Cdx1 promotes differentiation in a rat intestinal epithelial cell line Philippe Soubeyran, Frédéric André, Jean-Claude Lissitzky, Gustavo Vidal Mallo, Virginie Moucadel, Monique Roccabianca, Hocine Rechreche, Jacques Marvaldi, Ivan Dikic, Jean-Charles Dagorn, Juan Lucio Iovanna  Gastroenterology  Volume 117, Issue 6, Pages 1326-1338 (December 1999) DOI: 10.1016/S0016-5085(99)70283-0 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Cdx1 and Cdx2 mRNA expression in IEC-6 Cdx1-transfected cells. Total RNA (20 μg) from IEC-6/CAT and IEC-6/Cdx1 cells was subjected to electrophoresis, blotted, and hybridized with the Cdx1 and Cdx2 32P-labeled cDNAs as probes. As control, the filter was washed and hybridized with a 32P-labeled 28S ribosomal RNA probe. The autoradiographs were exposed at −80°C for 2 days. (B) Analysis of Cdx1 protein expression by transactivation of the Hoxa-7 promoter. The IEC-6/CAT and IEC-6/Cdx1 cells were transfected with constructs made of the Hoxa-7 or TK promoters driving the LacZ gene, using lipofectin reagents. Forty-eight hours later, the β-galactosidase activity was measured in cell extracts. The values of β-galactosidase activity generated by the Hoxa-7 LacZ, relative to TK LacZ, are shown (3 independent experiments). Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Cdx1 and Cdx2 mRNA expression in IEC-6 Cdx1-transfected cells. Total RNA (20 μg) from IEC-6/CAT and IEC-6/Cdx1 cells was subjected to electrophoresis, blotted, and hybridized with the Cdx1 and Cdx2 32P-labeled cDNAs as probes. As control, the filter was washed and hybridized with a 32P-labeled 28S ribosomal RNA probe. The autoradiographs were exposed at −80°C for 2 days. (B) Analysis of Cdx1 protein expression by transactivation of the Hoxa-7 promoter. The IEC-6/CAT and IEC-6/Cdx1 cells were transfected with constructs made of the Hoxa-7 or TK promoters driving the LacZ gene, using lipofectin reagents. Forty-eight hours later, the β-galactosidase activity was measured in cell extracts. The values of β-galactosidase activity generated by the Hoxa-7 LacZ, relative to TK LacZ, are shown (3 independent experiments). Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of Cdx1 expression on cell proliferation. (A) IEC-6/Cdx1, clone A3, clone H3, and IEC-6/CAT cells were grown in their standard medium. The IEC-6 Cdx1-expressing cells proliferated 2 times faster than IEC-6/CAT cells. (B) IEC-6/Cdx1 and IEC-6/CAT cells were grown in standard medium changed every other day. The growth rate of the 2 cell lines was similar. (C) IEC-6/Cdx1 cells were grown in a medium conditioned by the IEC-6/CAT cells; conversely, IEC-6/CAT cells were grown in a medium conditioned by IEC-6/Cdx1 cells. The medium was changed every other day. IEC-6/Cdx1 cells grew faster than IEC-6/CAT cells. Cell numbers are expressed as mean ± SD for 4 independent experiments. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of Cdx1 expression on cell proliferation. (A) IEC-6/Cdx1, clone A3, clone H3, and IEC-6/CAT cells were grown in their standard medium. The IEC-6 Cdx1-expressing cells proliferated 2 times faster than IEC-6/CAT cells. (B) IEC-6/Cdx1 and IEC-6/CAT cells were grown in standard medium changed every other day. The growth rate of the 2 cell lines was similar. (C) IEC-6/Cdx1 cells were grown in a medium conditioned by the IEC-6/CAT cells; conversely, IEC-6/CAT cells were grown in a medium conditioned by IEC-6/Cdx1 cells. The medium was changed every other day. IEC-6/Cdx1 cells grew faster than IEC-6/CAT cells. Cell numbers are expressed as mean ± SD for 4 independent experiments. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of Cdx1 expression on cell proliferation. (A) IEC-6/Cdx1, clone A3, clone H3, and IEC-6/CAT cells were grown in their standard medium. The IEC-6 Cdx1-expressing cells proliferated 2 times faster than IEC-6/CAT cells. (B) IEC-6/Cdx1 and IEC-6/CAT cells were grown in standard medium changed every other day. The growth rate of the 2 cell lines was similar. (C) IEC-6/Cdx1 cells were grown in a medium conditioned by the IEC-6/CAT cells; conversely, IEC-6/CAT cells were grown in a medium conditioned by IEC-6/Cdx1 cells. The medium was changed every other day. IEC-6/Cdx1 cells grew faster than IEC-6/CAT cells. Cell numbers are expressed as mean ± SD for 4 independent experiments. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of cell density on the rate of [3H]thymidine incorporation in DNA of IEC-6/Cdx1 and IEC-6/CAT cells. Radioactivity was normalized to the cell number and expressed as mean ± SD of 8 independent determinations. IEC-6/CAT cells showed inhibition of [3H]thymidine incorporation on cell contact. The Cdx1-expressing IEC-6 showed an opposite behavior; [3H]thymidine incorporation increased with the number of cells seeded. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of TGF-β1 on [3H]thymidine incorporation in DNA of IEC-6/Cdx1 and IEC-6/CAT cells. Radioactivity was normalized to the cell number and expressed as ± SD of 6 independent determinations. Experiments were repeated 3 times with similar results. IEC-6 cells were more sensitive to the TGF-β1 than Cdx1-expressing cells. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Sensitivity to apoptosis of Cdx1-transfected IEC-6 cells. The apoptosis rate was determined by flow-cytometric analysis of propidium iodide–stained IEC-6/Cdx1 and IEC-6/CAT cells in 2 different conditions of confluence. (A) IEC-6/CAT cells at 50% of confluence. (B) IEC-6/Cdx1 cells at 50% of confluence. (C) IEC-6/CAT cells confluent for 15 days. (D) IEC-6/Cdx1 cells confluent for 15 days. Whereas the pattern of propidium staining was similar for the 2 cell lines during the exponential phase of growth, the Cdx1-expressing IEC-6 cells, contrary to IEC-6/CAT cells, did not undergo spontaneous apoptosis in postconfluent conditions. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Migration of IEC-6/CAT and IEC-6 Cdx1-expressing cells 6 hours after wounding. IEC-6/CAT, IEC-6/Cdx1, and clones A3 and H3 were plated in 6-well culture plates and grown to confluence. The monolayers were wounded and cell migration was monitored. (A) To distinguish between the contributions of cell migration and cell proliferation to the wound closure process, migration assays were also carried out in the presence of mitomycin (5 μg/mL). Cells migrating across the wound margin were counted in 8 different areas chosen randomly. Values are means ± SD for 2 experiments performed in triplicate (n = 6). Cdx1-expressing IEC-6 cells treated or not by mitomycin showed a 3–4-fold greater mobility than control cells. (B) Typical wound picture taken during experiments shown in A. IEC-6/CAT cells (top left) and IEC-6/Cdx1 cells (top right), clone A3 (bottom left), and clone H3 (bottom right). Original magnification ×200. Wound position is shown by arrow. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Migration of IEC-6/CAT and IEC-6 Cdx1-expressing cells 6 hours after wounding. IEC-6/CAT, IEC-6/Cdx1, and clones A3 and H3 were plated in 6-well culture plates and grown to confluence. The monolayers were wounded and cell migration was monitored. (A) To distinguish between the contributions of cell migration and cell proliferation to the wound closure process, migration assays were also carried out in the presence of mitomycin (5 μg/mL). Cells migrating across the wound margin were counted in 8 different areas chosen randomly. Values are means ± SD for 2 experiments performed in triplicate (n = 6). Cdx1-expressing IEC-6 cells treated or not by mitomycin showed a 3–4-fold greater mobility than control cells. (B) Typical wound picture taken during experiments shown in A. IEC-6/CAT cells (top left) and IEC-6/Cdx1 cells (top right), clone A3 (bottom left), and clone H3 (bottom right). Original magnification ×200. Wound position is shown by arrow. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Cdx1 expression induces morphological changes in IEC-6 cells. Light morphology of the (A) IEC-6/CAT cells, (B) IEC-6/Cdx1 cells, (C) clone A3, and (D) clone H3. Original magnification 200×. (E) IEC-6/CAT control cells formed a simple monolayer composed of flat cells when grown at confluence. By contrast, the IEC-6/Cdx1 cells developed morphological changes characterized by multicellular and often multilayered structures. (F) The upper cells of domes developed a columnar-like epithelium. Original magnification 1000×; bar = 10 μm. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Cdx1 expression induces morphological changes in IEC-6 cells. Light morphology of the (A) IEC-6/CAT cells, (B) IEC-6/Cdx1 cells, (C) clone A3, and (D) clone H3. Original magnification 200×. (E) IEC-6/CAT control cells formed a simple monolayer composed of flat cells when grown at confluence. By contrast, the IEC-6/Cdx1 cells developed morphological changes characterized by multicellular and often multilayered structures. (F) The upper cells of domes developed a columnar-like epithelium. Original magnification 1000×; bar = 10 μm. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Cdx1 expression induces ultrastructural differentiation in IEC-6 cells. Electron-microscopic analysis was performed on cells harvested at confluence. (A) IEC-6/CAT cells (original magnification 3000×) showed only sparse microvilli but no feature of intestinal differentiation (bar = 2 μm). (B) IEC-6/Cdx1 cells (original magnification 25,000×) developed numerous microvilli covered with an abundant glycocalyx (arrowheads; bar = 200 nm). (C) Junctional complexes (arrowheads) and interdigitations of the lateral membrane (arrow) could be observed in IEC-6/Cdx1 cells only (original magnification 30,000×; bar = 200 nm). Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Immunolocalization of aminopeptidase N, villin, and F-actin in IEC-6/CAT and IEC-6/Cdx1 cells. (A, C, and E) IEC-6/CAT and (B, D, and F) IEC-6/Cdx1 cells were grown 10 days after confluence in glass chamber slides. Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100, and stained for (A and B) aminopeptidase N, (C and D) villin, and (E and F) F-actin. Localization of aminopeptidase N and villin was obtained by the binding to polyclonal antibodies, followed by revelation with biotinylated anti-rabbit immunoglobulins and fluorescein-conjugated streptavidin. F-actin distribution was observed after binding of rhodamine-phalloidin. (A) Aminopeptidase N was localized to the cytoplasm of IEC-6/CAT control cells. (B) The enzyme was translocated to the basolateral membrane in Cdx1-expressing cells. (C) A very weak staining for villin was obtained in IEC-6/CAT control cells, whereas (D) a strong cytoplasmic signal was observed in IEC-6/Cdx1 cells. (E) Most of the microfilaments appeared as stress fibers in IEC-6/CAT cells. (F) By contrast, in IEC-6/Cdx1 cells, actin fibers were mainly located at the cellular cortex. Gastroenterology 1999 117, 1326-1338DOI: (10.1016/S0016-5085(99)70283-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions