1. Volume 123, Issue 5, Pages 1565-1577 (November 2002)
CDX2 regulates liver intestine–cadherin expression in normal and malignant colon epithelium and intestinal metaplasia 
Takao Hinoi, *, Peter C. Lucas, ‡, Rork Kuick, §, Samir Hanash, §,∥, Kathleen R. Cho, *,‡,∥, Eric R. Fearon, *,‡,∥,¶ 
Gastroenterology 
Volume 123, Issue 5, Pages (November 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
CDX2 activates LI-cadherin expression in HT-29 cells. (A) Western blot analysis of CDX2 protein expression in HT-29/PGS-CDX2 and HT-29/PGS-neo. A monoclonal CDX2 antibody detects the roughly 40-kilodalton CDX2 protein in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. The membrane was stripped and probed with a rabbit polyclonal antibody against actin to verify loading and transfer. (B) Relative levels of LI-cadherin gene expression in HT-29/PGS-CDX2 and HT-29/PGS-neo cells in Affymetrix oligonucleotide array studies. (C) Northern blot analysis of LI-cadherin transcripts of roughly 3.7 kilobases in HT-29/PGS-CDX2 and HT-29/PGS-neo cells. After probing with a LI-cadherin cDNA probe, the membrane was stripped and probed with a GAPDH cDNA probe to confirm equivalent loading and transfer of the RNA. (D) Western blot analysis of LI-cadherin protein expression in HT-29/PGS-CDX2 and HT-29/PGS-neo cells. A goat polyclonal anti-human LI-cadherin antibody detects the roughly 120-kilodalton LI-cadherin protein in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. The membrane was stripped and probed with a monoclonal antibody against β-actin to verify loading and transfer.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Expression of CDX2 and LI-cadherin in colorectal cancer cell lines. In the indicated 13 colorectal cancer lines, Western blot analyses of LI-cadherin and CDX2 expression were performed using a goat polyclonal antibody against human LI-cadherin and a mouse monoclonal antibody against human CDX2. The membranes were stripped and reprobed with a monoclonal antibody against β-actin to verify loading and transfer. Northern blot analysis of LI-cadherin expression using an LI-cadherin cDNA probe. The membrane was stripped and reprobed with a GAPDH cDNA probe to verify loading and transfer.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
The LI-cadherin gene is a primary target of CDX2 action. (A) Time course of LI-cadherin gene induction in response to activation of a CDX2-ER fusion protein by 4-OHT. Northern blot analysis of LI-cadherin expression was performed on total RNA isolated from HT-29/CDX2-ER or HT-29 cells at various time points following exposure of the cells to 4-OHT. Following detection of the approximately 3.7-kilobase LI-cadherin transcripts, the blot was stripped and hybridized to a GAPDH cDNA probe to control for loading and transfer. (B) Induction of LI-cadherin transcripts by CDX2 is not inhibited by the protein synthesis inhibitor cycloheximide. Northern blot analysis of LI-cadherin expression was performed on total RNA isolated from HT-29/CDX2-ER cells treated for 12 hours with cycloheximide, 4-OHT, or both reagents as well as from HT-29 cells treated for 12 hours with cycloheximide and 4-OHT. The blot was stripped and rehybridized to a GAPDH cDNA probe. (C) LI-cadherin protein expression is induced in HT-29/CDX2-ER cells by 4-OHT treatment, but the induction of LI-cadherin protein is blocked by cycloheximide treatment. Western blot analysis of LI-cadherin expression was performed with a goat polyclonal anti-human LI-cadherin antibody on lysates from HT-29/CDX2-ER cells treated for 3 days with cycloheximide, 4-OHT, or both reagents. Probing of the blot with a monoclonal antibody against β-actin confirmed equal loading and transfer of the lanes.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Localization of regulatory elements and CDX2 binding sites in the 5'-flanking region of the LI-cadherin gene. (A) Schematic representation of the 5'-flanking region of the LI-cadherin gene and LI-cadherin reporter gene constructs constructed. The location and sequence of 4 consensus CDX2-binding sites in the 5'-flanking region of LI-cadherin is indicated, with +1 assigned to the most 5' base in the LI-cadherin cDNA sequence present in the 5' RACE analysis. The direction of the arrows indicates the strand on which the candidate CDX2-binding element was found (i.e., sense or antisense). The LI-cadherin genomic DNA sequences present in the reporter gene vectors are indicated. Localized mutations in the candidate CDX2-binding sites (i.e., sites A, B, C, and D) were introduced into the −500/+48 construct as noted at the bottom of the panel, and the series of constructs generated is shown. (B) Key sequences for LI-cadherin transcription in CDX2-expressing cell lines reside between base pairs −500 