Unexpected Formation of 11(9→7)-abeo-Steroid Skeleton in Synthetic Studies toward Batrachotoxin.

Batrachotoxin (1) is a potent cardio- and neurotoxic steroid isolated from certain species of frogs, birds, and beetles. We previously disclosed two synthetic routes to 1. During our synthetic studies toward 1, we explored an alternative strategy for efficiently assembling its 6/6/6/5-membered steroidal skeleton (ABCD-ring). Here we report the application of intermolecular Weix and intramolecular pinacol coupling reactions. While Pd/Ni-promoted Weix coupling linked the AB-ring and D-ring fragments, SmI2-mediated pinacol coupling did not cyclize the C-ring. Instead, we discovered that SmI2 promoted a 1,4-addition of the α-alkoxy radical intermediate to produce the unusual 11(9→7)-abeo-steroid skeleton. Thus, this study demonstrates the convergent assembly of the skeleton of the natural product matsutakone in 11 steps from 2-allyl-3-hydroxycyclopent-2-en-1-one.

Total Synthesis of (-)-Batrachotoxin Enabled by a Pd/Ag-Promoted Suzuki-Miyaura Coupling Reaction.

Batrachotoxin is an extremely potent cardio- and neurotoxic steroidal alkaloid found in certain species of frogs, birds, and beetles. The steroidal 6/6/6/5-membered carbocycle (ABCD-ring) is U-shaped and functionalized with two double bonds, a six-membered C3-hemiacetal across the AB-ring, a seven-membered oxazepane on the CD-ring, and a dimethylpyrrolecarboxy group at the D-ring carbon chain. These structural features present an unusual and formidable synthetic challenge. Herein we report a total synthesis of batrachotoxin based on a newly devised convergent strategy through a 22-step sequence. Enantiopure AB-ring and D-ring fragments were prepared and subjected to a crucial C(sp2 )-C(sp2 ) coupling reaction. Although both C(sp2 ) centers were sterically encumbered by proximal tetrasubstituted carbon atoms, Ag2 O strongly promoted the Pd(PPh3 )4 -catalyzed Suzuki-Miyaura coupling reaction at room temperature, thereby connecting the two fragments without damaging their preexisting functionalities. Subsequent treatment with t-BuOK induced Dieckmann condensation to cyclize the C-ring. The judiciously optimized functionalizations realized oxazepane formation, carbon chain extension, and pyrrole carboxylic acid condensation to deliver batrachotoxin.

Construction of Pentacyclic Limonoid Skeletons via Radical Cascade Reactions.

Limonoids 1 and 2 share a 6/6/6/5-membered ABCD-ring system and a six-membered oxacycle and differ in their C9-stereochemistries. A new radical-based strategy was devised to construct the pentacyclic skeletons of 1 and 2. An oxacycle-fused A-ring and enyne fragments were coupled to produce radical precursors 4a-4c with different C7-oxygen functionalities. The bridgehead tertiary bromide of 4a-4c participated in a radical cascade reaction with the three unsaturated bonds to cyclize the C9-diastereomeric BCD-rings.

The total synthesis and functional evaluation of fourteen stereoisomers of yaku'amide B. The importance of stereochemistry for hydrophobicity and cytotoxicity.

Yaku'amide B is a highly unsaturated linear tridecapeptide and an extremely potent cytotoxin. Herein, we describe the synthesis of fourteen new stereoisomers of yaku'amide B using a unified assembly strategy. The hydrophobicities and cytotoxicities of these analogues were analyzed, along with those of four previously prepared isomers. Although all of the analogues share a common planar structure, their log D values varied significantly (3.39-5.32), presumably reflecting their distinct three-dimensional shapes. Subnanomolar-level cytotoxicity was observed for the natural yaku'amide B and its epimer of the N-terminal acyl group, whereas the other sixteen isomers exhibited 13- to 1200-fold weaker activities than that of the natural isomer. These data indicated the importance of the overall stereostructure of the 13-mer sequence of yaku'amide B for exerting its potent toxicity.

Elucidation and Total Synthesis of the Correct Structures of Tridecapeptides Yaku'amides A and B. Synthesis-Driven Stereochemical Reassignment of Four Amino Acid Residues.

Yaku'amides A (1) and B (2) possess four α,ß-dehydroamino acid residues in their linear tridecapeptide sequence and differ in their residue-3 (Gly for 1 and Ala for 2). The highly unsaturated peptide structure, characteristic cytotoxicity profile, and extreme scarcity from natural sources motivated us to launch synthetic studies of 1 and 2. Here, we report the total synthesis of the originally proposed structure of yaku'amide B (2a) by applying the route to 1a, which was previously established in our group. However, this accomplishment only proved that 2a and natural 2 were structurally different and prompted investigations directed toward determining the true structure of 2. Extensive Marfey's analyses of minute amounts of natural 2 and its degradation products presented us the possible stereoisomers, all of which were synthetically prepared for chromatographic comparison with the authentic fragments of 2. Based on this detective work, we proposed a corrected structure for yaku'amide B (2c), in which the orders of residues-7 and -8 and residues-11 and -12 are reversed. Finally, the total synthesis of 2c led to confirmation of its structural identity. Moreover, the revised structure of yaku'amide A (1c) was constructed by switching Ala-3 to Gly-3 and was found to be chromatographically matched with the re-isolated natural 1. The present work demonstrated the high reliability and sensitivity of the MS- and LC-based structural analyses and the indispensable role of chemical synthesis in structural elucidation of scarce natural products.

CDX1 confers intestinal phenotype on gastric epithelial cells via induction of stemness-associated reprogramming factors SALL4 and KLF5.

Intestinal metaplasia of the stomach, a mucosal change characterized by the conversion of gastric epithelium into an intestinal phenotype, is a precancerous lesion from which intestinal-type gastric adenocarcinoma arises. Chronic infection with Helicobacter pylori is a major cause of gastric intestinal metaplasia, and aberrant induction by H. pylori of the intestine-specific caudal-related homeobox (CDX) transcription factors, CDX1 and CDX2, plays a key role in this metaplastic change. As such, a critical issue arises as to how these factors govern the cell- and tissue-type switching. In this study, we explored genes directly activated by CDX1 in gastric epithelial cells and identified stemness-associated reprogramming factors SALL4 and KLF5. Indeed, SALL4 and KLF5 were aberrantly expressed in the CDX1(+) intestinal metaplasia of the stomach in both humans and mice. In cultured gastric epithelial cells, sustained expression of CDX1 gave rise to the induction of early intestinal-stemness markers, followed by the expression of intestinal-differentiation markers. Furthermore, the induction of these markers was suppressed by inhibiting either SALL4 or KLF5 expression, indicating that CDX1-induced SALL4 and KLF5 converted gastric epithelial cells into tissue stem-like progenitor cells, which then transdifferentiated into intestinal epithelial cells. Our study places the stemness-related reprogramming factors as critical components of CDX1-directed transcriptional circuitries that promote intestinal metaplasia. Requirement of a transit through dedifferentiated stem/progenitor-like cells, which share properties in common with cancer stem cells, may underlie predisposition of intestinal metaplasia to neoplastic transformation.

Endoscopic ultrasound with double-balloon endoscopy in the evaluation of small-bowel disease.

