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.     

Short-term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29-D4 cells.

Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation.     

Impaired carcinoembryonic antigen release during the process of suramin-induced differentiation of the human colonic adenocarcinoma cell clone HT29-D4

The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29-D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.: Biology of the Cell 65:163-169, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: Journal of Biological Chemistry 264:10282-10286, 1989). We investigated the release of CEA in the culture medium of glucose-deprived HT29-D4 cells (HT29-D4-Gal) and studied its alteration in suramin-treated HT29-D4 cells (HT29-D4-S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29-D4-Glu cells was very low (5 to 8 ng/10(6) cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/10(6) cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29-D4-S cells remained very low and comparable with the one of HT29-D4-Glu cells. Moreover suramin, when added to CEA-producing HT29-D4-Gal cells, strongly inhibited its release. Radioiodination of cell surface proteins followed by immunoprecipitation using an anti-CEA monoclonal antibody showed the presence of a 180 kDa polypeptide, i.e., CEA, predominantly labeled in HT29-D4-Gal and -S cells. The total CEA cellular content was higher in HT29-D4-Glu and HT29-D4-S cells than in HT29-D4-Gal cells. When HT29-D4-Gal or -S cells were treated with the bacterial phosphatidylinositol phospholipase C (Pl-PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29-D4-Glu and -S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.     

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29-D4 cells

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.     

Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation

A clone HT29-18 has been isolated from the parent cell line HT-29, which derived from a human colon adenocarcinoma (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141). This clone is able to differentiate as the parent cell line does. Differentiation occurs when glucose is replaced by galactose in the culture medium (Pinto, M., M.D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196). We demonstrate here that the differentiated cloned population HT29-18/gal is heterogenous: although 90% of the cells show morphological characteristics of "absorptive cells", only 20-30% of them display sucrase-isomaltase in their apical microvillar membranes. About 10% of the entire cell population consists of cells containing mucous granules similar to intestinal goblet cells. We have isolated two subclones, HT29-18-C1 and HT29-18-N2, from the differentiated HT29-18/gal cells. HT29-18-C1 cells show morphological characteristics of polarized absorptive cells, when growing either in glucose- or in galactose-containing media, but the sucrase-isomaltase is not expressed in the cells grown in glucose-containing medium. The clone HT29-18-N2 is also polarized in both culture conditions and is similar to globlet cells in vivo. It grows as a monolayer, exhibits tight junctions, and contains numerous mucous granules whose exocytosis can be triggered by carbachol, a parasympathomimetic drug. We conclude that the clone HT29-18 first isolated was a multipotent cell population from which we isolated several subclones that differentiate either as absorptive (HT29-18-C1) or as mucous (HT29-18-N2) cells. In contrast to the parent HT-29 cell line, the subclones retain most of their differentiated properties in glucose-containing medium.     

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.     

Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.     

Aldosterone binding in the human colon carcinoma cell line HT29: correlation with cell differentiation

The purpose of the present study is to investigate aldosterone binding to human colon carcinoma HT29 cells. These cells grow as undifferentiated epithelial cells when cultured under standard conditions (Dulbecco's MEM; 10% FCS). Modification of the culture medium induced two types of differentiation: (1) enterocytic differentiation when glucose (25 mM) is replaced by galactose (5 mM) (2) mucus secreting cells in FCS free medium. Aldosterone specific binding was detected in the cytosolic fraction of enterocyte-differentiated cells. This binding corresponded to a single class of sites with affinity, specificity and anion-exchange chromatographic behaviour similar to those observed in the epithelial crypts of human colon. These putative aldosterone receptors were also detected in mucus secreting cells that are derived from the enterocyte-differentiated cells. Enterocytic differentiation appears thus to be a necessary step for the "induction" of aldosterone receptors in HT29 cells.     

In vitro differentiated HT 29-D4 clonal cell line generates leakproof and electrically active monolayers when cultured in porous-bottom culture dishes

HT 29-D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (Fantini et al. [1986], J. Cell Sci. 83, 235-249). Both undifferentiated and differentiated HT 29-D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29-D4 cells grown in glucose-free, galactose-containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial-like cells with a well organized apical brush border. Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29-D4 monolayers cultured in glucose-free, galactose-containing medium were electrically active, with a transepithelial resistance R = 172 +/- 46 omega.cm2, a potential difference PD = 0.35 +/- 0.05 mV, apical negative and a short-circuit current Isc = 2.0 +/- 0.4 microA.cm-2. Apical addition of amphotericin B induced a rapid and considerable increase in Isc and PD, which was abolished by basal ouabain. In contrast, HT 29-D4 cells grown in glucose-containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 +/- 0.9 omega.cm2). It is concluded from these studies that HT 29-D4 cells grown in glucose-free, galactose-containing medium acquire functional characteristics of epithelia, compared to HT 29-D4 cells grown in glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.     

The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line

The human colonic cancer cell line HT29, when grown in DMEM, forms a morphologically unpolarized cell culture in which the cells are covered with irregular microvilli and devoid of belt zonula occludens type tight junctions. However, by modifying the culture medium and growing the cells in RPMI, a different morphology was obtained. A large number of intracellular luminae appeared and at late confluency 90–95% of cells exhibited an epical brush border after four subsequent passages. Junctional complexes and a well developed zonula occludens were revealed under the apical brush border. Immuno-electron microscopical localization of specific markers, sucrase isomaltase (SI), secretory components (SC) and β2 microglobulin (β2M) showed that SI was limited to the apical surface, whereas 2M and SC were located at the basolateral surfaces. These results indicate that modification of culture conditions affects the ability of HT29 cells to express epithelial cell polarity.     

Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells.

The gene defective in cystic fibrosis has recently been shown to code for a membrane protein designated the "cystic fibrosis transmembrane conductance regulator" (CFTR) protein. While it has been shown that detectable levels of the mRNA for the normal CFTR protein are present in epithelial cells from different tissues, factors which regulate CFTR expression have not been identified. A clonal cell line originating from a human colon adenocarcinoma (HT29-18) differentiates to multiple epithelial cell types when deprived of glucose in the culture medium. In these studies, mRNA isolated from these cells was examined by hybridization to a 1.45-kilobase cDNA probe which encodes transmembrane portions of the CFTR protein between exons 13 and 19. Cellular differentiation of HT29-18 causes a 9-18-fold increase in CFTR mRNA abundance versus the mRNA for the structural proteins actin and tubulin. Cellular differentiation also causes a 5-fold increase in second messenger-regulated Cl- transport which is sensitive to a Cl- channel blocker (diphenylamine 2-carboxylate). Subclones of HT29-18 which are committed to differentiate to either a mucin-secreting (HT29-18-N2) or an "enterocyte-like" (HT29-18-C1) phenotype have also been examined. In both subclones, elevated levels of CFTR mRNA are observed when compared with undifferentiated HT29-18 cells. However, during cellular differentiation, the regulation of CFTR mRNA abundance and membrane enzyme expression by the subclones is different from HT29-18. The results show that elevated CFTR mRNA occurs in multiple differentiated intestinal epithelial cell types, despite a phenotype-specific regulation of membrane protein expression. This suggests that CFTR expression plays a role in the differentiated functions of multiple epithelial phenotypes and that both cellular differentiation and cellular phenotypes are factors which regulate CFTR expression.     

Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.     

Colonic cancer cell (HT29) adhesion to laminin is altered by differentiation: Adhesion may involve galactosyltransferase

The effects of cell differentiation on cell adhesion to laminin were studied using the human colon tumor cell line, HT29. HT29 cells were induced to differentiate either by glucose deprivation (HT29glc- vs HT29glc+) or by 2 mM butyrate (HT29glc-+B+). Adhesion was assayed after incubating cell suspensions in microtiter wells previously coated with laminin or other substrates. HT29glc+ cells adhered preferentially to laminin over BSA, fibronectin, and ovalbumin. The adhesion to laminin was greater than 50% of maximum within 15 min. HT29glc- cell adhesion to laminin was consistently lower than that for HT29glc+ or HT29glc+B+ cells. alpha-Lactalbumin (ALA), a modifier of galactosyltransferase (GT) substrate specificity, caused a significant reduction (greater than 50%) in HT29glc+ cell adhesion to laminin when ALA was added to the adhesion incubation mixture. Addition of glucose+ALA to the suspension restored adhesion to laminin. Ovalbumin, a GT substrate, increased adhesion of HT29glc+ and HT29glc- cells to laminin, but lactose, a GT product, did not. The data show that undifferentiated HT29 cells adhere preferentially to laminin over fibronectin and collagen IV and that differentiation of HT29 cells reduces adhesion to laminin. In addition, the data imply that cell adhesion to laminin may be mediated by factors that also modify galactosyltransferase activity.     

