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• W2154684225 abstract "In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas. In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas. There is consistent data indicating that in human stomach as well as in other organs mucin genes are expressed in a regulated cell- and tissue-specific manner and that altered mucin gene expression occurs in cancer and precancerous lesions (1Van Seuningen I. Pigny P. Perrais M. Porchet N. Aubert J.-P. Front. Biosc. 2001; 6: d1234-d1246Google Scholar). In normal gastric mucosa most studies show little or no expression of the intestinal mucin MUC2 (2Ho S.B. Shekels L. Toribara N.W. Kim Y.S. Lyftogt C. Cherwitz D.L. Niehans G.A. Cancer Res. 1995; 55: 2681-2690PubMed Google Scholar, 3Xing P.-X. Prenzoska J. Layton G.T. Devine P.L. McKenzie I.F.C. J. Natl. Cancer Inst. 1992; 84: 699-703Crossref PubMed Scopus (79) Google Scholar, 4Audié J.P. Janin A. Porchet N. Copin M.C. Gosselin B. Aubert J.P. J. Histochem. Cytochem. 1993; 41: 1479-1485Crossref PubMed Scopus (407) Google Scholar, 5Devine P.L. McGuckin M.A. Birrell G.W. Whitehead R.H. Sachdev G.P. Shield P. Ward B.G. Br. J. Cancer. 1993; 67: 1182-1188Crossref PubMed Scopus (46) Google Scholar, 6Gambus G. de Bolós C. Andreu D. Francy C. Egea G. Real F.X. Gastroenterology. 1993; 104: 3-102Crossref PubMed Scopus (0) Google Scholar, 7Ho S. Niehans G.A. Lyftogt C. Yan P.S. Cherwitz P.L. Gum E.T. Dahiya R. Kim Y.S. Cancer Res. 1993; 53: 641-651PubMed Google Scholar, 8Carrato C. Balagué C. de Bolós C. Gonzalez E. Gambus G. Planas J. Perini J.M. Andreu D. Real F.X. Gastroenterology. 1994; 107: 160-172Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 9Reis C.A. Sørensen T. Mandel U. David L. Mirgorodskaya E. Roepstorff P. Kihlberg J. Stig-Hansen J.E. Clausen H. Glycoconj. J. 1998; 15: 51-62Crossref PubMed Scopus (66) Google Scholar). In intestinal metaplasia, a preneoplastic lesion of the stomach characterized by the transdifferentiation of the gastric mucosa to an intestinal phenotype, there are alterations in the mucin expression pattern including de novo expression of MUC2, mostly in goblet cells (10Reis C.A. David L. Correa P. Carneiro F. de Bolós C. Garcia E. Mandel U. Clausen H. Sobrinho-Simões M. Cancer Res. 1999; 59: 1003-1007PubMed Google Scholar). Thirty percent of gastric carcinomas, including all carcinomas of the mucinous type, also aberrantly express MUC2 intestinal mucin (11Reis C.A. David L. Carvalho F. Mandel U. de Bolos C. Mirgorodskaya E. Clausen H. Sobrinho-Simões M. J. Histochem. Cytochem. 2000; 48: 377-388Crossref PubMed Scopus (132) Google Scholar, 12Pinto-de-Sousa J. David L. Reis C.A. Gomes R. Silva L. Pimenta A. Virchows Arch. 2002; 440: 304-310Crossref PubMed Scopus (81) Google Scholar). The molecular mechanisms responsible for the regulation of MUC2 transcription and expression are beginning to be elucidated. The structure of MUC2 promoter was characterized (13Gum Jr., J.R. Hicks J.W. Kim Y.S. Biochem. J. 1997; 325: 259-267Crossref PubMed Scopus (82) Google Scholar, 14Velcich A. Palumbo L. Seilleri L. Evans G. Augenlicht L. J. Biol. Chem. 1997; 272: 7968-7976Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and MUC2 expression was reported to be regulated by methylation of the promoter (15Hanski C. Riede E. Gratchev A. Foss H.D. Bohm C. Klumann E. Hummel M. Mann B. Buhr H.J. Stein H. Kim Y.S. Gum J. Riecken E.O. Lab. Invest. 1997; 77: 685-695PubMed Google Scholar, 16Gratchev A. Siedow A. Bumke-Vogt C. Hummel M. Foss H-D Hanski M-L Kobalz U. Mann B. Lammert H. Stein H. Riecken E.O. Hanski C. Mansmann U. Cancer Lett. 2001; 168: 71-80Crossref PubMed Scopus (39) Google Scholar, 17Mesquita P. Peixoto A.J. Seruca R. Hanski C. Almeida R. Silva F. Reis C. David L. Cancer Lett. 2003; 189: 129-136Crossref PubMed Scopus (37) Google Scholar) and by the Sp1 family of transcription factors (13Gum Jr., J.R. Hicks J.W. Kim Y.S. Biochem. J. 1997; 325: 259-267Crossref PubMed Scopus (82) Google Scholar, 18Aslam F. Palumbo L. Augenlicht L.H. Velcich A. Cancer Res. 2001; 61: 570-576PubMed Google Scholar, 19Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). It has also been described that MUC2 is transcriptionally activated by p53 (20Ookawa K. Kudo T. Aizawa S. Saito H. Shigeki T. J. Biol. Chem. 2002; 277: 48270-48275Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and, in tracheobronchial epithelial cells, by lipopolysaccharide from Pseudomonas aeruginosa (21Li J-D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Crossref PubMed Scopus (255) Google Scholar, 22Li J-D. Feng W. Gallup M. Miyata S. Kim J-H. Gum J. Kim Y. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5718-5723Crossref PubMed Scopus (287) Google Scholar) and epidermal growth factor (19Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). However, information on MUC2 transcriptional regulation in gastric cells, in relation with the overexpression of MUC2 in intestinal metaplasia and gastric carcinoma, is essentially unknown.The intestine-specific homeobox genes Cdx-1 1The abbreviations used are: Cdx-1caudal-related homeobox factor 1GAPDHglyceraldehyde-3-phosphate dehydrogenaseSIF1sucrase-isomaltase footprint 15′-UTR5′-untranslated regionEMSAelectrophoretic mobility shift assayRTreverse transcriptase.1The abbreviations used are: Cdx-1caudal-related homeobox factor 1GAPDHglyceraldehyde-3-phosphate dehydrogenaseSIF1sucrase-isomaltase footprint 15′-UTR5′-untranslated regionEMSAelectrophoretic mobility shift assayRTreverse transcriptase. and Cdx-2 were also recently shown to be aberrantly expressed in intestinal metaplasia and in a subset of gastric carcinomas. Both in intestinal metaplasia and in gastric carcinoma, expression of Cdx-1 and Cdx-2 is closely associated with the expression of mucin MUC2 (23Almeida R. Silva E. Santos-Silva F. Silberg D.G. Wang J. de Bolós C. David L. J. Pathol. 2003; 199: 36-40Crossref PubMed Scopus (233) Google Scholar). Altogether, these observations suggest that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and some gastric carcinomas, partly due to MUC2 regulation at the transcriptional level (23Almeida R. Silva E. Santos-Silva F. Silberg D.G. Wang J. de Bolós C. David L. J. Pathol. 2003; 199: 36-40Crossref PubMed Scopus (233) Google Scholar). This hypothesis is further supported by the direct involvement of Cdx-1 and Cdx-2 in the differentiation of intestinal epithelial cells (24Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar), namely in transgenic models (25Silberg D.G. Sullivan J. Kang E. Swain G.P. Moffett J. Sund N.J. Sackett S.D. Kaestner K.H. Gastroenterology. 2002; 122: 689-696Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar) and by the evidence showing that they act as transcription factors for several intestinal genes such as sucrase-isomaltase (24Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar, 26Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar, 27Traber P.G. Silberg D.G. Annu. Rev. Physiol. 1996; 58: 275-297Crossref PubMed Scopus (135) Google Scholar), lactase-phlorizin hydrolase (27Traber P.G. Silberg D.G. Annu. Rev. Physiol. 1996; 58: 275-297Crossref PubMed Scopus (135) Google Scholar, 28Mitchelmore C. Troelsen J.T. Spodsberg N. Sjostrom H. Noren O. Biochem. J. 2000; 346: 529-535Crossref PubMed Scopus (102) Google Scholar, 29Fang R. Santiago N.E. Olds L.C. Sibley E. Gastroenterology. 