fetal calf serum. PC-3 cells that stably overexpress human LRAT (line RL-PC-3/LRAT-4, generated in our laboratory) were maintained in the same medium as the PC-3 parental cells. For radiolabeling studies, RT-PCR, and Western blot analyses, PrECs and PC-3 cells were cultured in a mixture of 50% prostate epithelial cell growth medium and 50% RPMI 1640 supplemented with 10% fetal calf serum only during the periods of drug treatment, because retinoids are not stable in serum-free medium.Stable transfection of the human LRAT in PC-3 cellsAn 800 bp PCR fragment of the human LRAT cDNA was cloned into the EcoRI and XhoI sites of the pcDNA3.1/V5-His A vector (Invitrogen). DNA sequencing confirmed that no mutations were introduced during the PCR cloning process and that the cDNA contained the correct sequence. Electroporation was performed as described previously (52Boylan J.F. Lufkin T. Achkar C.C. Taneja R. Chambon P. Gudas L.J. Targeted disruption of retinoic acid receptor a (RAR a) and RAR g results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism..Mol. Cell. Biol. 1995; 15: 843-851Crossref PubMed Google Scholar). PC-3 cells were electroporated with 20 μg of pcDNA3.1/V5-His A-LRAT at 200 mV and 960 μF. After 48 h, cells were selected with G418 (300 μg/ml active drug; Sigma) for 2 weeks. Several independent cell colonies were picked, expanded, frozen, and tested for LRAT activity. These stably transfected colonies exhibited ∼5- to 10-fold more LRAT activity, as measured by retinol esterification in living cells, than that in the parental PC-3 cells (data not shown).Cell proliferation assaysPC-3 and RL-PC-3/LRAT-4 cells that stably overexpress human LRAT were seeded on 24-well plates in triplicate at a density of 10,000 cells/ml/well. The next day, cells were cultured in the presence of 1 μM ATRA, 5 μM RXR agonist (BMS 188649), or different concentrations of PA or DHA in the absence or presence of 1 μM ROH. Fresh drugs were added every 2 days. After 72 and 120 h of culture in the presence or absence of the drugs, the cells were counted in triplicate using a Coulter counter.[3H]retinol radiolabeling, retinoid extraction, and HPLCPrECs and PC-3 cells were plated in 60 mm2 dishes at a density of ∼4 × 105 cells/dish. PrECs and PC-3 cells were treated with various drugs in the presence of 100 nM [3H]retinol for 6, 12, and 24 h, and cell samples were harvested. The metabolism of [3H]retinol was determined by HPLC analyses of retinoids extracted from harvested cells. Alternatively, cells were plated in 150 mm dishes and cultured with or without drugs in the presence or absence of 1 μM nonradiolabeled retinol for 48 h. Cell samples were then harvested and retinoids were extracted. Cell numbers were counted in duplicate dishes, and the metabolism data were normalized to the cell number.The extraction of retinoids and the HPLC conditions were as described previously (25Guo X. Gudas L.J. Metabolism of all-trans-retinol in normal human cell strains and squamous cell carcinoma lines from the oral cavity and skin: reduced esterification of retinol in SCC lines..Cancer Res. 1998; 58: 166-176PubMed Google Scholar). For the radiolabeled samples, nonradiolabeled retinoid standards [ATRA, ROH, and retinyl palmitate (RP)] were added to the radiolabeled samples before extraction. For nonradiolabeled samples, standards only were run as a separate sample in each experiment. For extraction, 350 μl of acetonitrile-butanol (50:50, v/v) and 0.1% butylated hydroxytoluene were added to a total volume of 0.5 ml of cell suspension in PBS. The mixtures were vortexed thoroughly for 60 s. After the addition of 300 μl of a saturated (1.3 kg/l) K2HPO4 solution and thorough mixing, the samples were centrifuged for 10 min at 12,000 g at room temperature. The upper organic layers were collected and transferred to injector vials for automated HPLC analysis. Retinoids from nonradiolabeled samples were extracted in the same manner except that only one standard, retinyl acetate, was added to each sample. (Human cells do not synthesize retinyl acetate, so retinyl acetate is not one of the endogenous retinoids.)The HPLC analysis was performed using a Waters Millennium system (Waters Corp., Milford, MA) to separate the various retinoids. Samples were applied to an analytical 5 μm reverse-phase C18 column (Vydac, Hesperia, CA) at a flow rate of 1.5 ml/min (25Guo X. Gudas L.J. Metabolism of all-trans-retinol in normal human cell strains and squamous cell carcinoma lines from the oral cavity and skin: reduced esterification of retinol in SCC lines..Cancer Res. 1998; 58: 166-176PubMed Google Scholar). Retinoids were identified by HPLC based on two criteria: an exact match of the retention times of unknown peaks with those of authentic retinoid standards, and identical ultraviolet light spectra (220–400 nm) of unknowns against spectra from authentic retinoid standards during HPLC by the use of a photodiode array detector.Mass spectrometric analysis of retinoidsTo identify REs generated during the drug treatments, mass spectrometry (MS) was used as described previously (53Suh M.J. Tang X.H. Gudas L.J. Structure elucidation of retinoids in biological samples using postsource decay laser desorption/ionization mass spectrometry after high-performance liquid chromatography separation..Anal. Chem. 2006; 78: 5719-5728Crossref PubMed Scopus (6) Google Scholar). PC-3 cells that stably overexpress exogenous human LRAT (RL-PC-3/LRAT-4) were plated in 150 mm2 dishes and cultured with various drugs in the presence or absence of 1 μM nonradiolabeled retinol for 48 h, and the cell samples were then harvested. The extracted retinoids were separated by HPLC, and appropriate fractions were collected. The HPLC fractions were dried by SpeedVac and redissolved in 5 μl of absolute ethanol. One microliter of the redissolved sample was spotted onto the surface of a stainless-steel target. Mass spectrometry was performed on a Voyager-DE PRO time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a nitrogen laser emitting at 337 nm. Mass spectra were obtained in the positive ion mode at an acceleration voltage of 20 kV by accumulating 300 laser shots. Laser power was adjusted to slightly above the threshold to obtain good resolution and signal-to-noise ratios. Laser desorption ionization (LDI) mass spectra were present in the mass range m/z 250∼700. In the postsource decay (PSD) experiments, the precursor ion of interest was isolated, using a timed ion selector, for further structural characterization. Data Explorer 4.0 software, provided by Applied Biosystems, was used for data acquisition and processing. Calibration of mass spectra was done using the standards ROH and RP as external calibrants.Real-time RT-PCRCells were plated in 35 mm dishes and were treated with fresh PA or DHA, respectively, in the presence or absence of 1 μM ATRA or ROH for 6 and 24 h, and total RNA was extracted by using TRIzol reagent. Total RNA (3 μg per sample) was reverse-transcribed to cDNA using Superscript III reverse transcriptase at 53°C for 1 h. Real-time PCR was performed using gene-specific oligonucleotide primers. These primers were designed to generate cDNA fragments that cross an intron-exon boundary in the genomic DNA. The cDNA generated from 30 ng of total RNA was used for real-time PCR. Real-time PCR was performed on a DNA Engine Opticon system (MJ Research, Boston, MA) with a SYBR Green I Quantitect kit. For every primer set, a series of dilutions of a sample with the highest gene expression, from semiquantitative RT-PCR, was used to generate a standard curve. The conditions for the PCR were as follows: 95°C for 10 min to activate the DNA polymerase, followed by 50 cycles of 94°C for 30 s, primer annealing at 58°C for 30 s, and product extension at 72°C for 30 s. After each cycle, fluorescence was read at 80°C. The primer sequences were as follows. For human LRAT, forward primer, 5′-TGG AAC AAC TGC GAG CAC TTC GTG-3′; reverse primer, 5′-GCA GGA AGG GTA GTG TAT GAT ACC-3′. For human hypoxanthine guanine phosphoribosyl transferase (HPRT), a constitutively expressed enzyme, forward primer, 5′-TGC TCG AGA TGT GAT GAA GG-3′; reverse primer, 5′-TCC CCT GTT GAC TGG TCA TT-3′." @default.
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