BACKGROUND: Double-balloon endoscopy (DBE) has become a new standard in enteroscopy. However, it may be difficult to make a diagnosis or plan treatment strategy with endoscopic visualization alone. The addition of endoscopic ultrasonography (EUS) has the potential to improve the ability to establish the diagnosis and develop a treatment strategy. The present study was conducted to assess the feasibility and usefulness of EUS with DBE. METHODS: EUS with DBE was performed in 31 of 891 patients who underwent DBE from July 2004 to March 2011 at Jichi Medical University Hospital. We analyzed the EUS findings for lesions and evaluated the usefulness of EUS considering the following three factors: qualitative diagnostic value for lesions, depth grading of lesions, and evaluation of the structure of severe strictures prior to endoscopic balloon dilation. RESULTS: EUS was performed for 31/32 lesions (97%) in 31 patients. EUS findings were informative for 29/32 lesions (91%). EUS findings were useful for establishing a qualitative diagnosis in 15/25 lesions (60%). EUS findings for depth grading provided useful information for determining the therapeutic strategy in 11/13 lesions (85%). EUS with DBE was useful in the evaluation of strictures for all six lesions (100%). The overall usefulness of EUS with DBE on decision making was 72% (23/32) in this study. CONCLUSIONS: EUS with DBE is feasible and useful. It provides additional information on small-bowel disease and contributes to establishing a precise diagnosis and selection of an appropriate therapeutic strategy.

Sox2 expression is maintained while gastric phenotype is completely lost in Cdx2-induced intestinal metaplastic mucosa.

Sox2 is closely related to the gastric phenotype. Sox2 plays a pivotal role in gastric epithelial differentiation in the adult. Sox2 expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium. The gastric mucosa is replaced by intestinal metaplastic mucosa in the stomach of caudal type homeobox 2 (Cdx2)-transgenic mice. The aim of this study was to use Cdx2-transgenic mice to investigate: (i) Sox2 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Sox2 and Cdx2. Quantitative real-time PCR was performed to determine Sox2, Cdx2, Muc5Ac, and alkaline phosphatase mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of Sox2, Cdx2, gastric mucin and alkaline phosphatase activity. We determined that Sox2 mRNA in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach was expressed 3.5-fold compared to the normal mouse stomach. Immunohistochemical analysis showed that the same cells in the intestinal metaplastic mucosa expressed both Cdx2 and Sox2. Gastric mucin was not expressed while alkaline phosphatase activity was recognized in the intestinal metaplastic mucosa in spite of the Sox2 expression. Cdx2 increased the transcriptional activity of the Sox2 gene, and Sox2 increased the transcriptional activity of the Muc5Ac gene, which was reduced by cotransfecion of Cdx2 together with Sox2 in the human gastric carcinoma cell line AGS. In conclusion, Sox2 expression is maintained while gastric phenotype is completely lost in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Direct repression of Sonic Hedgehog expression in the stomach by Cdx2 leads to intestinal transformation.

Shh (Sonic Hedgehog) is a morphogen involved in gastric fundic gland differentiation in the adult. Shh expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium and mice that lack Shh show intestinal transformation of the gastric mucosa. Similarly, in the stomach of Cdx2 (caudal-type homeobox 2)-transgenic mice, the gastric mucosa is replaced by intestinal metaplastic mucosa. The aim of the present study was to use Cdx2-transgenic mice to investigate: (i) Shh expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Shh and Cdx2. We determined that Shh mRNA levels were dramatically reduced in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach compared with the normal (wild-type) mouse stomach. This was not due to hypermethylation of the Shh promoter, but instead we showed that Cdx2 directly bound to the TATA box region of the Shh promoter. Cdx2 also down-regulated transcription of the Shh gene in the human gastric carcinoma cell lines AGS, MKN45 and MKN74. In conclusion, Cdx2 reduced Shh expression by binding to the unmethylated Shh promoter in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Monocyte chemoattractant protein-1 is generated via TGF-beta by myofibroblasts in gastric intestinal metaplasia and carcinoma without H. pylori infection.

