Bile Acids Induce Ectopic Expression of Intestinal Guanylyl Cyclase C Through Nuclear Factor-κB and Cdx2 in Human Esophageal Cells
2006; Elsevier BV; Volume: 130; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2005.12.032
ISSN1528-0012
AutoresPhilip R. Debruyne, Matthew E. Witek, Li Gong, Ruth Birbe, Inna Chervoneva, Tianru Jin, Claire Domon–Cell, Juan P. Palazzo, Jean‐Noël Freund, Peng Li, Giovanni M. Pitari, Stephanie Schulz, Scott A. Waldman,
Tópico(s)Esophageal and GI Pathology
ResumoBackground & Aims: Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. Methods: Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. Results: GC-C mRNA and protein were ectopically expressed in ∼80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor κB (NF-κB). Furthermore, deoxycholate increased NF-κB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-κB subunit p50. Moreover, a dominant negative construct for NF-κB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. Conclusions: Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-κB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C. Background & Aims: Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. Methods: Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. Results: GC-C mRNA and protein were ectopically expressed in ∼80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor κB (NF-κB). Furthermore, deoxycholate increased NF-κB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-κB subunit p50. Moreover, a dominant negative construct for NF-κB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. Conclusions: Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-κB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C. Adenocarcinomas of the stomach (ACS) and esophagus (ACE) are leading causes of cancer and cancer-related mortality worldwide. The incidence of ACE has risen steadily in both the United States and Europe, and in white males in the United States has become the solid tumor with one of the most rapidly increasing incidences over the past 30 years.1Devesa S.S. Blot W.J. Fraumeni Jr, J.F. Changing patterns in the incidence of esophageal and gastric carcinoma in the United States.Cancer. 1998; 83: 2049-2053Crossref PubMed Scopus (1997) Google Scholar, 2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J Clin. 2004; 54: 8-29Crossref PubMed Scopus (3943) Google Scholar ACS is the second most frequent cause of cancer death worldwide, and rates of adenocarcinoma below the gastroesophageal junction in the gastric cardia also have increased during the last 30 years.2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J Clin. 2004; 54: 8-29Crossref PubMed Scopus (3943) Google Scholar Virtually all ACEs arise from Barrett's metaplasia, in which normal squamous epithelial cells of the esophagus are replaced by intestinal epithelial and goblet cells, associated with gastroesophageal reflux disease (GERD).3Wild C.P. Hardie L.J. Reflux, Barrett's oesophagus and adenocarcinoma burning questions.Nat Rev Cancer. 2003; 3: 676-684Crossref PubMed Scopus (244) Google Scholar, 4Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer. 2003; 3: 592-600Crossref PubMed Scopus (286) Google Scholar Most ACSs are initiated by infection by Helicobacter pylori, which also progresses to intestinal metaplasia preceding neoplastic transformation.4Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer. 2003; 3: 592-600Crossref PubMed Scopus (286) Google Scholar Neoplastic transformation above and below the gastroesophageal junction, in part, reflects gastroduodenal reflux.3Wild C.P. Hardie L.J. Reflux, Barrett's oesophagus and adenocarcinoma burning questions.Nat Rev Cancer. 2003; 3: 676-684Crossref PubMed Scopus (244) Google Scholar, 5Paulson T.G. Reid B.J. Focus on Barrett's esophagus and esophageal adenocarcinoma.Cancer Cell. 2004; 6: 11-16Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 6Spechler S.J. The role of gastric carditis in metaplasia and neoplasia at the gastroesophageal junction.Gastroenterology. 1999; 117: 218-228Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar There is a strong association between the concentration of acid and bile acid in the refluxate and the degree of GERD, and bile reflux is a main predisposing factor for Barrett's esophagus.7Nehra D. Howell P. Williams C.P. Pye J.K. Beynon J. Toxic bile acids in gastro-oesophageal reflux disease influence of gastric acidity.Gut. 1999; 44: 598-602Crossref PubMed Scopus (354) Google Scholar, 8Vaezi M.F. Richter J.E. Role of acid and duodenogastroesophageal reflux in gastroesophageal reflux disease.Gastroenterology. 