Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 activates MDR1 expression in HT-29 cells. (A, upper) A monoclonal anti-CDX2 antibody detects the roughly 40-kD CDX2 protein in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. (A, lower) Relative level of MDR1 gene expression in HT-29/PGS-CDX2 and HT-29/PGS-neo in Affymetrix microarray studies. (B) Northern and Western blot analysis detects MDR1 transcripts and products in HT-29/PGS-CDX2 with low or absent MDR1 expression in HT-29/PGS-neo cells. In Western blot analysis, a mouse monoclonal anti-MDR1 antibody detects the roughly 170-kD MDR1 product in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. (C) Expression of CDX2 and MDR1 in 13 CRC cell lines. In the indicated 13 colorectal cancer cell lines, Western blot analyses of MDR1 and CDX2 expression were performed using a mouse monoclonal antibody against human MDR1 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 MDR1 expression was performed using an MDR1 cDNA probe. The membrane was stripped and reprobed with a GAPDH cDNA probe to verify loading and transfer.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Expressing, Gene Expression, Microarray, Northern Blot, Western Blot, Membrane

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: The MDR1 gene is a primary target of CDX2 action. (A) Time course of MDR1 gene induction in response to activation of a CDX2-ER fusion protein by 4-OHT. (B) Induction of MDR1 transcript in response to activation of a CDX2-ER fusion protein by 4-OHT is not inhibited by the protein synthesis inhibitor cycloheximide, but protein synthesis is blocked. (C) Inhibition of CDX2 expression by siRNA targeting leads to decreased MDR1 expression in CRC cell line DLD1. Assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Activation Assay, Inhibition, Expressing

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: Localization of regulatory elements and CDX2 binding sites in the 5′-flanking region of the MDR1 gene. (A) Schematic representation of the 5′-flanking region of the MDR1 gene and MDR1 reporter gene constructs constructed. The location and sequence of four consensus CDX2-binding sites in the 5′-flanking region of MDR1 is indicated. The direction of the arrows indicates the strand on which the candidate CDX2-binding element was found (i.e., sense or antisense). The MDR1 genomic DNA sequences present in the reporter gene vectors are indicated. Localized mutations in the candidate CDX2-binding sites (i.e., site A and B) were introduced into the -4003/+50 construct as noted at the bottom of the panel, and the series of constructs generated is shown. (B) Key sequences for MDR1 transcription in CDX2-expressing cell lines reside between base pairs -4003 and -3414. Mean and SD values of the luciferase activity ratio in HT29/PGS-CDX2 cells to that in HT29/PGS-neo cells are shown. (C) CDX2 candidate binding sites “A” and “B” play critical roles in MDR1 transcription. All assays were performed in triplicate, and mean and SD values of luciferase activity ratio are shown.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Binding Assay, Construct, Sequencing, Generated, Expressing, Luciferase, Activity Assay

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 binding to MDR1 promoter region shown by ChIP. (A) 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. (B) Specificity of recovery of DNA fragments of MDR1 promoter region following ChIP with anti-CDX2 antibody was confirmed in Caco2, which has endogenous strong CDX2 expression. Assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Binding Assay, Expressing

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 and MDR1 expressions are well correlated in human colon epithelium and stomach cancer tissues. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues with anti-CDX2 monoclonal antibody (A, B, left) and with anti-MDR1 monoclonal antibody, C494 (A, B, right) in (A) human colon epithelium and (B) stomach cancer tissue.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Immunohistochemistry

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: HT29 cells ectopically expressing CDX2 have MDR1-dependent drug resistance. (A) Effect of chemotherapeutic drugs on HT29/PGS-CDX2 (open circle) and HT29/PGS-neo (closed circle) cell lines. (B) Effect of additional verapamil on vincristine and paclitaxel in HT29/PGS-CDX2 (open circle) and HT29/PGS-neo (closed circle) cell lines. (C) [IC50 (72h)] determined by MTT assay on HT29/PGS-CDX2 and HT29/PGS-neo cells. Co-treatment with verapamil significantly recovered the sensitivity of vincristine and paclitaxel on HT-29/PGS-CDX2 cells. The cytotoxic assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Expressing, MTT Assay