Helicobacter pylori (H. pylori) stimulates secretion of monocyte chemoattractant protein 1 (MCP-1) from gastric mucosa. Monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration are recognized in human gastric carcinoma. We have previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia. Both chronic H. pylori-associated gastritis and Cdx2-transgenic mouse stomach develop intestinal metaplasia and finally gastric carcinoma. In this study we have directed our attention to MCP-1 expression in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. Quantitative real-time PCR was performed to determine MCP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of MCP-1, TGF-beta type I receptor, and alpha-smooth muscle actin (alphaSMA). We determined that MCP-1 mRNA dramatically increased in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach, compared with normal mouse stomach. Both MCP-1 and TGF-beta type I receptor were co-expressed in the alphaSMA-positive myofibroblasts of intestinal metaplastic mucosa and gastric carcinoma. Exogenous application of TGF-beta1 increased MCP-1 mRNA expression levels in the intestinal metaplastic tissue. Furthermore, TGF-beta1 was overexpressed and macrophage was strongly infiltrated in the gastric carcinoma. In conclusion, MCP-1 expression, which was stimulated by TGF-beta1, was recognized in the TGF-beta type I receptor-expressing myofibroblasts of the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. The present results suggest that intestinal metaplasia and gastric carcinoma themselves induce MCP-1 expression independently of H. pylori infection.

Expression of Claudin-2 in intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

OBJECTIVE: Cdx2 is expressed in human intestinal metaplastic mucosa and induces intestinal metaplastic mucosa in Cdx2-transgenic mouse stomach. Claudin-2 is a structural component of tight junctions in the intestine and Cdx2 activates the Claudin-2 promoter in the human intestinal epithelial cell line Caco-2. Our aim is to evaluate the expression of Claudin-2 in intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach. MATERIAL AND METHODS: The Claudin-2 expression in the normal gastric mucosa and normal intestinal mucosa of wild type mice and the intestinal metaplastic mucosa of Cdx2-transgenic mice was analyzed by immunohistochemistry, Western blotting and quantitative real-time PCR (qRT-PCR). RESULTS: Claudin-2 was expressed in the base of the glands in intestine and intestinal metaplasia while it was not expressed in the body of stomach. Claudin-2 expression was found in the antrum of stomach, while it was weaker than that in the intestine and the intestinal metaplasia. Claudin-2 was also detected in intestinal metaplasia, colon and ileum by both Western blotting and qRT-PCR while it was not detected in gastric body. CONCLUSION: These results suggest that Cdx2 plays an important role in the expression of Claudin-2 in vivo.

A close relationship between intestinal metaplasia and Cdx2 expression in human gallbladders with cholelithiasis.

We previously reported a case of a human gallbladder with cholelithiasis consisting of intestinal metaplasia with the expression of caudal-related homeobox transcription factor (Cdx2). However, it is unclear how often intestinal metaplasia and Cdx2 expression occur in human, nontumorous gallbladders with cholelithiasis. We studied the incidence of intestinal metaplasia and Cdx2 expression in human gallbladders with cholelithiasis. Gallbladders were resected under laparoscopy from 103 patients with cholelithiasis between September 2003 and March 2005. The mean age of the patients was 59.6 +/- 15.0 years (range, 22-92 years). We retrospectively reviewed these cases to look for the presence of intestinal metaplasia and the expression of Cdx2. In addition, the characteristics of intestinal metaplasia were examined by immunostaining for Muc2, chromogranin A, and serotonin. Intestinal metaplasia was found in 11.7% (12/103) of the gallbladders with cholelithiasis. The mean ages of patients with and without intestinal metaplasia were 60.8 +/- 15.4 and 59.4 +/- 14.9 years, respectively. Cdx2, Muc2, chromogranin A, and serotonin were expressed in 91.7% (11/12), 91.7% (11/12), 83.3% (10/12), and 50.0% (6/12) in intestinal metaplastic mucosa, respectively. Only one case (1.1%) that expressed Cdx2 without intestinal metaplasia did not express Muc2, chromogranin A, and serotonin. We found that 10.7% (11/103) of nontumorous gallbladders resected because of cholelithiasis under laparoscopy revealed intestinal metaplasia with Cdx2 expression.

Homeobox protein CDX2 reduces Cox-2 transcription by inactivating the DNA-binding capacity of nuclear factor-kappaB.

BACKGROUND: While cyclooxygenase-2 (COX-2) is not normally expressed by epithelial cells lining the human colon, COX-2 protein is aberrantly overexpressed in premalignant adenomatous polyps and carcinomas of the human colon. On the other hand, Cdx2 has been identified as a colonic tumor-suppressor gene, besides its role in cell differentiation. However, the relationship between CDX2 attenuation and COX-2 overexpression in colorectal carcinoma has not been established. Here, we investigated the mechanistic link between CDX2 downregulation and COX-2 upregulation. METHODS: Gene expression was examined by immunoblotting, reverse transcription-polymerase chain reaction, and promoter analysis. Promoter transactivation was quantified by using a luciferase construct. DNA binding of nuclear factor-kappaB (NF-kappaB) was examined by electromobility shift analysis. RESULTS: CDX2 decreased expression of COX-2 mRNA and protein at the transcriptional level in the human colon cancer Caco-2 cell line. Though p50/p65 NF-kappaB translocated into nucleus in the presence of CDX2, CDX2 interacted with p50/p65 NF-kappaB and impeded the formation of an NF-kappaB-DNA complex, required for promotion of Cox-2 transcription. CONCLUSION: The results indicate that CDX2 inhibits transcription of Cox-2 by interfering with the binding of NF-kappaB on the NF-kappaB binding site.

Differential gene expression between flat adenoma and normal mucosa in the colon in a microarray analysis.

BACKGROUND: Flat adenomas in the colon are associated with a relatively higher potential for malignancy. Distinct genes may be involved in the development of flat adenoma. The aim of this study was to profile gene expression changes in flat adenomas in the colon. METHODS: A genomewide expression analysis was carried out by using flat adenoma and adjacent normal mucosa in the colon to detect differences in gene expression. Because the right and left colon have different embryonic origins, each sample was classified according to its location, and the gene expression levels between flat adenoma and adjacent normal mucosa were also compared among samples derived from the right or left colon. RESULTS: A total of 180 genes were differentially expressed between flat adenoma and normal mucosa in the colon, including matrix metalloproteinase 7 (MMP7), cadherin 3 (CDH3), S100P, and dual oxidase 2 (DUOX2). In addition, a total of 89 and 49 genes were differentially expressed between flat adenoma and normal mucosa among the samples from the right and left colon, respectively. Subsequent quantitative real-time reverse transcriptase-polymerase chain reaction supported the reliability of the expression analysis. Immunohistochemical analysis confirmed differential CDH3 and MMP7 protein expression. CONCLUSIONS: This is the first report characterizing the genes differentially expressed in flat adenomas using a microarray analysis. Considerable differences in the gene expression profiles of flat adenomas also exist between the right and left colon. These data should lead to new insights into the pathogenesis of flat adenomas in the colon as well as to new therapeutic strategies.

Development of gastric carcinoma from intestinal metaplasia in Cdx2-transgenic mice.

In the progression of chronic gastritis, gastric mucosal cells deviate from the normal pathway of gastric differentiation to an intestinal phenotype. Many epidemiologic studies have found an association between the formation of intestinal metaplasia and the development of gastric carcinoma. However, there is no direct evidence that shows intestinal metaplasia is a precursor lesion of gastric carcinoma, to date. We periodically examined the intestinal metaplastic mucosa of Cdx2-transgenic mice we have previously generated. Gastric polyps developed from intestinal metaplastic mucosa in all stomachs of Cdx2-transgenic mice examined. These gastric polyps consisted of intestinal-type adenocarcinoma that invaded the submucosa and muscularis propria and occasionally spread into the subserosa. p53 and APC gene mutations were recognized in the adenocarcinomas. The participation of APC and p53 gene mutations in gastric carcinogenesis from the intestinal metaplasia was verified by the Cdx2-transgenic mice, carrying Apc(Min) mutation or p53 deficiency, that developed gastric polyps much earlier than Cdx2 alone. We successfully showed that long-term intestinal metaplasia induces invasive gastric carcinoma. These results indicate that intestinal metaplasia itself plays a significant role in the genesis and progression of gastric carcinoma.