1996; 111: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar Similarly, the risk of intestinal metaplasia in the stomach is related to the concentration of bile acid in gastric juice, independently of H pylori infection.9Sobala G.M. O'Connor H.J. Dewar E.P. King R.F. Axon A.T. Dixon M.F. Bile reflux and intestinal metaplasia in gastric mucosa.J Clin Pathol. 1993; 46: 235-240Crossref PubMed Scopus (197) Google Scholar Moreover, bile acid reflux promotes the development of intestinal metaplasia and progression to neoplasia in the upper gastrointestinal tract in rodents.10Newberne P.M. Charnley G. Adams K. Cantor M. Roth D. Supharkarn V. Fong L. Gastric and oesophageal carcinogenesis models for the identification of risk and protective factors.Food Chem Toxicol. 1986; 24: 1111-1119Crossref PubMed Scopus (27) Google Scholar, 11Kobori O. Shimizu T. Maeda M. Atomi Y. Watanabe J. Shoji M. Morioka Y. Enhancing effect of bile and bile acid on stomach tumorigenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.J Natl Cancer Inst. 1984; 73: 853-861PubMed Google Scholar Although important etiologic factors associated with neoplastic transformation at the gastroesophageal junction, the precise mechanisms by which acid and bile acid induce neoplastic transformation in the esophagus and stomach remain undefined.3Wild C.P. Hardie L.J. Reflux, Barrett's oesophagus and adenocarcinoma burning questions.Nat Rev Cancer. 2003; 3: 676-684Crossref PubMed Scopus (244) Google Scholar, 4Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer. 2003; 3: 592-600Crossref PubMed Scopus (286) Google Scholar Cdx2, a member of the homeobox family of transcription factors that are homologs of the Drosophila caudal gene product, is important in establishing and maintaining the intestinal epithelium by regulating enterocyte-specific transcription in humans.12Lorentz O. Duluc I. Arcangelis A.D. Simon-Assmann P. Kedinger M. Freund J.N. Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.J Cell Biol. 1997; 139: 1553-1565Crossref PubMed Scopus (260) Google Scholar, 13Suh E. Traber P.G. An intestine-specific homeobox gene regulates proliferation and differentiation.Mol Cell Biol. 1996; 16: 619-625Crossref PubMed Scopus (459) Google Scholar Although Cdx2 is not expressed in normal epithelial cells of the esophagus or stomach, it is nearly ubiquitously expressed in intestinal metaplasia and frequently expressed in adenocarcinomas arising therein, suggesting an important role for this tissue-specific transcription factor in neoplastic transformation at the gastroesophageal junction.9Sobala G.M. O'Connor H.J. Dewar E.P. King R.F. Axon A.T. Dixon M.F. Bile reflux and intestinal metaplasia in gastric mucosa.J Clin Pathol. 1993; 46: 235-240Crossref PubMed Scopus (197) Google Scholar, 14Phillips R.W. Frierson Jr, H.F. Moskaluk C.A. Cdx2 as a marker of epithelial intestinal differentiation in the esophagus.Am J Surg Pathol. 2003; 27: 1442-1447Crossref PubMed Scopus (214) Google Scholar, 15Guo R.J. Suh E.R. Lynch J.P. The Role of Cdx proteins in intestinal development and cancer.Cancer Biol Ther. 2004; 3: 593-601Crossref PubMed Scopus (213) Google Scholar This causal relationship is underscored by the development of intestinal metaplasia followed by invasive carcinoma in transgenic mice that express ectopic Cdx2 in the foregut.16Mutoh H. Sakurai S. Satoh K. Tamada K. Kita H. Osawa H. Tomiyama T. Sato Y. Yamamoto H. Isoda N. Yoshida T. Ido K. Sugano K. Development of gastric carcinoma from intestinal metaplasia in Cdx2-transgenic mice.Cancer Res. 2004; 64: 7740-7747Crossref PubMed Scopus (154) Google Scholar, 17Silberg D.G. Sullivan J. Kang E. Swain G.P. Moffett J. Sund N.J. Sackett S.D. Kaestner K.H. Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.Gastroenterology. 2002; 122: 689-696Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar Indeed, Cdx2 is one of the most likely contributing factors in the induction of intestinal metaplasia in the upper gastrointestinal tract.4Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer. 2003; 3: 592-600Crossref PubMed Scopus (286) Google Scholar Delineating the molecular events underlying the pathogenesis of neoplastic transformation offers the unprecedented opportunity to identify novel molecules that could serve as diagnostic markers and therapeutic targets for managing patients at various stages of premalignant and malignant progression at the gastroesophageal junction.3Wild C.P. Hardie L.J. Reflux, Barrett's oesophagus and adenocarcinoma burning questions.Nat Rev Cancer. 2003; 3: 676-684Crossref PubMed Scopus (244) Google Scholar, 4Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer. 2003; 3: 592-600Crossref PubMed Scopus (286) Google Scholar Guanylyl cyclase C (GC-C), encoded by GUCY2C, is a member of the guanylyl cyclase family of single transmembrane receptors that is expressed selectively in apical membranes of intestinal epithelial cells.18Park J. Schulz S. Waldman S.A. Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.Gastroenterology. 2000; 119: 89-96Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Lucas K.A. Pitari G.M. Kazerounian S. Ruiz-Stewart I. Park J. Schulz S. Chepenik K.P. Waldman S.A. Guanylyl cyclases and signaling by cyclic GMP.Pharmacol Rev. 2000; 52: 375-414PubMed Google Scholar Activation of GC-C by the endogenous peptides guanylin and uroguanylin or diarrheagenic bacterial heat-stable enterotoxins (STs) induces the accumulation of guanosine 3′, 5′ cyclic monophosphate (cGMP). This cyclic nucleotide stimulates the opening of the cystic fibrosis transmembrane conductance regulator chloride channel and inhibits a sodium-hydrogen exchanger, and the resulting efflux of fluid and electrolytes can manifest as secretory diarrhea. Furthermore, GC-C signaling regulates proliferation of epithelial cells along the crypt-villus axis, induces cytostasis in human colon cancer cells, and suppresses intestinal tumor formation.20Steinbrecher K.A. Wowk S.A. Rudolph J.A. Witte D.P. Cohen M.B. Targeted inactivation of the mouse guanylin gene results in altered dynamics of colonic epithelial proliferation.Am J Pathol. 2002; 161: 2169-2178Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 21Pitari G.M. Di Guglielmo M.D. Park J. Schulz S. Waldman S.A. Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells.Proc Natl Acad Sci U S A. 2001; 98: 7846-7851Crossref PubMed Scopus (139) Google Scholar, 22Pitari G.M. Zingman L.V. Hodgson D.M. Alekseev A.E. Kazerounian S. Bienengraeber M. Hajnoczky G. Terzic A. Waldman S.A. Bacterial enterotoxins are associated with resistance to colon cancer.Proc Natl Acad Sci U S A. 2003; 100: 2695-2699Crossref PubMed Scopus (131) Google Scholar, 23Shailubhai K. Yu H.H. Karunanandaa K. Wang J.Y. Eber S.L. Wang Y. Joo N.S. Kim H.D. Miedema B.W. Abbas S.Z. Boddupalli S.S. Currie M.G. Forte L.R. Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP.Cancer Res. 2000; 60: 5151-5157PubMed Google Scholar Of significance, intestine-specific expression of GC-C is mediated by Cdx2.18Park J. Schulz S. Waldman S.A. Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.Gastroenterology. 2000; 119: 89-96Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Moreover, ectopic expression of GC-C mRNA was detected in a limited series of primary and metastatic ACE and ACS.24Birbe R. Palazzo J.P. Walters R. Weinberg D. Schulz S. Waldman S.A. Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia and adenocarcinoma of the gastrointestinal tract.Hum Pathol. 2005; 36: 170-179Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 25Park J. Schulz S. Haaf J. Kairys J.C. Waldman S.A. Ectopic expression of guanylyl cyclase C in adenocarcinomas of the esophagus and stomach.Cancer Epidemiol Biomarkers Prev. 2002; 11: 739-744PubMed Google Scholar These data imply that ectopically expressed GC-C might be a unique disease-selective mechanism-based diagnostic marker and therapeutic target for cells progressing from intestinal metaplasia through adenocarcinoma at the gastroesophageal junction.18Park J. Schulz S. Waldman S.A. Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.Gastroenterology. 