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.     

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.     

Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.     

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.     

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 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)     

Restricted localization of functional vasoactive intestinal peptide (VIP) receptors in in vitro differentiated human colonic adenocarcinoma cells (HT29-D4)

HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor number of differentiated cells was twice that of undifferentiated cells. Leakproof differentiated cell monolayers grown on permeable substratum produced cAMP in response to VIP only when the peptide was present in the lower chamber of the culture wells. Taking these data altogether, we conclude that the localization of functional VIP receptors is restricted to the basolateral domain in differentiated post-confluent HT29-D4 cells.     

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.     

Cell polarity regulates the release of secretory component, the epithelial receptor for polymeric immunoglobulins, from the surface of HT-29 colon carcinoma cells.

The HT-29 human colon carcinoma cell line differentiates in glucose-free medium to an enterocytic phenotype. We previously isolated a series of HT-29 subclones selected for high levels of expression of secretory component (SC), the epithelial receptor for polymeric immunoglobulins. To develop a model system for studying effects of cell polarity on SC expression and release from the cell surface, the HT-29.74 subclone was induced to differentiate in glucose-free medium. Expression of SC was induced by glucose deprivation in both the parental HT-29 cell line and, to an even greater extent, in the HT-29.74 subclone. Prolonged glucose deprivation of HT-29.74 cells resulted in morphological changes consistent with enterocytic differentiation. Metabolic radiolabeling of SC in differentiated HT-29.74 cells indicated that proteolytic cleavage of membrane-bound to free SC occurred both on the cell surface and intracellularly, possibly in a vacuolar apical compartment or intrapeithelial lumen. To study effects of cell polarity on SC release, differentiated HT-29.74 cells were depolarized by culturing in low calcium medium. Within 2 hours after transfer of the cells into low calcium medium, a burst of SC release was observed concomitant with cell depolarization. Subsequently, release of SC declined significantly and remained low as long as cells were maintained in a depolarized state. The extent of cell depolarization could be controlled by varying the extracellular calcium concentration or by substituting the divalent cation Sr++, which partially prevents depolarization, for Ca++. In either case, the magnitude of the initial burst and subsequent decline in release of SC was proportional to the extent of cell depolarization. We conclude that cell polarity plays an important role in controlling the release of SC in intestinal epithelial cells, most likely by regulating the distribution of membrane-bound SC and SC protease, which are on the basolateral and apical cell surfaces, respectively, in differentiated cells.     

Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells

Human colon adenocarcinoma cells (HT29-ATCC) and the clone HT29-5F7 were cultured under conditions that differentiate cells to a polarized intestinal phenotype. Differentiated cells showed the presence of junctional complexes and intercellular lumina bordered by microvilli. Intestinal brush border hydrolase activities (sucrase, aminopeptidase N, lactase and maltase) were detected mainly in differentiated HT29-ATCC cells compared with the differentiated clone, HT29-5F7. The presence of non-GM1 receptors of Escherichia coli heat-labile enterotoxin (LT-I) on both types of differentiated HT29 cells was indicated by the inability of cholera toxin B subunit to block LT-I binding to the cells. Binding of LT-I to cells, when GM1 was blocked by the cholera toxin B subunit, was characterized by an increased number of LT-I receptors with respect to undifferentiated control cells. Moreover, both types of differentiated cells accumulated higher amounts of cyclic AMP in response to LT-I than undifferentiated cells. Helix pomatia lectin inhibited the binding of LT-I to cells and the subsequent production of cyclic AMP. LT-I recognized blood group A-active glycosphingolipids as functional receptors in both HT29 cell lines and the active pro-sucrase form of the glycoprotein carrying A-blood group activity present in HT29-ATCC cells. These results strongly suggest that LT-I can elicit an enhanced functional response using blood group A-active glycoconjugates as additional receptors on polarized intestinal epithelial cells.     