Cdx2 specifies the differentiation of morphological as well as functional absorptive enterocytes of the small intestine.

Many transcription factors are involved in the molecular control of intestinal epithelial cell differentiation. We report in this study that the transcription factor Cdx2 functions to define absorptive enterocytes during intestinal epithelial differentiation. Cdx2 is expressed in the villi of the normal small intestine. Intestinal metaplasia, which expresses Cdx2, occurs as a pathological condition in gastric mucosa. We have previously established Cdx2 transgenic mice expressing Cdx2 exclusively in the gastric epithelium. In this study using Cdx2 transgenic mice, we show that Cdx2 plays a key role in the differentiation of intestinal absorptive enterocytes. The gastric mucosa of Cdx2 transgenic mice was morphologically completely changed into intestinal metaplastic mucosa. Absorptive enterocytes had microvilli which were observed by electron microscope. The intestinal metaplastic mucosa of Cdx2 transgenic mice expressed sucrase and peptide transporter PepT1. Disaccharidase and leucine aminopeptidase activities were observed in the intestinal metaplastic mucosa. Glucose and amino acids were absorbed from Cdx2 transgenic mouse stomach with intestinal metaplasia. Finally we generated mice whose intestine was extensively excised. Cdx2 transgenic mice with intestinal metaplasia survived even after extensive intestinal excision. We successfully demonstrated that Cdx2 induced not only morphological but also functional absorptive enterocytes in the intestinal metaplastic mucosa in vivo. Our results suggest that Cdx2 is necessary and sufficient by itself to specify the development of intestinal absorptive enterocytes, whereas other factors which are expressed in the small intestine are not always necessary for the differentiation of functional absorptive enterocytes.

[Expression of transcription factor in Barrett's esophagus].

Barrett's esophagus is the result of chronic injury which is usually caused by gastroesophageal reflux. NF-kappaB is expressed in the reflux esophagitis. Specialized columnar epithelium (SCE) is characteristic of Barrett's esophagus and has a malignant predisposition. SCE expresses Cdx1 and Cdx2. Adenocarcinoma in Barrett's esophagus is believed to develop through the metaplasia-dysplasia-carcinoma sequence. P53, beta-catenin, PPARgamma, and estrogen receptor beta are closely related to the development of esophageal carcinogenesis.

Comparative analysis of gastrointestinal microbiota between normal and caudal-related homeobox 2 (cdx2) transgenic mice.

BACKGROUND/AIMS: Caudal-related homeobox 2 (Cdx2) is expressed in the human intestinal metaplastic mucosa and induces intestinal metaplastic mucosa in the Cdx2 transgenic mouse stomach. Atrophic gastritis and intestinal metaplasia commonly lead to gastric achlorhydria, which predisposes the stomach to bacterial overgrowth. In the present study, we determined the differences in gut microbiota between normal and Cdx2 transgenic mice, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). METHODS: Twelve normal (control) and 12 Cdx2 transgenic mice were sacrificed, and the gastric, jejunal, ileac, cecal and colonic mucosa, and feces were collected. To quantitate bacterial microbiota, we used real-time qRTPCR with 16S rRNA gene-targeted, species-specific primers. RESULTS: The total numbers of bacteria in the gastric, jejunal, ileac, cecal, and colonic mucosa of the Cdx2 transgenic mice were significantly higher than those of the normal mice. The Bacteroides fragilis group and also Prevotella were not detected in the stomach of the normal mice, although they were detected in the Cdx2 transgenic mice. Moreover, the Clostridium coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group, and Prevotella were not detected in the jejunum or ileum of the normal mice, although they were detected in the Cdx2 transgenic mice. The fecal microbiota of the normal mice was similar to that of the Cdx2 transgenic mice. CONCLUSIONS: Our results showed the differences in composition of gut microbiota between normal and Cdx2 transgenic mice, which may be caused by the development of gastric achlorhydria and intestinal metaplasia in Cdx2 transgenic mice.

Aberrant expression of CDX2 in Barrett's epithelium and inflammatory esophageal mucosa.

BACKGROUND: There have been no detailed reports directly comparing the expression of CDX1 with that of CDX2 in the inflammatory esophageal mucosa and Barrett's epithelium. The present study was designed to examine the expression of CDX 1/2 in inflammatory esophageal mucosa with or without Barrett's epithelium. METHODS: The expression of CDX1/2 genes was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) in 34 human esophageal biopsy specimens, and CDX2 expression was also evaluated immunohistochemically, using anti-human CDX2 monoclonal antibody. The biopsy specimens for RNA extraction were taken endoscopically from esophageal mucosa with mucosal break due to gastroesophageal reflux disease (GERD), Barrett's epithelium, and normal epithelium. The expressions of mucin markers (MUC2) and intestine-specific genes (sucrase-isomal-tase, human defensin-5, alkaline phosphatase) were also comparatively analyzed. RESULTS: CDX1/2 expression was not found in the normal esophageal mucosa. The prevalence of CDX1/2 mRNA expression was significantly higher in the mucosa with Barrett's epithelium than in the mucosa without Barrett's epithelium. It is noteworthy, however, that the CDX2 mRNA expression was initiated at the stage of esophagitis, when neither CDX1 nor intestine-specific genes had emerged yet. In contrast to CDX2, CDX1 was expressed only in Barrett's epithelium. Immunohistochemical study demonstrated strong and extensive nuclear immunoreactivity for CDX2 in Barrett's epithelium. Furthermore, fine granular cytoplasmic staining was also observed in the cytoplasm in Barrett's epithelium, as well as in inflammatory esophageal mucosa. CONCLUSIONS: We report here, for the first time, that CDX2 is expressed in patients with Barrett's epithelium and inflammatory esophageal mucosa. These findings imply that the expression of CDX2 may be an early event leading to the development of Barrett's esophagus.

Expression of homeobox gene CDX2 precedes that of CDX1 during the progression of intestinal metaplasia.

BACKGROUND: The CDX1 and CDX2 genes are intestinal transcription factors that may be involved in the regulation of proliferation and differentiation of intestinal epithelial cells. There have been no detailed reports directly comparing the expression of CDX1 with that of CDX2 in chronic gastritis and intestinal metaplasia. Accordingly, we examined the expression of CDX1/2 and its association with the expression of other intestinal metaplasia-associated genes during the development of intestinal metaplasia. METHODS: The expression of CDX1/2 genes was analyzed, using the reverse transcriptase-polymerase chain reaction, in 44 human gastric tissue samples obtained endoscopically. The expressions of mucin markers (MUC2, MUC5AC), intestine-specific genes (sucrase-isomaltase, human defensin-5, alkaline phosphatase), a gene marker for fundic gland area (H+/K+ATPase beta subunit), and a gene for entire gastric glands (pepsinogen C) were also comparatively analyzed. RESULTS: There was no expression of CDX1/2 in gastric mucosa not infected by Helicobacter pylori. The prevalence of CDX1 mRNA expression was significantly higher in mucosa with intestinal metaplasia than in mucosa without intestinal metaplasia. It is noteworthy that CDX2 was expressed in the antral and fundic mucosa in the absence of the expression of CDX1 and gene markers for intestinal metaplasia. CONCLUSIONS: The expression of CDX2 precedes those of CDX1, sucrase-isomaltase, other intestine-specific genes (human defensin-5, alkaline phosphatase), and MUC2 during the progression of intestinal metaplasia. These findings imply that the expression of CDX2 may trigger the initiation and development of intestinal metaplasia.