2000; 119: 89-96Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The working hypothesis suggests that novel GC-C expression at the gastroesophageal junction reflects ectopic expression of Cdx2 and the associated transdifferentiation to an intestinal phenotype induced by etiologic agents implicated in carcinogenesis at the gastroesophageal junction. Here, the expression of GC-C in normal esophagus and stomach is compared with that in a series of specimens from patients with ACE and ACS. Moreover, studies in a human esophageal adenocarcinoma cell line reveal for the first time a molecular mechanism by which bile acid and acid could induce intestinal transformation at the gastroesophageal junction. Indeed, novel expression in esophageal cells of GC-C, normally a marker of differentiated intestinal epithelium, is mediated by ectopic Cdx2 expression induced by bile acid through transcriptional regulation by mculear factor κB (NF-κB). T84 human colon cancer cells (ATCC, Manassas, VA) were maintained as described.18Park J. Schulz S. Waldman S.A. Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.Gastroenterology. 2000; 119: 89-96Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar TE12b cells derived from human squamous cell carcinoma of the esophagus (SCE)26Nishihira T. Hashimoto Y. Katayama M. Mori S. Kuroki T. Molecular and cellular features of esophageal cancer cells.J Cancer Res Clin Oncol. 1993; 119: 441-449Crossref PubMed Scopus (219) Google Scholar were obtained from Dr K. Huebner (Kimmel Cancer Center, Thomas Jefferson University) and maintained in minimal essential medium supplemented with 10% fetal bovine serum and penicillin and streptomycin. OE33 (JROECL33; European Collection of Cell Cultures; ECACC, Wiltshire, UK) and OE19 human ACE cells (JROECL19; ECACC)27de Both N.J. Wijnhoven B.P. Sleddens H.F. Tilanus H.W. Dinjens W.N. Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro.Virchows Arch. 2001; 438: 451-456Crossref PubMed Scopus (32) Google Scholar were obtained from Dr Winand Dinjens (Department Pathology, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands) through Dr Marc Mareel (Department of Radiotherapy & Nuclear Medicine, Ghent University, Ghent, Belgium) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/L-glucose and L-glutamine (Mediatech Inc, Herndon, VA) and with 10% fetal bovine serum and penicillin and streptomycin. TE7 human ACE cells26Nishihira T. Hashimoto Y. Katayama M. Mori S. Kuroki T. Molecular and cellular features of esophageal cancer cells.J Cancer Res Clin Oncol. 1993; 119: 441-449Crossref PubMed Scopus (219) Google Scholar were obtained from Dr T. Kudo (Tohoku University School of Medicine, Sendai, Japan) through Dr H. Kubokura (Department of Surgery, Washington University School of Medicine, St. Louis, MO) and maintained in RPMI 1640 with L-glutamine (Mediatech Inc) supplemented with 10% fetal bovine serum and penicillin and streptomycin. Cells derived from ACS (AGS, KATO III, SNU-1, SNU-5, and RF-1) were obtained from ATCC through Dr R. Baffa (Kimmel Cancer Center, Thomas Jefferson University) and maintained in RPMI 1640 with L-glutamine (Mediatech Inc) supplemented with 10% fetal bovine serum and penicillin and streptomycin. All cell lines were mycoplasma free (MycoAlert mycoplasma detection kit; Cambrex Corp, East Rutherford, NJ). Deoxylic acid (DCA) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were from Sigma (St. Louis, MO). ST was a gift from Targeted Diagnostics and Therapeutics, Inc (Exton, PA). For acidic culture conditions, the medium was acidified with 0.5 N HCl to achieve pH 6.5. In these experiments, an equal volume of water or 1 mol/L glucose was added to control medium (pH 7.4) to control for osmolality or glucose effects. Treatments (DCA and acidified medium) were conducted in medium containing 1% fetal bovine serum after starvation in serum-free media for 24 hours. For DNA synthesis, cells were synchronized by serum starvation for 48 hours, followed by stimulation for 24 hours in DMEM containing 10% fetal bovine serum for 24 hours. ST (1 μmol/L) or 8-Br-cGMP (5 mmol/L) were added to cells 15 min before 0.2 μCi/well of [methyl-3H]thymidine, which was added for 3 hours, followed by quantification of 3H-thymidine incorporation into DNA.21Pitari G.M. Di Guglielmo M.D. Park J. Schulz S. Waldman S.A. Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells.Proc Natl Acad Sci U S A. 2001; 98: 7846-7851Crossref PubMed Scopus (139) Google Scholar, 22Pitari G.M. Zingman L.V. Hodgson D.M. Alekseev A.E. Kazerounian S. Bienengraeber M. Hajnoczky G. Terzic A. Waldman S.A. Bacterial enterotoxins are associated with resistance to colon cancer.Proc Natl Acad Sci U S A. 2003; 100: 2695-2699Crossref PubMed Scopus (131) Google Scholar Fresh frozen and formalin-fixed paraffin-embedded specimens were obtained from the Cooperative Human Tissue Network (Philadelphia, PA) and Thomas Jefferson University under a protocol approved by the Thomas Jefferson University Institutional Review Board. Total RNA from cell lines was extracted by using the RNeasy kit (Qiagen Inc, Valencia, CA), and total RNA from fresh frozen clinical samples was extracted with TRI Reagent (Molecular Research Center, Inc, Cincinnati, OH) according to the manufacturers' instructions. Samples were homogenized by using ceramic beads and a Mini Beadbeater-8 (Biospec Products Inc, Bartlesville, OK). The concentration and purity of RNA of samples were determined by UV spectrophotometry (Ultrospec 3000, Amersham Bioscience Corp., Piscataway, NJ). Total RNA was isolated from formalin-fixed paraffin-embedded tissues by using the Paraffin Block RNA Isolation Kit and RNeasy Mini kit (RNA Cleanup protocol; Ambion, Inc, Austin, TX) with deoxyribonuclease treatment according to the manufacturer's instructions with the exception that xylene was replaced by CitriSolv (Fisher Scientific International Inc, Hampton, NH). Only specimens yielding RNA for ≥2 analyses (2 × 1 μg) were included. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was conducted by using the 5′ nuclease assay (1 tube, 1 enzyme protocol; TaqMan EZ RT-PCR Kit; ABI, Foster City, CA) and an Applied Biosystems 7000 Sequence Detection Instrument according to the manufacturer's instructions (ABI, Foster City, CA). The sequences of primers used for GUCY2C were 5′-ATTCTAGTGGATCTTTTCAATGACCA-3′ (forward) and 5′-CGTCAGAACAAGGACATTTTTCAT-3′ (reverse) and for the β-actin gene ACTB were 5′-CCACACTGTGCCCATCTACG-3′ (forward) and 5′-AGGATCTTCATGAG-GTAGTCAGTCAG-3′ (reverse). The TaqMan probe sequences were 5′-FAM-TACTTGGAGGACAATGTCACAGCCCCTG-TAMRA-3′ and 5′-FAM-ATGCCC-X(TAMRA)-CCCCCATGCCATCCTGCGTp-3′ for GUCY2C and ACTB, respectively. CDX2 primer/probe mix (Assay on Demand) was obtained from ABI. All RT-PCR reactions were conducted with 1 μg of total RNA with the following thermocycler conditions: before reverse transcription the RNA template was heated at 50°C for 2 minutes in the presence of 0.01 U/L uracil N-glycosylase, 60°C for 30 minutes (reverse transcription), 95°C for 5 minutes, followed by 45 cycles of 94°C for 20 seconds and 62°C for 1 minute. Data were analyzed by using Sequence Detection Software (Applied Biosystems) with thresholds set at 0.2. Accurate quantification was achieved through the generation of standard curves by serial dilution of GUCY2C, CDX2, and ACTB RNA transcribed by T7 RNA polymerase. Template-negative controls were run on each PCR plate. For experiments using cell cultures, the GUCY2C or CDX2 mRNA were normalized to ACTB (encoding β-actin) mRNA levels in order to control for unequal loading or degradation of mRNA samples. For clinical specimens, mRNA levels were not routinely normalized to a housekeeping gene, reflecting the heterogeneity of contribution of epithelial and stromal cells to different specimens; the variability of expression of housekeeping genes in different cells, tissues, and patients; and the general acceptance of normalization to amount of total RNA analyzed.28Bustin S.A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR) trends and problems.J Mol Endocrinol. 2002; 29: 23-39Crossref PubMed Scopus (2074) Google Scholar Clinical specimens yielding <2 × 103 copies of β-actin mRNA per microgram of total RNA were deemed insufficient and omitted from further analysis. Formalin-fixed paraffin-embedded blocks were obtained from the pathology archives under an institutional review board–approved protocol at Thomas Jefferson University Hospital. Two pathologists (R.B. and J.P.P.) reviewed all hematoxylin and eosin-stained slides to confirm the diagnosis and ensure the presence of representative tissue before immunostains were performed. Immunohistochemistry for GC-C was performed using a previously characterized rabbit polyclonal antibody generated to the structurally unique carboxyl terminus of human GC-C.24Birbe R. Palazzo J.P. Walters R. Weinberg D. Schulz S. Waldman S.A. Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia and adenocarcinoma of the gastrointestinal tract.Hum Pathol. 