Cdx1 promotes differentiation in a rat intestinal epithelial cell line.

BACKGROUND & AIMS: Homeobox genes are involved in establishing and maintaining differentiated patterns in adult tissues. Cdx1 might carry out that function in the intestinal epithelium because its expression is specific to that tissue and increases during development. METHODS: Cdx1 expression was induced in IEC-6 intestinal epithelial cells by stable transfection, and subsequent changes in cell growth, resistance to apoptosis, migration, and differentiation were monitored. RESULTS: Compared with control, IEC-6/Cdx1 cells proliferated more rapidly, were more resistant to apoptosis, and migrated 3-4 times faster, as shown by an in vitro wound assay. IEC-6/Cdx1 cells in culture formed multilayers. Morphology of the top layer was similar to that of columnar epithelium, with cells showing typical features of differentiated enterocytes, including complex junctions and well-developed microvilli with glycocalix. Expression of 2 markers of enterocyte differentiation, aminopeptidase N and villin, was induced in IEC-6/Cdx1 cells. Aminopeptidase N was targeted to the basolateral membrane, and villin was localized to the cytoplasm. Actin filaments, which were mostly present in transcytoplasmic stress fibers in control cells, were redistributed to the cortex in Cdx1-transfected cells. CONCLUSIONS: Cdx1 expression in IEC-6 cells induces phenotypic changes characteristic of differentiating enterocytes, suggesting an important role for Cdx1 in the transition from stem cells to proliferating/transit cells.

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).

Convergent effects of growth factors, hormones, and fibronectin are necessary for the enterocyte differentiation of a colon adenocarcinoma cell line (HT29-D4).

The aim of this work was to show in serum-free medium a convergent effect of physiological factors and extracellular matrix proteins on the differentiation process of enterocytes by taking as a model the HT29-D4 clone that has the feature of differentiating when subcultured in fetal bovine serum glucose-free medium. We show that triiodothyronine (T3) as well as insulin promotes limited cell growth and differentiation, whereas fibronectin or bovine serum albumin (BSA) induces cell growth and a low level of differentiation. However, insulin, T3, fibronectin, and BSA together with epidermal growth factor and transferrin promoted satisfactory growth and enterocyte morphology with epithelial electrophysiological properties in HT29-D4 cells. With these factors adequate protein targeting was achieved since cells apically expressed the carcinoembryonic antigen, and basolaterally transferrin and insulin receptors, beta 1 and alpha v beta 6 integrins, talin, vinculin, and focal adhesion kinase (FAK). Talin, vinculin, FAK, and alpha v beta 6 integrin, the fibronectin receptor, were clustered in focal contacts, which agrees with a possible role of fibronectin in final cell growth, the latter process mediating the final phase of differentiation. This level of differentiation can be maintained for a long time. Thus HT29-D4 cells appear to be a suitable model to study the implication of integrins in the differentiation process of human enterocytes.

[Modification of the response to paclitaxel of human differentiated colon cancer line after long-term treatment at very low dose]. / Modification de la réponse au paclitaxel d'une lignée cancéreuse humaine de côlon différenciée après traitement continu à très faible dose.

Taxoids (paclitaxel, Taxol and docetaxel, Taxotere), drugs which stabilize microtubules, demonstrate marked activity against ovarian, brain and lung cancer. We investigated, on the human colon adenocarcinoma HT29-D4 cell line, firstly the effects of taxoids in function of the differentiation state and secondly the effects of a low dose of paclitaxel on the differentiation process. The cellular parameters studied were microtubular network, cell cycle and cell tubulin content. Undifferentiated cells treated with paclitaxel or docetaxel (10-100 nM) classically induced bundles in interphasic cells and pseudoasters in mitotic cells, arrest in cell cycle in G2M and increase in tubulin content. Inversely, no modification was observed in differentiated cells after similar taxoid treatment. Long-term low-dose pretreatment of differentiated cells restitutes some sensitivity to taxoids since these cells become responsive to further taxoid treatment, as reflected by modifications of the microtubule network.

des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.

HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.

Potential autocrine role of insulin-like growth factor II during suramin-induced differentiation of HT29-D4 human colonic adenocarcinoma cell line.

Suramin, a drug that binds to several types of growth factors, has been previously shown to induce the enterocyte-like differentiation of HT29-D4 human colonic adenocarcinoma cells, suggesting that growth factors are involved in such a process. Undifferentiated HT29-D4 cells release insulin-like growth factor II (IGF-II) into the culture medium that is totally complexed to heterogeneous IGF binding proteins (IGFBP) expressing high affinities for this growth factor (Kda = 0.02 nM and Kdb = 1.4 nM). These complexes do not allow IGF-II to bind to HT29-D4 cell surface type I IGF receptors, as evidenced by using 125I-IGF-II-IGFBP complexes. However, the addition of 40-100 micrograms/ml suramin, i.e., concentrations identical to the ones that are able to induce HT29-D4 cell differentiation, induces the release of IGF-II from IGF-II-IGFBP complexes, thereby allowing IGF-II to bind to the cell surface receptors. At such concentrations, suramin is indeed unable to alter IGF-II binding to HT29-D4 cells, a capacity that is observed only for concentrations higher than 200 micrograms/ml. Thus, suramin might have the unusual capacity to allow the establishment of an IGF-II autocrine loop involved in HT29-D4 cell differentiation. Consistent with this hypothesis is the fact that exogenously applied IGF-I (2.5 micrograms/ml) or agonist monoclonal antibody alpha IR-3 (2.5 micrograms/ml), which can bypass IGFBP present in the culture medium, induces part of HT29-D4 cell differentiation that is characterized by an important carcinoembryonic antigen release and the induction of numerous intercellular cysts with microvilli.

