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• W1969959095 abstract "Development of normal colon epithelial cells proceeds through a systematic differentiation of cells that emerge from stem cells within the base of colon crypts. Genetic mutations in the adenomatous polyposis coli (APC) gene are thought to cause colon adenoma and carcinoma formation by enhancing colonocyte proliferation and impairing differentiation. We currently have a limited understanding of the cellular mechanisms that promote colonocyte differentiation. Herein, we present evidence supporting a lack of retinoic acid biosynthesis as a mechanism contributing to the development of colon adenomas and carcinomas. Microarray and reverse transcriptase-PCR analyses revealed reduced expression of two retinoid biosynthesis genes: retinol dehydrogenase 5 (RDH5) and retinol dehydrogenase L (RDHL) in colon adenomas and carcinomas as compared with normal colon. Consistent with the adenoma and carcinomas samples, seven colon carcinoma cell lines also lacked expression of RDH5 and RDHL. Assessment of RDH enzymatic activity within these seven cell lines showed poor conversion of retinol into retinoic acid when compared with normal cells such as normal human mammary epithelial cells. Reintroduction of wild type APC into an APC-deficient colon carcinoma cell line (HT29) resulted in increased expression of RDHL without affecting RDH5. APC-mediated induction of RDHL was paralleled by increased production of retinoic acid. Investigations into the mechanism responsible for APC induction of RDHL indicated that β-catenin fails to repress RDHL. The colon-specific transcription factor CDX2, however, activated an RDHL promoter construct and induced endogenous RDHL. Finally, the induction of RDHL by APC appears dependent on the presence of CDX2. We propose a novel role for APC and CDX2 in controlling retinoic acid biosynthesis and in promoting a retinoid-induced program of colonocyte differentiation. Development of normal colon epithelial cells proceeds through a systematic differentiation of cells that emerge from stem cells within the base of colon crypts. Genetic mutations in the adenomatous polyposis coli (APC) gene are thought to cause colon adenoma and carcinoma formation by enhancing colonocyte proliferation and impairing differentiation. We currently have a limited understanding of the cellular mechanisms that promote colonocyte differentiation. Herein, we present evidence supporting a lack of retinoic acid biosynthesis as a mechanism contributing to the development of colon adenomas and carcinomas. Microarray and reverse transcriptase-PCR analyses revealed reduced expression of two retinoid biosynthesis genes: retinol dehydrogenase 5 (RDH5) and retinol dehydrogenase L (RDHL) in colon adenomas and carcinomas as compared with normal colon. Consistent with the adenoma and carcinomas samples, seven colon carcinoma cell lines also lacked expression of RDH5 and RDHL. Assessment of RDH enzymatic activity within these seven cell lines showed poor conversion of retinol into retinoic acid when compared with normal cells such as normal human mammary epithelial cells. Reintroduction of wild type APC into an APC-deficient colon carcinoma cell line (HT29) resulted in increased expression of RDHL without affecting RDH5. APC-mediated induction of RDHL was paralleled by increased production of retinoic acid. Investigations into the mechanism responsible for APC induction of RDHL indicated that β-catenin fails to repress RDHL. The colon-specific transcription factor CDX2, however, activated an RDHL promoter construct and induced endogenous RDHL. Finally, the induction of RDHL by APC appears dependent on the presence of CDX2. We propose a novel role for APC and CDX2 in controlling retinoic acid biosynthesis and in promoting a retinoid-induced program of colonocyte differentiation. Colon cancer arises from distinct genetic events that initiate and promote tumor formation. An inherited colon cancer predisposition, familial adenomatous polyposis, results from mutations in a single gene known as adenomatous polyposis coli (APC). 1The abbreviations used are: APC, adenomatous polyposis coli; RA, retinoic acid; RT, reverse transcription; TGF, transforming growth factor; RDH5, retinol dehydrogenase 5; RDHL, retinol dehydrogenase L; HPLC, high pressure liquid chromatography; RSV, Rous sarcoma virus; TCF, T cell factor; LEF, lymphoid enhancer factor; TTNPB, 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid. 1The abbreviations used are: APC, adenomatous polyposis coli; RA, retinoic acid; RT, reverse transcription; TGF, transforming growth factor; RDH5, retinol dehydrogenase 5; RDHL, retinol dehydrogenase L; HPLC, high pressure liquid chromatography; RSV, Rous sarcoma virus; TCF, T cell factor; LEF, lymphoid enhancer factor; TTNPB, 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid. This syndrome is characterized by the appearance of hundreds to thousands of colon adenomas in affected individuals. Recent investigations have generated a model describing downstream events controlled by APC. In the current model, APC regulates the activity of a transcriptional pathway that may control colonocyte proliferation (for a review, see Refs. 1Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (811) Google Scholar and 2Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (604) Google Scholar). It does so by regulating the levels of β-catenin, a protein found initially to function as a link between extracellular adhesion molecules and the cytoskeleton. It appears, however, that β-catenin also regulates transcription through a partnership with TCF/LEF transcription factors. In cells expressing functional APC, APC acts to repress β-catenin levels through regulation of ubiquitin-mediated proteolysis. Low levels of β-catenin prevent activation of TCF/LEF. In cells harboring mutated APC, β-catenin accumulates. This accumulation allows assembly of β-catenin·TCF/LEF complexes and activation of the transcriptional capabilities of TCF/LEF (1Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (811) Google Scholar, 2Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (604) Google Scholar). β-catenin·TCF/LEF-dependent transcriptional activation of specific cell cycle regulatory genes, like c-myc (3He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4048) Google Scholar) and cyclin D1 (4Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3237) Google Scholar), may underlie the development of colon adenomas and colon carcinomas (1Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (811) Google Scholar, 2Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (604) Google Scholar, 3He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4048) Google Scholar, 5Sparks A.B. Morin P.J. Vogelstein B. Kinzler K.W. Cancer Res. 1998; 58: 1130-1134PubMed Google Scholar, 6Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar, 7Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar, 8Polakis P. Hart M. Rubinfeld B. Adv. Exp. Med. Biol. 1999; 470: 23-32Crossref PubMed Google Scholar). In addition to sustained cell proliferation, colonocytes within adenomas and carcinomas display differentiation defects (9Potten C.S. Booth C. Pritchard D.M. Int. J. Exp. Pathol. 1997; 78: 219-243Crossref PubMed Scopus (399) Google Scholar, 10Karam S.M. Front. Biosci. 1999; 4: D286-298Crossref PubMed Google Scholar, 11Wright N.A. Int. J. Exp. Pathol. 2000; 81: 117-143Crossref PubMed Scopus (135) Google Scholar, 12Booth C. Potten C.S. J. Clin. Invest. 2000; 105: 1493-1499Crossref PubMed Scopus (292) Google Scholar, 13Clatworthy J.P. Subramanian V. Mech. Dev. 2001; 101: 3-9Crossref PubMed Scopus (101) Google Scholar). For example, crypts from colon adenomas are deficient in mucin-producing goblet cells (14Otori K. Sugiyama K. Hasebe T. Fukushima S. Esumi H. Cancer Res. 1995; 55: 4743-4746PubMed Google Scholar), one of the three predominant, terminally differentiated cell types seen within normal colon crypts. Although APC/β-catenin pathway target genes such as c-myc and cyclin D1 offer mechanistic insights into disregulation of colonocyte proliferation, few of the current APC pathway target genes have easily identifiable roles in cellular differentiation.Retinoids are a class of small lipid mediators derived from vitamin A that have important roles in vision, cell growth, and embryonic development (15Noy N. Nau H. Blaner W.S. Retinoids: The Biochemical Basis of Vitamin A and Retinoid Action. Springer-Verlag, Berlin1999: 3-24Google Scholar, 16De Luca L.M. Darwiche N. Celli G. Kosa K. Jones C. Ross S. Chen L.C. Nutr. Rev. 1994; 52: S45-S52Crossref PubMed Scopus (41) Google Scholar). A number of studies implicate retinoids in normal colonocyte function and in the development of colon neoplasms. For example, vitamin A-deficient animals display abnormalities in many epithelial tissues, including colon (17Narisawa T. Reddy B.S. Wong C.Q. Weisburger J.H. Cancer Res. 1976; 36: 1379-1383PubMed Google Scholar, 18Nzegwu H. Levin R.J. Gut. 1991; 32: 1324-1328Crossref PubMed Scopus (11) Google Scholar, 19Nzegwu H.C. Levin R.J. Gut. 1992; 33: 794-800Crossref PubMed Scopus (12) Google Scholar, 20Perumal A.S. Samy T.S. Lakshmanan M.R. Jungalwala F.B. Rao P.B. Biochim. Biophys. Acta. 1966; 124: 95-100Crossref PubMed Scopus (9) Google Scholar, 21Perumal A.S. Lakshmanan M.R. Cama H.R. Biochim. Biophys. Acta. 1968; 170: 399-408Crossref PubMed Scopus (13) Google Scholar, 22Zile M. Deluca H.F. Arch. Biochem. Biophys. 1970; 140: 210-214Crossref PubMed Scopus (26) Google Scholar, 23Zile M. Bunge C. Deluca H.F. J. Nutr. 1977; 107: 552-560Crossref PubMed Scopus (57) Google Scholar). These abnormalities include decreased mucus production, expansion of proliferation zones within the crypt, and ion flux alterations (17Narisawa T. Reddy B.S. Wong C.Q. Weisburger J.H. Cancer Res. 1976; 36: 1379-1383PubMed Google Scholar, 18Nzegwu H. Levin R.J. Gut. 1991; 32: 1324-1328Crossref PubMed Scopus (11) Google Scholar, 19Nzegwu H.C. Levin R.J. Gut. 1992; 33: 794-800Crossref PubMed Scopus (12) Google Scholar, 20Perumal A.S. Samy T.S. Lakshmanan M.R. Jungalwala F.B. Rao P.B. Biochim. Biophys. Acta. 1966; 124: 95-100Crossref PubMed Scopus (9) Google Scholar, 21Perumal A.S. Lakshmanan M.R. Cama H.R. Biochim. Biophys. Acta. 1968; 170: 399-408Crossref PubMed Scopus (13) Google Scholar, 22Zile M. Deluca H.F. Arch. Biochem. Biophys. 1970; 140: 210-214Crossref PubMed Scopus (26) Google Scholar, 23Zile M. Bunge C. Deluca H.F. J. Nutr. 1977; 107: 552-560Crossref PubMed Scopus (57) Google Scholar). At a molecular level, vitamin A-deficient rats showed diminished expression of TGF-β2 in certain epithelial tissues, including intestinal mucosa (24Glick A.B. McCune B.K. Abdulkarem N. Flanders K.C. Lumadue J.A. Smith J.M. Sporn M.B. Development. 1991; 111: 1081-1086PubMed Google Scholar). Systemic administration of retinoic acid (RA) induced the expression of TGF-β2 and TGF-β3 in these animals. In other studies, retinoid analogs have proven effective in preventing 5-azoxymethane-induced colon carcinoma formation in rats (17Narisawa T. Reddy B.S. Wong C.Q. Weisburger J.H. Cancer Res. 1976; 36: 1379-1383PubMed Google Scholar, 25Zheng Y. Kramer P.M. Olson G. Lubet R.A. Steele V.E. Kelloff G.J. Pereira M.A. Carcinogenesis. 1997; 18: 2119-2125Crossref PubMed Scopus (59) Google Scholar, 26Zheng Y. Kramer P.M. Lubet R.A. Steele V.E. Kelloff G.J. Pereira M.A. Carcinogenesis. 1999; 20: 255-260Crossref PubMed Scopus (102) Google Scholar, 27Pereira M.A. Adv. Exp. Med. Biol. 1999; 470: 55-63Crossref PubMed Google Scholar, 28Wargovich M.J. Jimenez A. McKee K. Steele V.E. Velasco M. Woods J. Price R. Gray K. Kelloff G.J. Carcinogenesis. 2000; 21: 1149-1155Crossref PubMed Scopus (234) Google Scholar). Retinoids also induce markers of differentiation, inhibit cell growth, increase cell adhesion, reduce colony formation, block anchorage-independent growth, and suppress invasiveness in colon cancer cells (29Niles R.M. Wilhelm S.A. Thomas P. Zamcheck N. Cancer Invest. 1988; 6: 39-45Crossref PubMed Scopus (53) Google Scholar, 30Reynolds S. Rajagopal S. Chakrabarty S. Cancer Lett. 1998; 134: 53-60Crossref PubMed Scopus (29) Google Scholar, 31Adachi Y. Itoh F. Yamamoto H. Iku S. Matsuno K. Arimura Y. Imai K. Tumour Biol. 2001; 22: 247-253Crossref PubMed Scopus (53) Google Scholar, 32Nicke B. Kaiser A. Wiedenmann B. Riecken E.O. Rosewicz S. Biochem. Biophys. Res. Commun. 