2000; 118: 115-127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 30Krasinski S.D. Van Wering H.M. Tannemaat M.R. Grand R.J. Am. J. Physiol. 2001; 281: G69-G84Crossref PubMed Google Scholar), intestine phospholipaseA/lysophospholipase (31Taylor J.K. Boll W. Levy T. Suh E. Siang S. Mantei N. Traber P.G. DNA Cell Biol. 1997; 16: 1419-1428Crossref PubMed Scopus (60) Google Scholar), claudin-2 (32Sakaguchi T. Gu X. Golden H.M. Suh E. Rhoads D.B. Reinecker H.C. J. Biol. Chem. 2002; 277: 21361-21370Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and more recently β-1,3-galactosyltransferase T5 (33Isshiki S. Kudo T. Nishihara S. Ikehara Y. Togayachi A. Furuya A. Shitara K. Kubota T. Watanabe M. Kitajima M. Narimatsu H. J. Biol. Chem. 2003; 278: 36611-36620Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Since the promoter of MUC2 contains some Cdx putative binding sites, we hypothesized that Cdx-1 or Cdx-2 might function as transcriptional regulators of MUC2. In this study we show that Cdx-2 is a regulator of MUC2 expression both in gastric and intestinal cancer cells whereas Cdx-1 only transactivates MUC2 in intestinal cells. The implications in intestinal metaplasia and gastrointestinal cell differentiation are discussed.EXPERIMENTAL PROCEDURESCell Culture—Human gastric carcinoma cell lines were cultured at 37 °C in a humidified 5% CO2 incubator. GP202, GP220, and MKN45 cells were maintained in RPMI 1640 medium (with Glutamax and 25 mm Hepes) supplemented with 10% fetal bovine serum and gentamicin (50 μg/ml). KATOIII cell line was maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum and gentamicin (50 μg/ml). AGS cell line was cultured in Nutrient Mixture Ham's F12 (with l-Glutamine) supplemented with 10% fetal bovine serum and gentamicin (50 μg/ml). Human colon carcinoma cell lines HT-29 STD, Caco-2 and LS174T were cultured as described in Van Seuningen et al. (34Van Seuningen I. Perrais M. Pigny P. Porchet N. Aubert J.P. Biochem. J. 2000; 348: 675-686Crossref PubMed Scopus (81) Google Scholar). Cell lines GP202 and GP220 were established at IPATIMUP (35Gärtner F. David L. Seruca R. Machado J.C. Sobrinho-Simões M. Virchows Arch. 1996; 428: 91-98Crossref PubMed Google Scholar).Reverse Trancriptase-PCR Analysis—Total RNA from gastric carcinoma cell lines was isolated using Purescript RNA isolation kit (Gentra systems, Minneapolis) and treated with DNase. 5 μg of RNA were primed with random hexamers and reverse transcribed using Superscript III (Invitrogen) in a final volume of 20 μl. Four microliters of this mixture was PCR-amplified in a 25-μl reaction using AmpliTaq DNA polymerase (Applied Biosystems). The sequences of the primers used to amplify MUC2, Cdx-1 and Cdx-2 are indicated in Table I. GAPDH was used as an internal standard. The PCR reaction mixture was denatured at 94 °C for 2 min followed by 35 cycles at 94 °C for 45 s, 55 °C for 15 s, and 72 °C for 45 s for MUC2, Cdx-1 and Cdx-2 (25 cycles for GAPDH).Table ISequences of the oligonucleotides used for RT-PCR, site-directed mutagenesis, and EMSAsPrimers used for RT-PCRSequence (5′→3′)MUC2aS: CGA AAC CAC GGC CAC AAC GTAS: GAC CAC GGC CCC GTT AAG CACdx-1aS: GGA CCA AGG ACA AGT ACC GCAS: GGT GTT GCT GGG ACA CAG GCdx-2aS: CCA GGA CGA AAG ACA AAT ATC GAAS: CTC TGG GAC ACT TCT CAG AGGGAPDHaS: ACC ATC TTC CAG GAG CGA GAS: GGA TGA CCT TGC CCA CAGMUC2bS: CTG CAC CAA GAC CGT CCT CAT GAS: GCA AGG ACT GAA CAA AGA CTC AGA CCdx-1bS: CGG GCA CAC CGT CCT CGC CCGAS: CAT TGG AGA GGA GGT GGC CAG GCdx-2bS: CGG CTG GAG CTG GAG AAG GAS: TCA GCC TGG AAT TGC TCT GC28SbS: GCA GGG CGA AGC AGA AGG AAA CTAS: TGA GAT CGT TTC GGC CCC AAPrimers used for site-directed mutagenesisSequence (5′→3′)Cdx-2 site-191/-187AAG AAG GCT GCG GCG TGC CAG GGA GCC ATA AAG AGA TGA CCT CCGCGG AGG TCA TCT CTT TAT GGC TCC CTG GCA CGC CGC AGC CTT CTT-177/-171AAG AAG GCT GCG TTT ACC CAG GGA GCC GCG TGG AGA TGA CCT CCGCGG AGG TCA TCT CCA CGC GGC TCC CTG GGT AAA CGC AGC CTT CTTDouble mutationCTG AAG AAG GCT GCG GCG TCC CAG GGA GCC GCG TAG AGA TGA CCT CCGCGG AGG TCA TCT CTA CGC GGC TCC CTG GGA CGC CGC AGC CTT CTT CAGOligonucleotides used for EMSAsSequence (5′→3′)ProbeSIF1GGG TGC AAT AAA ACT TTA TGA GTAwt -201/-161GAA GGC TGC GTT TAC CCA GGG AGC CAT AAA GAG ATG ACC Tdm -201/-161GAA GGC TGC GGC GTG CCA GGG AGC CGC GTG GAG ATG ACC T Open table in a new tab Total RNAs from colon cancer cells were prepared using the RNeasy mini-kit from Qiagen. Cells were harvested at 100% of confluence and 1.5 μg of total RNA was used to prepare cDNA (Advantage™ RT-for-PCR kit, Clontech) as described before (34Van Seuningen I. Perrais M. Pigny P. Porchet N. Aubert J.P. Biochem. J. 2000; 348: 675-686Crossref PubMed Scopus (81) Google Scholar). PCR was performed on 2 μl of cDNA using specific pairs of primers (MWG-Biotech, Germany) for MUC2, Cdx-1, Cdx-2, and 28 S rRNA as described in Table I. PCR reactions were carried out in 50-μl final solutions (5 μl of 10× PCR buffer containing MgCl2, 4 μl 2.5 mm dNTPs, 10 pmol of each primer, 1 unit of Taq polymerase (Roche Applied Science)). Cycling conditions were as follows: 1) denaturation: 94 °C, 2 min for one cycle; 2) denaturation: 94 °C, 45 s; annealing: 60 °C, 1 min; and extension: 72 °C, 1 min for 30 cycles; and 3) final extension: 72 °C, 10 min. PCR products were analyzed on 2% ethidium bromide-agarose gels run in 1× Tris borate-EDTA buffer.MUC2/Luciferase Plasmid Construction and Transient Transfection Assays—pGL3-MUC2 promoter constructs covering the –947/–1, –2096/+27, and –2627/–1 regions of MUC2 promoter were previously described and used to study MUC2 regulation in the mucoepidermoid NCI-H292 lung cancer cell line (19Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). –371/+27 and –947/+27 deletion mutants used in this study were prepared as described in Perrais et al. (19Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). For the transient transfection assays, gastric carcinoma cell lines were seeded at 2.5 × 105/well in 24-well plates. Transfections and co-transfections were performed the next day by mixing 0.8 μg of the pGL3 construct of interest, and 0.4 μg of expression vector in the co-transfections, with tfx-50 reagent (Promega) (tfx-50:DNA ratio of 4:1) in 200 μl of serum-free and antibiotic-free medium. Cells were incubated with the transfection mixture for 1 h at 37 °C followed by the addition of 1 ml of complete medium. Total cell extracts were prepared after a 48 h incubation at 37 °C using 1× reporter lysis buffer (Promega), as described in the manufacturer's instruction manual. 20 μl of cell extract were mixed with 100 μl of luciferase assay reagent (Promega) to determine luciferase activity in a 1450 Microbeta luminescence counter (Wallac). The β-galactosidase activity was measured using 50 μl of cell extract. The luciferase activity of test plasmids is expressed as fold of induction of the test plasmid activity compared with that of the corresponding empty vector (pGL3 basic, Promega), after correction for transfection efficiency as measured by the β-galactosidase activity. Each plasmid was assayed in triplicate in two separate experiments. Transfection of