Vectorial release of carcinoembryonic antigen induced by IFN-gamma in human colon cancer cells cultured in serum-free medium.

Confluent monolayers of intestinal cell lines are useful models for studies of intestinal epithelial structure and function. Three cell lines have retained morphological and functional properties of intestinal epithelial cells compatible with such studies: Caco-2, T84 and HT29. However, the requirement of fetal bovine serum for the culture of these cells does not facilitate the design of experiments dealing with growth factors or hormonal regulation. The clonal intestinal cell line HT29-D4 can be cultured as fully differentiated epithelial monolayers in a synthetic medium containing transferrin, selenous acid, epidermal growth factor and suramin, a potent differentiation inducer. In the present study it is shown that HT29-D4 cells grown on permeable substratum in this synthetic medium developed electrically active monolayers consisting of columnar cells with morphological characteristics of normal enterocytes. After metabolic labelling with [35S]-methionine, HT29-D4 monolayers released most of their radiolabelled secretory proteins preferentially in the basal compartment of the cell culture chamber. However, the carcinoembryonic antigen, shown to be present in the apical plasma membrane, was exclusively released apically. This oriented release was stimulated by recombinant gamma-interferon (IFN-gamma) added only in the basal chamber, suggesting a basolateral restriction for IFN-gamma receptors.

The concentration of glucose in the culture medium determines the effect of suramin on the growth and differentiation of the human colonic adenocarcinoma cell clone HT29-D4.

Suramin, a drug currently used for advanced malignancy, induces the differentiation of the human colonic adenocarcinoma cell clone HT29-D4 and this process is correlated with a decreased glycolytic activity. We investigated the effects of suramin on HT29-D4 cells in the presence of various glucose concentrations. The main result of this study is that suramin has only an effect on HT29-D4 cell growth and differentiation when the concentration of glucose is above 10 mM. Therefore the efficiency of suramin as an anticancer drug may be greater on poorly differentiated tumoral cells with a high proliferative capacity.

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose.

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.

Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT29-D4.

Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis inhibits growth factor-induced mitogenesis. In the present report, we show that suramin inhibits the growth of human colic adenocarcinoma cells HT29-D4 and rapidly induces their differentiation into enterocyte-like cells. As soon as 6 days after the addition of suramin (100 micrograms/ml) in the culture medium, the cells form a polarized monolayer of regular columnar cells with occluding junctions delimiting two distinct membrane domains (apical and basolateral) and an apical brush-border expressing alkaline phosphatase and sucrase-isomaltase. The process of differentiation is fully reversible when the drug is removed from the culture medium. We also show that suramin inhibits both glucose consumption and lactate production so that the glycolytic activity of the treated cells is lowered by 42%. This observation would shed some light on the complex mechanisms involved during the induction of HT29 cell differentiation when glucose is removed from the culture medium.

Down regulation of hypertrophied follicular cell volume in thyroid hyperplastic gland.

In the present study, changes in thyroid follicular cell volume and its regulation have been investigated during the early involution of a hyperplastic goitre. Male Wistar rats were administered an iodine deficient diet for 6 months with propylthiouracil (PTU, 0.15%) during the last two months. At the end of iodine deficiency (day 0), some rats were killed and the others received a normal iodine diet. These rats were killed after different periods of iodine refeeding. Thyroid follicular cell volume was very high in hyperplastic gland whereas thyroid protein concentration was low. Thyroid follicular cell volume quickly decreased when rats were normally iodine refed, whereas thyroid protein concentration increased. Electron microscopal observations showed that thyroid follicular cells retained their endocrine aspect in hyperplastic state and throughout the iodine refeeding period. Using concomitant stereological and biochemical techniques, it is shown that the amount of cellular iodide and an unknown iodinated compound strongly increased during the early iodine refeeding. Plasma TSH was high on day 0 and remained at this level until day 8 whereas plasma T3 and T4 were low on day 0 and remained at this low level until day 4. The present data show that the involution of thyroid follicular cell volume is induced by iodide and mediated by an iodinated compound at least in the initial phase, and is independent of plasma TSH, T3, T4, so indicating the involvement of a thyroid autoregulatory mechanism. These changes in cell volume may be of importance in ion transport, i.e. in the metabolism of thyroid follicular cell during the early involution of the hyperplastic goitre.