1999; 261: 572-577Crossref PubMed Scopus (25) Google Scholar). Finally, recent studies have demonstrated that RA can not only inhibit the transcriptional activity of β-catenin (33Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar) but can also independently promote the translocation of β-catenin from the cytoplasm to the membrane (34Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These effects of RA on β-catenin function are thought to inhibit proliferation and promote differentiation, respectively (34Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Although these studies offer evidence supporting a role for retinoids in colonocyte function, the specific cellular controls governing retinoid response pathways remain undefined in colon tissues.In addition to the nuclear hormone receptors, retinoid responsiveness within cells is governed by retinoid availability (35Blaner W.S. Piantedosi R. Sykes A. Vogel S. Nau H. Blaner W.S. Retinoids: The Biochemical Basis of Vitamin A and Retinoid Action. Springer-Verlag, Berlin1999: 117-144Google Scholar, 36Vogel S. Gamble M.V. Blaner W.S. Nau H. Blaner W.S. Retinoids: The Biochemical Basis of Vitamin A and Retinoid Action. Springer-Verlag, Berlin1999: 31-84Google Scholar). For the most part, cells acquire retinoids in the form of retinol, an inactive precursor. Tissues must, therefore, convert retinol into RA in order to activate the network of nuclear receptors required to evoke retinoid transcriptional responses. The enzymes that catalyze these conversions fall into three distinct classes that include the alcohol dehydrogenases, the short-chain dehydrogenases/reductases, and the aldehyde dehydrogenases. Alcohol dehydrogenases and short-chain dehydrogenase/reductase enzymes convert retinol into the aldehyde, retinal. Further conversion of retinal into RA is carried out by the aldehyde dehydrogenase enzyme family. Enzymes in each class have broad substrate specificities and can oxidize or reduce many physiologically important alcohols or aldehydes including ethanol, steroids, and retinoids. The actions of RA, in turn, can be limited by catabolism via cytochrome P450 enzymes (35Blaner W.S. Piantedosi R. Sykes A. Vogel S. Nau H. Blaner W.S. Retinoids: The Biochemical Basis of Vitamin A and Retinoid Action. Springer-Verlag, Berlin1999: 117-144Google Scholar, 36Vogel S. Gamble M.V. Blaner W.S. Nau H. Blaner W.S. Retinoids: The Biochemical Basis of Vitamin A and Retinoid Action. Springer-Verlag, Berlin1999: 31-84Google Scholar). Although the biochemistry of these retinoid biosynthetic and metabolic enzymes is emerging, little is known about the regulation of these enzymes within tissues or specific cell types.In order to define the molecular pathways that may govern APC-dependent differentiation of colonocytes, we have analyzed gene expression profiles in colon adenomas and carcinomas compared with normal colon. Our analyses revealed that colon adenomas and carcinomas show consistent down-regulation of the RA biosynthetic enzymes retinol dehydrogenase 5 (RDH5) and retinol dehydrogenase-like (RDHL). Given that loss of RA biosynthetic genes may contribute to the lack of differentiation observed in colon adenomas and carcinomas, we investigated the regulatory mechanisms that control the expression of RDH5 and RDHL. We found in a survey of normal human tissues that, whereas both RDHL and RDH5 were expressed in the colon, RDHL expression appears relatively restricted to the colon. Reintroduction of APC into APC-deficient colon cancer cells induced RDHL expression. Furthermore, the intestinal specific transcription factor, CDX2, targets RDHL and acts synergistically with APC in the induction of RDHL. Since RA holds known differentiation properties, our data create a model wherein APC controls RA biosynthesis as a potential mechanism for regulating colonocyte differentiation.EXPERIMENTAL PROCEDURESMicroarray Analysis—Microarray analyses were performed as described previously (37Karpf A.R. Peterson P.W. Rawlins J.T. Dalley B.K. Yang Q. Albertsen H. Jones D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14007-14012Crossref PubMed Scopus (172) Google Scholar). Briefly, total RNA was extracted from surgically excised and microdissected colon tissue samples using Trizol reagent (Invitrogen). Poly(A) RNA was selected using an Oligotex Kit (Qiagen). First-strand cDNA probes were generated by reverse transcription of 1 μg of purified