colon carcinoma cell lines was performed as described in Perrais et al. (19Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar).Stable Transfections with Cdx-2—The gastric carcinoma cell line, GP202, was transfected at confluence with 10 μg of the Cdx-2 expression vector (pRC/CMV-Cdx-2), or the empty vector pRC/CMV (mock cells). Transfections were performed with tfx-50 reagent (Promega) (tfx-50:DNA ratio of 4:1) and the procedure was as previously described for transient transfections. After 48 h, transfected cells were selected with the antibiotic G418 (Sigma) at a final concentration of 0.5 mg/ml.Site-directed Mutagenesis—QuickChange site-directed mutagenesis kit (Stratagene) was used to generate site-specific mutations in the two Cdx-2 binding sites in the MUC2 promoter constructs –371/+27 and –947/+27. Single and double mutations were realized for each construct. Oligonucleotides containing the desired mutations were designed according to the manufacturer's instructions and their sequences are depicted in Table I.Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts from GP220 cells were prepared as described by Van Seuningen et al. (37Van Seuningen I. Ostrowski J. Bustelo X.R. Sleath P. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and kept at –80 °C until use. Protein content (2 μl of cell extracts) was measured using the bicinchoninic acid method (Pierce), as described in the manufacturer's instruction manual. The sequences of the oligonucleotides used for EMSAs are indicated in Table I. They were synthesized by MWG-Biotech (Germany). SIF1 (sucrase-isomaltase footprint 1) probe, corresponding to an evolutionarily conserved Cdx-2 cis-element of the sucrase isomaltase gene (26Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar), was used as a positive control. Equimolar amounts of single-stranded oligonucleotides were annealed and radiolabeled using T4 polynucleotide kinase (Promega) and [γ-32P]dATP. Radiolabeled probes were purified by chromatography on a Bio-Gel P-6 column (Bio-Rad). Nuclear proteins (8 μg) were preincubated for 20 min on ice in 20 μl of binding buffer with 1 μg of poly(dI-dC) (Sigma) and 1 μg of sonicated salmon sperm DNA. Radiolabeled DNA probe was added (60,000 cpm), and the reaction was left for another 20 min on ice. For competition experiments 50× excess of the cold probe was added 20 min before adding the radiolabeled probe. For supershift analyses, 1 μl of the Cdx-2 antibody (Biogenex) was added to the proteins and left for 30 min at room temperature before adding the radiolabeled probe. Reactions were stopped by adding 2 μl of loading buffer. Samples were loaded onto a 4% non-denaturing polyacrylamide gel, and electrophoresis conditions were as described in Van Seuningen et al. (34Van Seuningen I. Perrais M. Pigny P. Porchet N. Aubert J.P. Biochem. J. 2000; 348: 675-686Crossref PubMed Scopus (81) Google Scholar). Gels were vacuum-dried and autoradiographed for 48 h at –80 °C.Immunofluorescence—GP220 and GP202 cells, Cdx-2 transfected GP202 clones and GP202 mock cells were trypsinized, fixed in Preserv-Cyt solution (Cytyc) and layed on slides in a ThinPrep2000, according to the manufacturer. Cells were treated with neuraminidase, for 2 h at 37 °C, and blocked with non immune serum diluted at 1:5 in a 10% bovine serum albumin solution for 20 min. Excess normal serum was removed and replaced with anti-MUC2 PMH1 antibody (10Reis C.A. David L. Correa P. Carneiro F. de Bolós C. Garcia E. Mandel U. Clausen H. Sobrinho-Simões M. Cancer Res. 1999; 59: 1003-1007PubMed Google Scholar). Slides were incubated overnight at 4 °C. After three washes with 1× phosphate-buffered saline, the slides were incubated at room temperature with fluorescein isothiocyanate-labeled secondary antibody (DAKO) diluted at 1:200 with a 5% bovine serum albumin solution, for 30 min in the dark, mounted in vectashield with DAPI (Vector, Burlingame, CA), and analyzed with a Leica DMIRE2 fluorescent microscope.RESULTSExpression of MUC2, Cdx-1, and Cdx-2 in Human Gastric and Colon Carcinoma Cell Lines—Expression of MUC2, Cdx-1, and Cdx-2 was studied by RT-PCR. As shown in Fig. 1A, MUC2 mRNA is expressed in all gastric carcinoma cell lines, except for GP202. Cdx-1 is expressed in GP220 gastric carcinoma cell line. These cells also express low levels of Cdx-2 (Fig. 1A). In colon carcinoma cell lines, MUC2 is expressed in mucus-secreting LS174T cells and these cells also express Cdx-1 and Cdx-2. Caco-2 enterocytes and HT-29 STD undifferentiated cells do not express MUC2 or Cdx-1. Cdx-2 is expressed in Caco-2 cells and to a lower extent in HT-29 STD cells (Fig. 1B).Characterization of the Promoter Activity of MUC2 Gene in Gastric Cancer Cells—A panel of deletion mutants covering 2.6 kb of the promoter of MUC2 were constructed in promoterless pGL3 basic vector (Fig. 2A). They were used in transient transfection experiments in four gastric carcinoma cell lines (KATOIII, MKN45, GP220, and AGS) (Fig. 2B). Transient transfection of a pGL3 basic reporter construct containing nucleotides –371 to +27 of the MUC2 gene resulted in low levels of luciferase activity. Addition of the next 576 nucleotides up to nucleotide –947 (–947/+27) led to a significant increase in promoter activity in all cell lines (about 4-fold activation on average) (Fig. 2B). Addition of the distal region up to nucleotide –2096 (–2096/+27) increased normalized luciferase activity by 2-fold in AGS cells and in MKN45 cells to a lower extent (1.2-fold). No further increase in activity was observed when the –2627/–1 construct was used in either of the cell lines tested (Fig. 2B). The presence of the 5′-UTR in construct –947/+27 did not modify the activity of the corresponding construct devoid of the 5′-UTR (–947/–1). This suggests that the 5′-UTR does not play a major role in regulating MUC2 gene in gastric cancer cells.Fig. 2Analysis of the activity of the promoter of MUC2 gene by transient transfections.A, schematic representation and localization of the different constructs covering 2627 nucleotides of the MUC2 promoter. B, transcriptional activity in gastric carcinoma cell lines (KATOIII, MKN45, GP220, and AGS). The values obtained in cells transfected with the empty vector were referred as to 1. The results are means ± S.D. and represent two separate experiments in triplicate for each fragment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, these results suggest that, in the four gastric cancer cell lines studied, essential positive regulatory elements for MUC2 promoter activity are present within the –947/–372 region. In AGS cells, a second distal active region, stretching over the –2096/–948 nucleotides, contains enhancer elements.Cdx-1 and Cdx-2 Transactivate the MUC2 Promoter in a Cell-specific Manner—The involvement of Cdx-1 and Cdx-2 in the intestinal development and differentiation and their role as transcription factors for several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 could regulate MUC2 transcription. Adding to this, four putative binding sites for Cdx-1 and Cdx-2 were identified in MUC2 promoter at –177/–171, –191/–187, –1010/–1006, and –2614/–2610 that all contain Cdx consensus sequence TTTAT/C (Fig. 3A). Co-transfection experiments were performed to study the