Differential mucin expression in colon carcinoma HT-29 clones with variable resistance to 5-fluorouracil and methotrexate

A current challenge is to define the biological characteristics of colon tumor cells resistant to chemotherapy. Distinct sub-populations of mucus-secreting cells were previously obtained from the colon cancer cell line HT-29 after long-term treatment with the anti-cancer drugs, 5-fluorouracil (5-FU) and methotrexate (MTX). Since mucins are increasingly implicated as playing a role in carcinogenesis, we studied the pattern of mucin expression in two HT-29 clones of mucus-secreting and two clones of enterocyte-like phenotype which differ in their capacity to resist to 5-FU and/or MTX. The expression of both transmembrane (MUC1, MUC3, MUC4) and secreted gel-forming (MUC2, MUC5AC, MUC5B, MUC6) mucins in clones was studied by northern and/or western blotting. The four HT-29 clones showed three cellular phenotypes: (1) The mucus-secreting clone HT29-5F12 consists of unpolarized cells with mucus secretions that have anti-colonic mucin immunoreactivity, and mainly expresses MUC2 and is resistant to 5-FU and sensitive to MTX; (2) The mucus-secreting clone HT29-5M21 forms a monolayer of polarized cells with strong anti-gastric mucin immunoreactivity and mainly expresses MUC5AC and MUC5B and is resistant to MTX and sensitive to 5-FU; (3) The two enterocyte-like clones, HT29-5F7 and HT29-5M12 are resistant to both MTX and 5-FU and express mainly MUC1 and MUC5B, respectively. These clones which originate from a same colorectal tumour and display different patterns of mucin expression as well as differing resistance to MTX and 5-FU will make useful in vitro models for studying the potential role of mucins or other biological markers in drug resistance pathways.     

The posttranslational processing of sucrase-isomaltase in HT-29 cells is a function of their state of enterocytic differentiation

The biosynthesis of sucrase-isomaltase was compared in enterocyte-like differentiated (i.e., grown in the absence of glucose) and undifferentiated (i.e., grown in the presence of glucose) HT-29 cells. Unlike differentiated cells, in which the enzyme is easily detectable and active, undifferentiated cells display almost no enzyme activity and the protein cannot be detected by means of cell surface immunofluorescence or immunodetection in membrane-enriched fractions or cell homogenates. Pulse experiments with L-[35S]-methionine show that the enzyme is, however, synthesized in these undifferentiated cells. As compared with the corresponding molecular forms in differentiated cells, the high-mannose form of the enzyme in undifferentiated cells is similarly synthesized and has the same apparent Mr. However, its complex form is less labeled and has a lower apparent Mr. Pulse-chase experiments with L-[35S]methionine show that, although the enzyme is synthesized to the same extent in both situations, the high-mannose and complex forms are rapidly degraded in undifferentiated cells, with an apparent half-life of 6 h, in contrast to differentiated cells in which the enzyme is stable for at least 48 h. A comparison of the processing of the enzyme in both situations shows that the conversion of the high-mannose to the complex form is markedly decreased in undifferentiated cells. These results indicate that the absence of sucrase-isomaltase expression in undifferentiated cells is not the consequence of an absence of biosynthesis but rather the result of both an impaired glycosylation and a rapid degradation of the enzyme.     

Casein phosphopeptides modulate proliferation and apoptosis in HT-29 cell line through their interaction with voltage-operated L-type calcium channels

At the intestinal level, proliferation and apoptosis are modulated by the extracellular calcium concentration; thus, dietary calcium may exert a chemoprotective role on normal differentiated intestinal cells, while it may behave as a carcinogenesis promoter in transformed cells. Calcium in milk is associated with casein and casein phosphopeptides (CPPs), hence is preserved from precipitation. CPPs were demonstrated to induce uptake of extracellular calcium ions by in vitro intestinal tumor HT-29 cells but only upon differentiation. Here, the hypothesis that CPPs could differently affect proliferation and apoptosis in undifferentiated and differentiated HT-29 cells through their binding with calcium ions was investigated. Results showed that CPPs protect differentiated intestinal cells from calcium overload toxicity and prevent their apoptosis favoring proliferation while inducing apoptosis in undifferentiated tumor cells. The CPP effect on undifferentiated HT-29 cells, similar to that exerted by ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA), is presumably due to the ability in binding the extracellular calcium. The effect on differentiated HT-29 cells is coupled to the interaction of CPPs with the voltage-operated L-type calcium channels, known to activate calcium entry into the cells under depolarization and to exert a mitogenic effect: the use of an agonist potentiates the cell response to CPPs, while the antagonists abolish the response to CPPs (36% of examined cells) or reduce both the percentage of responsive cells and the increase of intracellular calcium concentration. Taken together, these results confirm the potentialities of CPPs as nutraceuticals/functional food and also as modulators of cellular processes connected to the expression of a cancer phenotype.     

In vitro isolation and establishment of new embryonal cell lines from rat nephroblastoma

Summary Two mixed cell lines designated REN-1 and REN-2 were established successfully in continuous in vitro culture from subcutaneously propagated transplant tissue derived from a nephroblastoma which had occurred spontaneously in an Nb hooded rat. In monolayer culture the lines consisted of clumps, islands, and cords of densely crowded, small basophilic cells of epithelioid character, together with mesenchymelike cells occupying the intervening spaces. The proportion of epithelioid cells to mesenchyme could be enhanced by high seeding densities and the intermittent application ofcishydroxyproline to the medium. A third cell form with the appearance of more mature epithelium was observed as a later development in one of the monolayer cultures (REN-1). This larger epithelial cell and a homogeneous mesenchymal population were isolated as cloned cell lines from REN-1 (REN-1-C/2 and REN-1-C/1, respectively), but attempts to clone the dominant basophilic epithelioid cell were not successful. Light and electron microscopy indicated the small, basophilic epithelioid cells to be morphologically consistent with the undifferentiated embryonal blast cells of the parent tumor. They were a distinctive population unlike known malignant cell lines representative of chemically transformed rat kidney mesenchyme and epithelium. The mesenchymelike cells present in the uncloned cell lines, and cloned in REN-1-C/1, were fibroblasts by ultrastructural criteria and therefore distinct from the epithelioid moiety. Ultrastructurally the larger epithelium cloned in REN-1-C/2 displayed the features of differentiated renal epithelium. Subcutaneous transplantation in syngeneic and allogeneic recipients showed the uncloned parent cell lines containing the basophilic epithelioid population to be highly tumorigenic, producing rapidly growing tumors that were unequivocal nephoblastomas. The mesenchymal clone, REN-1-C/1, was also tumorigenic but, consistent with its fibroblastic nature, produced only fibrosarcomas on transplantation. The mature epithelial clone, REN-1-C/2, transplanted as an anaplastic carcinoma with limited tubule formation. Because of their distinctive morphology and growth behavior, and their ability to proliferate into nephroblastomas on transplantation, the dominant population of basophilic epithelioid cells in the parent uncloned cell lines is considered to represent neoplastic kidney cells of undifferentiated embryonal type. The possible host origin of the mesenchymal population from the supporting stroma of the original transplantation tumor is suggested in discussion. This investigation was supported by research grants CA-24216 and CA-12227 awarded by the National Cancer Institute, Department of Health and Human Services, and in part by the National Cancer Institute of Canada.     

Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose

In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT-29 cells was selected for its capacity to grow in the total absence of sugar. These cells (Glc-cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase-isomaltase. The differentiation of Glc- cells is reversible: the addition of glucose to postconfluent cultures of Glc- cells results in an inhibiting effect on the expression of sucrase-isomaltase; switching growing cultures of Glc- cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage-related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc- medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc- cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT-29 cells.