mRNA with SuperScript II (Invitrogen) after the addition of Cy3-dCTP or Cy5-dCTP (Amersham Biosciences). Probes were purified and reconstituted in 30 μl of 5× SSC, 0.1% SDS, 0.1 μg/ml salmon sperm DNA, and 50% formamide. After denaturation at 94 °C, the hybridization mix was deposited onto the slide under a coverslip.Hybridizations were performed overnight at 42 °C in a humidified chamber on glass slides displaying 4608 human genes in duplicate. In every case, RNA from adenoma or carcinoma tissue was compared with a pool of normal tissue RNA from six separate donors. Since RNA availability of most samples was limiting, most samples were only surveyed on our first slide (containing 4608 genes), which contained the RDH5 gene but not the RDHL gene. Since some of the samples (numbers 3, 6, 18, 22, and 25) contained plentiful RNA, we were able to survey their gene expression on our entire gene set (totaling 38,000 genes and including the RDHL gene). Following hybridization, slides were washed for 10 min in 1× SSC, 0.2% SDS and then for 20 min in 0.1× SSC, 0.2% SDS. Slides were dipped in distilled water and dried with compressed air, and the fluorescent hybridization signatures were captured using the “Avalanche” dual laser confocal scanner (Amersham Biosciences). Fluorescent intensities were quantified using ArrayVision 4.0 (Imaging Research). Differentially expressed genes were selected when the Log2 (Cy5 signal/Cy3 signal) exceeded 1.85-fold. This cut-off was selected based on the control comparisons examining normal colon versus normal colon samples. This value exceeds the variability seen in these control comparisons. Differential expression was determined using Student's t test for each gene in each comparison. The p value must not have exceeded 0.05.Quantitative RT-PCR—For colon samples, total RNA was isolated from 10 matched sets of frozen normal and adenoma or carcinoma tissue samples using an RNeasy kit (Qiagen). For HT29 APC- and LacZ-inducible cell lines, total RNA was harvested using Trizol (Invitrogen). cDNA was synthesized from 2 μg of total RNA using Superscript III (Invitrogen). PCR was performed using the Roche Light Cycler instrument and software, version 3.5 (Roche Applied Science). Intron-spanning primers (RDHL, forward (5′-TGGAAACTTGGCAGCCAGAA-3′) and reverse (5′-CCAGAGACCTTTCTCCCCAA-3′); RDH5, forward (5′-TTCTCTGACAGCCTGAGGCG-3′) and reverse (5′-TGCGCTGTTGCATTTTCAGG-3′); APC, forward (5′-AAAACGAGCACAGCGAAGAATAGC-3′) and reverse (5′-TCGTGTAGTTGAACCCTGACCAT-3′); 18 S rRNA, forward (5′-GGTGAAATTCTTGGACCGGC-3′) and reverse (5′-GACTTTGGTTTCCCGGAAGC-3′)) were designed to amplify 200-bp products and spanned at least one intron in order to rule out amplification from genomic DNA.PCR was performed in duplicate (or triplicate for 18 S rRNA) with a master mix consisting of cDNA template, buffer (500 mm Tris, pH 8.3, 2.5 mg/ml bovine serum albumin, 30 mm MgCl2), dNTPs (2 mm), TaqStart antibody (Clontech), Biolase DNA polymerase (Bioline), gene-specific forward and reverse primers (10 μm), and SYBR Green I (Molecular Probes, Inc., Eugene, OR). The PCR conditions are as follows: 35 cycles of amplification with 1-s denaturation at 95 °C and 5-s annealing at 54 °C for RDHL, 58 °C for RDH5, 60 °C for cyclin D, and 53 °C for 18 S rRNA. A template-free negative control was included in each experiment.The copy number was measured by comparing gene amplification with the amplification of standard samples that contained 103 to 107 copies of the gene or 105 to 109 for 18 S rRNA. The relative expression level of each gene was calculated by averaging the replicates and then dividing the average copy number of gene X by the average copy number of 18 S rRNA. The S.E. value of the ratios was calculated using a confidence interval.Cell Culture and Drug Treatments—HT29, HCT116, and RKO colon adenocarcinoma cells were cultured as recommended by the American Type Culture Collection. HT29 APC-inducible and LacZ-inducible cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD).Transfections and Luciferase Assays—Fugene 6 (Roche Applied Science) and LipofectAMINE Plus (Invitrogen) were used to transfect HCT116 cells and RKO cells, respectively, according to the manufacturers' protocols. Cells