biological effect of these transcription factors on MUC2 transcriptional activity using pGL3-MUC2 deletion constructs in the presence of expression vectors encoding Cdx-1 or Cdx-2. The luciferase activity was compared with the one obtained in the co-transfection experiments carried out with the corresponding empty vector. Cdx-1 did not have any significant effect on MUC2 promoter activity in any of the gastric carcinoma cell lines, except for a 30% inhibition of the luciferase activity observed with the –371/+27 fragment in AGS cells (Fig. 3B, black bar). In MKN45 (gray bars), co-transfection with Cdx-2 induced luciferase activity of the four MUC2 promoter constructs (4–7 fold induction) (Fig. 3B). In KATOIII cells (hatched bars), Cdx-2 co-transfection resulted in a 2.5-fold transcriptional activation of the –947/+27 construct. Cdx-2 had no effect on the other two gastric carcinoma cell lines tested (GP220 and AGS), except for a 70% inhibitory effect with the –371/+27 fragment in the AGS cell line (black bars).Fig. 3Regulation of the MUC2 promoter activity by Cdx-1 and Cdx-2 transcription factors.A, schematic representation and localization of the different constructs used in co-transfection experiments with Cdx-1 and Cdx-2 expression vectors. Cdx putative binding sites are indicated. B, co-transfection experiments were performed in the presence of the MUC2 promoter constructs –371/+27, –947/+27, –2096/+27, and –2627/–1 in gastric carcinoma cell lines. C, co-transfection experiments were performed in the presence of the MUC2 promoter constructs –371/+27 and –2627/–1 in colon carcinoma cell lines. The values obtained in cells transfected with the empty vector were referred as to 1. The results are means ± S.D. and represent two separate experiments in triplicate for each fragment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The same experiments performed in HT-29 STD, LS174T and Caco-2 colon carcinoma cell lines indicate that Cdx-2 transactivates MUC2 promoter in the three cell lines tested (Fig. 3C). In HT-29 STD (23-fold), LS174T (55-fold), and Caco-2 (15-fold), Cdx-2 transactivation is much stronger with the smaller construct (–371/+27) when compared with the longer construct (–2627/–1) (Fig. 3C). One can note that the levels of activation vary greatly between the different cell lines indicating cell-specific activity of Cdx-2. Unlike gastric cells, we observed transactivation of MUC2 promoter by Cdx-1 in colon cancer cells. Transactivation was more efficient on the short construct –371/+27 in HT-29 STD (4.5-fold) and LS174T (10-fold) cells whereas it was more active on the long construct –2627/–1 in Caco-2 cells (10-fold). The transactivation is however much less important than with Cdx-2. From these studies it can be concluded that the MUC2 promoter is strongly transactivated by Cdx-2 in KATO-III and MKN45 gastric carcinoma cell lines as well as in all colon carcinoma cell lines. Cdx-1 appears more specific as it only transactivates MUC2 promoter in colon cancer cells.Identification of Two Cdx-2 cis-Elements within the MUC2 Promoter—In order to demonstrate that the Cdx-2 transcription factor binds to the MUC2 promoter, EMSAs were performed in the presence of GP220 nuclear extracts and probe wild type –201/–161, that contains the two putative Cdx-2 binding sites at –177/–171" @default.
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• W2154684225 date "2003-12-01" @default.
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• W2154684225 title "Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines" @default.
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