2005; 36: 170-179Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Nuclear and cytoplasmic extracts were isolated by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) and dissolved in Laemmli buffer. Proteins (25 μg for nuclear extracts and 50 μg for total lysates and cytoplasmic extracts) were separated by 7.5% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose (Osmonics Inc, Minnetonka, MN). After incubation in 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.5% Tween 20 (Sigma) to block nonspecific protein binding, proteins of interest were identified by incubating with rabbit polyclonal antibodies against specific target proteins and, after a wash, antirabbit secondary antibodies conjugated to horseradish peroxidase. GC-C was identified by incubating with rabbit polyclonal antibody (1:500) raised against the structurally unique carboxyl terminus of human GC-C.24Birbe R. Palazzo J.P. Walters R. Weinberg D. Schulz S. Waldman S.A. Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia and adenocarcinoma of the gastrointestinal tract.Hum Pathol. 2005; 36: 170-179Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar In some experiments, primary antibody was preincubated with the antigenic peptide from GC-C (blocking peptide) or a control peptide. The p50 and p65 subunits of human NF-κB were identified by incubating with rabbit polyclonal antibodies (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA) to those specific subunits, respectively. GC-C, p50, and p65 staining was visualized with antirabbit antibody from donkey conjugated to horseradish peroxidase (NA934V; 1/5,000 Amersham Bioscience Corp). Cdx2 was identified by incubating with rabbit polyclonal antibodies to human Cdx2 (1:500; Quality Controlled Biochemical Biosource International, Hopkinton, MA), followed by antirabbit antibody from goat conjugated to horseradish peroxidase (1/50,000; Jackson ImmunoResearch Laboratories Inc, West Grove, PA). Secondary antibodies conjugated to horseradish peroxidase were visualized using the ECL staining reagent (Super Signal West Pico Chemiluminescent substrate; Pierce, Rockford, IL). Staining intensities were directly quantified with Kodak ID (V.3.5.4) software (Eastman Kodak, New Haven, CT) only in lanes receiving equivalent amounts of protein, determined after transfer by Ponceau S (Sigma) staining and confirmed by immunoblotting for β-actin (Santa Cruz Biotechnology). The role of Cdx2 in regulating GC-C expression was examined by using firefly luciferase reporter constructs including a pGL-3-Basic Luciferase Vector (Promega, Madison, WI; negative control), a pGL3 construct containing fragment −835 to +117 of the GUCY2C promoter (full length), and the identical construct with a TTT to CCC mutation in the Cdx2 consensus site (Cdx2MUT).18Park J. Schulz S. Waldman S.A. Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.Gastroenterology. 2000; 119: 89-96Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The role of NF-κB and AP-1 in regulating Cdx2 expression was examined by using reporter constructs including a pGL3 construct containing fragment −908/+119 of the murine CDX2 promoter (WT, previously denoted pCdx2-1Luc29Lorentz O. Cadoret A. Duluc I. Capeau J. Gespach C. Cherqui G. Freund J.N. Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras.Oncogene. 1999; 18: 87-92Crossref PubMed Scopus (74) Google Scholar), a pGL3 construct containing the full-length murine CDX2 promoter with mutations at the NF-κB–binding sites 1 (−15 to −6) and 2 (−103 to −93) (NF-κBMUT),30Kim S. Domon-Dell C. Wang Q. Chung D.H. Di Cristofano A. Pandolfi P.P. Freund J.N. Evers B.M. PTEN and TNF-alpha regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-kappaB-dependent pathway.Gastroenterology. 2002; 123: 1163-1178Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar a pGL3 construct that is truncated up to the AP-1 site (AP1WT; −195 to +119), and the identical construct with mutations in the AP-1 site (−186 to −179, AP1MUT; the TGTGTCAT sequence was changed to TGTGTTGT).29Lorentz O. Cadoret A. Duluc I. Capeau J. Gespach C. Cherqui G. Freund J.N. Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras.Oncogene. 1999; 18: 87-92Crossref PubMed Scopus (74) Google Scholar The role of Oct-1 in regulating Cdx2 expression was examined by using reporter constructs including a promoter-less luciferase construct SK-LUC (negative control) and the OCTWT and OCTMUT luciferase reporter constructs driven by the wild-type CDX2 promoter or that promoter containing a mutated Oct-1–binding site (−285/−10; this construct was
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