were seeded at a density of 100,000 cells/well in 12-well plates and transfected the next day. Transfections were performed using 0.6 μg of DNA (including 0.06 μg of normalization vector, 0.12 μg of reporter vector, and 0.42 μg of expression vector), and, in the absence of further treatment, cells were harvested 24 h after the start of transfection. In certain experiments, medium containing charcoal-stripped serum as well as either RA mixture (see above) or ethanol vehicle was added to cells 24 h after transfection, and cells were harvested 24 h after medium change. Luciferase values were analyzed using a Dual Luciferase Assay System (Promega). Transfection efficiencies were normalized by dividing the firefly luciferase activity by the Renilla luciferase activity for each sample. Data in each experiment are presented as the mean ± S.D. of duplicates from a representative experiment. All experiments were performed at least three times.Plasmids—Regions spanning –2228 to +1071 (in reference to the translational start site) of the RDHL promoter and –1637 to +83 of the RDH5 promoter were PCR-amplified from normal human genomic DNA (Clontech) using specific primers (RDHL, forward (5′-GAAGATACACTTGGGTAGAAG-3′) and reverse (5′-ACACCAGTTCCCATTTCCTACTC-3′); RDH5, forward (5′-GCTGCCTCCAGTCAGGTTAC-3′) and reverse (5′-TTACCTCTCTGTGGCGAAAGC-3′)). PCR products were then inserted upstream of the firefly luciferase gene in the pGL3basic vector (Promega) to create RDHL:LUC and RDH5:LUC, respectively. PRL:LUC contains –36 to +36 of the prolactin gene driving expression of the luciferase gene and was kindly provided by Dr. Andrew Thorburn (Wake Forest University, Winston-Salem, NC). The CDX1 and CDX2 expression vectors were constructed by RT-PCR from normal colon RNA using specific primers (CDX1, forward (5′-GCGCGGATCCATGTATGTGGGCTATGTGC-3′) and reverse (5′-GCGCGAATTCCTATGGCAGAAACTCCTCT-3′); CDX2, forward (5′-GCGCGGATCCATGTACGTGAGCTACCTC-3′) and reverse (5′-GCGCGAATTCTCACTGGGTGACGGTGG-3′)). The RT-PCR products were then cloned into a pCDNA3.1 His C vector (Invitrogen). The β-catenin S37A and DN-LEF expression vectors were kindly provided by Dr. Donald Ayer (University of Utah, Salt Lake City, UT). For luciferase assays, RDH5:LUC or RDHL:LUC reporters were co-transfected with a Rous sarcoma virus (RSV)-Renilla luciferase reporter plasmid that was used to normalize transfection efficiencies.Electrophoretic Mobility Shift Assays—22-Mer oligonucleotides representing –338 to –359 in the RDHL promoter containing putative CDX2 sites were annealed and 5′-end-labeled with [α-32P]ATP using T4 kinase (MBI Fermentas). The labeled oligonucleotides were then purified over a micro Bio-spin 6 column (Bio-Rad) to remove unincorporated nucleotides. For binding reactions, 6 μg of extract from 293 cells overexpressing CDX2 were incubated with 1 μl (50,000–200,000 cpm) of 32P-labeled probe in a 10-μl final volume of 1× binding buffer (20% glycerol, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, 250 mm NaCl, 50 mm Tris-HCl (pH 7.5), and 0.25 mg/ml poly(dI-dC)-poly(dI-dC)). For competition and supershift assays, a 10- or 50-fold molar excess of unlabeled oligonucleotide or 2 μg of anti-His tag antibody or anti-CDX2 antibody were added 15 min prior to the addition of labeled oligonucleotide. Complexes were separated on a 6% acrylamide gel in 1× TBE at 250 V for 3 h. The gel was then dried and exposed to a phosphor screen and visualized on a PhosphorImager (Amersham Biosciences).Northern Blotting—Total RNA was isolated using Trizol (Invitrogen) followed by poly(A) RNA selection using a Poly(A) Tract mRNA Isolation kit (Promega). Poly(A) RNA was fractionated through formaldehyde-containing agarose gels and transferred onto nylon membranes (Amersham Biosciences). Probes were generated using the Rediprime II random prime labeling system (Amersham Biosciences) supplemented with [α-32P]dCTP. Hybridizations with 32P-labeled probes were carried out using ULTRAhyb buffer (Ambion) as recommended by the manufacturer.RA Extraction and HPLC Analysis—Cells were treated with 100 nmol ATROL at 80–90% confluence for 12 h. Medium was removed, and cells were scraped into PBS for protein quantification. After the addition of 100 nmol of internal standard TTNPB, the medium was acidified with 6 n HCl (0.03× volume) and extracted with an equal volume of hexane containing 0.1 mg/ml butylated hydroxytoluene. The resulting solution was mixed vigorously and spun down at 11,500 rpm for 20 min. Following centrifugation, the organic phase was transferred to a glass vial, dried under nitrogen, and reconstituted in 100 μl of 1:1 Me2SO/MeOH for HPLC analysis. Extracted RA is expressed as pmol of RA/mg of protein/mmol of TTNPB.Retinoid analysis and quantification was performed using a reversed phase Phenomenex Luna C18, 4.6 × 250 mm, 5μ particle size analytical column. Retinoids were eluted with a gradient starting at 80% acetonitrile, 20% ammonium acetate, pH 5.0, to 100% acetonitrile in 40 min, with a flow rate of 1.5 ml/min at 350 nm. Identification of retinoid peaks from the extracts was done by comparing elution positions with matching retinoid standards. Quantification of extracted retinoids was performed by relating the area of the peak to areas obtained by the analysis of known quantities of retinoid standards.RESULTSRetinol Dehydrogenases Are Down-regulated in Neoplastic Colon—In order to identify signaling pathway alterations in neoplastic colon, we performed microarray expression analyses on colon adenomas and carcinomas in comparison with a pool of normal colon tissue. A striking feature of our colon tumor progression data was that ∼80% of the differentially expressed genes were down-regulated in adenoma and carcinoma tissues as compared with normal (data not shown). Two genes in particular showing down-regulation in colon adenomas and carcinomas caught our attention. The first gene encoded RDH5, an enzyme that catalyzes the conversion of retinol into retinal. The second gene encoded RDHL, a recently described, novel retinol dehydrogenase (described by Soref et al. (38Soref C.M. Di Y.P. Hayden L. Zhao Y.H. Satre M.A. Wu R. J. Biol. Chem. 2001; 276: 24194-24202Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), but referred to as hRDH-TBE). Each of these genes was down-regulated at least 2-fold in ∼70% of both adenoma and carcinoma tissues relative to normal (Fig. 1A).Due to small tissue sizes and correspondingly low yields of mRNA samples, we obtained limited data on RDHL by microarray (see “Experimental Procedures”). We thus used quantitative RT-PCR to assess the expression levels of RDH5 and RDHL in an additional 10 patient-matched normal and carcinoma colon tissues. Fig. 1B shows that expression of RDHL was decreased at least 2-fold in 9 of 10 carcinoma samples in comparison with the matched, normal appearing colon tissue, whereas RDH5 was decreased in 6 of 10 samples examined. As a positive marker for distinguishing colon carcinoma from normal, cyclin D1 expression levels were increased in each of the carcinoma samples relative to normal (data not shown) (39Bartkova J. Lukas J. Strauss M. Bartek J. Int. J. Cancer. 1994; 58: 568-573Crossref PubMed Scopus (146) Google Scholar, 40Arber N. Hibshoosh H. Moss S.F. Sutter T. Zhang Y. Begg M. Wang S. Weinstein I.B. Holt P.R. Gastroenterology. 1996; 110: 669-674Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). We also noted the loss of tags corresponding to RDHL in two colon carcinoma sample data sets deposited into the SAGE (serial analysis of gene expression) data base, an observation recently reported by Buckhaults et al. (41Buckhaults P. Rago C. St. Croix B. Romans K.E. Saha S. Zhang L. Vogelstein B. Kinzler K.W. Cancer Res. 2001; 61: 6996-7001PubMed Google Scholar).We examined the tissue distribution of RDH5 and RDHL by" @default.
• W1969959095 created "2016-06-24" @default.
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• W1969959095 creator A5018187188 @default.
• W1969959095 creator A5033203315 @default.
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• W1969959095 date "2004-08-01" @default.
• W1969959095 modified "2023-09-27" @default.
• W1969959095 title "The Tumor Suppressor Adenomatous Polyposis Coli and Caudal Related Homeodomain Protein Regulate Expression of Retinol Dehydrogenase L" @default.
• W1969959095 cites W1507061074 @default.
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