Growth in serum-free medium of human colonic adenocarcinoma cell lines on microcarriers: A two-step method allowing optimal cell spreading and growth

Summary Human colonic adenocarcinoma cells have been successfully grown on polystyrene microcarriers by modifying the culture conditions used in monolayer culture. The method can be divided into two culture phases: a) a phase of spreading, wherein cells were seeded in presence of serum-supplemented medium; b) a phase of active growth wherein spread cells on the beads were allowed to grow in a serum-free medium. Under these conditions, optimal spreading and growth of HT 29 and HRT 18 cells on the microcarriers were obtained. A differential propagation was observed between HT 29-D4 and HT 29-D9 cells (both clonal populations derived from HT 29 cells) on the microcarriers that is tentatively related to the discrepancy observed in the spreading efficiency of these clonal cells on serum-coated culture flasks. An index of spreading efficiency (IS index) has been defined to quantify the efficiency of spreading of each cell line on microcarriers. These data gave the opportunity to develop serum-free, scale-up methods to culture cells like HT 29 which release potentially useful products. This work was supported by CNRS (U.A. 202 and U.A. 1186), Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC), INSERM (CRE, n^o 847006), CNAMTS-INSERM (8386), MRT (GBM 85M0564) and l'Association pour la Recherche sur le Cancer (ARC 86-234).     

Carbohydrate metabolism in HT29 colon cancer cells cultured in a glucose free medium supplemented with inosine

1. Carbohydrate metabolism was studied in HT29 human colon cancer cells cultured in a glucose free medium supplemented with 2.8 mM inosine (HT29ino cells) in comparison with standard HT29 cells grown in the permanent presence of glucose (HT29Glc + cells) and with HT29Glc- cells which are adapted to grow permanently without glucose. 2. Inosine allows the standard cells to grow when glucose is lacking but surprisingly stops the growth of HT29Glc- cells. 3-mercaptopicolinate, an inhibitor of PEP-carboxykinase, does not hinder HT29ino cells to grow, which shows that gluconeogenesis from aspartate or pyruvate is not essential. It suggests that enough carbohydrate is supplied by the ribose moiety of inosine. 3. While standard HT29Glc + cells are highly glycolytic, it is not the case of HT29ino or HT29Glc- cells when glucose is given for few hours. When glucose is present for 24 hr or more, glycolytic rate increases in HT29ino cells and glycogen accumulates. 4. It is found that the pattern of enzymes activities related to carbohydrate metabolism in HT29ino cells is closer to that of HT29Glc + cells rather than to that of HT29Glc- cells. However, phosphofructokinase-1 activity, measured with saturating concentration of Fru-2,6-diP, is significantly lower in HT29ino cells. 5. Binding rate of hexokinase to mitochondria is similar in the three cell-lines. However, in HT29Glc- cells, bound hexokinase easily utilizes ATP generated by the mitochondria. By contrast, in HT29Glc+ and HT29ino cells, bound hexokinase is much more active with exogenous ATP, suggesting a functional defect in the mitochondria from these two latter 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+).     

Effect of glutamine deprivation and glutamate or ammonium chloride addition on growth rate, metabolism and differentiation of human colon cancer cell-line HT29

Effect of glutamine deprivation (GLN- medium) and of its replacement by 4mM ammonium chloride (GLN-/NH4+ medium) or by 4mM glutamate (GLN-/Gt+ medium) was studied on growth rate, morphology and metabolism of HT29 human colon cancer cells. Growth rates were modified as follows: at the first passage, growth of GLN- cells was strongly decreased (doubling time: 192 hr vs 32 hr in control cells grown in GLN+ medium); GLN-/NH4+ cells and GLN-/Gt+ cells were found to have doubling times of 72 and 70 hr, respectively. At the 8th passage, doubling times were decreased in all cases, being: 144 hr for GLN- cells, 60 hr for GLN-/NH4+ cells and 24 hr for GLN-/Gt+ cells, which indicates a capacity of adaptation of the cell-line to new culture conditions. GLN- cells and GLN-/NH4+ cells were found to exhibit an enterocytic type of differentiation (polarization of the cell layer with apical and cystic brush border and tight junctions); GLN-/Gt+ cells remained undifferentiated and comparable to control GLN+ cells. Glycogen level varied according to the phases of the culture, with a trend to lower level in glutamine deprived cells; glucose uptake and lactate production varied as a function of the medium composition and of the phases of the culture. At the 8th passage, all the glutamine deprived cells produced less lactate than control; GLN-/Gt+ cells were found to utilize less glucose than others.     

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.     

CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29-D4)

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29-D4) derived from a human colon adenocarcinoma cell line. HT29-D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti-CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29-D4 cells can be induced to differentiate into enterocyte-like cells by removing glucose from the culture medium. Under these conditions, HT29-D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29-D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29-D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.