and −300. Reporter assays with the series of LI-cadherin deletion constructs were performed in 2 CDX2-expressing colorectal cancer cell lines (Caco-2 and LS 174T, left) as well as a CDX2-negative colorectal cancer cell line (RKO) and a CDX2-negative breast cancer cell line (MCF-7) (right). The luciferase activity of the empty pGL3 basic vector was assigned a value of 1. The reporter assays were performed in triplicate, and mean and SD values of luciferase activity are shown. The control β-galactosidase–expressing vector pCH110 was used to correct for differences in transfection efficiency. (C and D) CDX2 candidate binding sites “A” and “D” play critical roles in LI-cadherin transcription. Reporter assays were performed in the (C) CDX2-expressing LS 174T colorectal cancer cell line and (D) HEK293 with minimal level of endogenous CDX2 expression stably overexpressing CDX2, using the series of LI-cadherin reporter constructs with mutations in candidate CDX2-binding sites. The activity of the pGL3 basic vector was assigned a value of 1 in LS 174T. The relative activity in D indicates the ratio of activity in HEK293/PGS-CDX2 cells to that in HEK293/PGS-neo cells. Assays were performed in triplicate, and mean and SD values of luciferase activity are shown. The pCH110 vector was used to correct for differences in transfection efficiency.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
CDX2 binding to LI-cadherin 5'-flanking region shown by ChIP. CDX2 function was activated in HT-29/CDX2-ER cells by treatment of the cells with 4-OHT, and the cells were harvested at the indicated time points. Bulk (input) DNA was prepared as well as DNA isolated from ChIP with anti-CDX2 monoclonal antibody or no antibody. PCR was performed for 35 cycles with oligos specific for amplification of a 1.0-kilobase LI-cadherin 5'-flanking region DNA fragment or a 0.5-kilobase fragment containing exon 3 of CDX1. The PCR products were detected by electrophoresis through agarose gels and ethidium bromide staining. The negative images of the PCR products in ethidium bromide–stained gels are shown.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
CDX2 and LI-cadherin expression are tightly coupled in normal and malignant colorectal tissues. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues using a mouse monoclonal antibody against human CDX2 and a goat polyclonal antibody against human LI-cadherin. (A and B) CDX2 and LI-cadherin expression in normal colon tissue. (C and D) CDX2 and LI-cadherin expression in a well-differentiated colonic adenocarcinoma. (E and F) Absence of CDX2 and LI-cadherin expression in an LCMDC of the colon. (G and H) Reduced CDX2 and LI-cadherin expression in an LCMDC of the colon. (Original magnification: A and B, 200×; C–H, 400×.)
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Aberrant expression of CDX2 and LI-cadherin in intestinal metaplasia arising in the stomach. A–C are low-power images (original magnification 25×), and D–F are high-power images (original magnification 200×). Immunohistochemistry on serial sections of formalin-fixed and paraffin-embedded tissue was performed with a mouse monoclonal antibody against CDX2 and a goat polyclonal antibody against LI-cadherin. (A and D) H&E-stained stomach tissue showing chronic gastritis and intestinal metaplasia. (B and E) CDX2 expression in intestinal metaplasia with arrows indicating normal gastric epithelium and arrowheads indicating intestinal metaplasia. In E, note that nuclear staining for CDX2 (arrowheads) is seen only in intestinal metaplasia, whereas weak cytoplasmic staining for CDX2 is seen in the normal-appearing gastric epithelium (arrows) near the metaplastic glands. (C and F) LI-cadherin expression in intestinal metaplasia. In F, note that LI-cadherin expression is not seen in the normal-appearing gastric epithelium with weak cytoplasmic staining for CDX2, but LI-cadherin expression is strong in the CDX2-expressing metaplastic glands.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Cdx2 and LI-cadherin expression are tightly coupled in the colonic polyps of Cdx2 (+/−) knockout mice. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded colon polyp tissue from 18-month-old Cdx2 (+/−) knockout mice using mouse monoclonal anti-CDX2 and anti–β-catenin antibodies and a goat polyclonal anti-rat LI-cadherin antibody. (A) H&E-stained colonic polyps. (B) Control staining by anti–β-catenin antibody. (C and D) Cdx2 expression is suppressed in colonic polyps, presumably because of the biallelic Cdx2 inactivation as previously studied. LI-cadherin is down-regulated concordantly with Cdx2, suggesting that Cdx2 regulates LI-cadherin in mouse colonic tissue. (Original magnification 100×.)
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions