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)     

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.     

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.     

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.     

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.     

Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.     

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.     

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.     

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.     

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.     

Characterization of a 36,000-dalton protein from the surface of Madin- Darby canine kidney cells involved in cell attachment and spreading

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.     

Assembly and functional analysis of tight junction in a colon adenocarcinoma cell line: effect of glucose depletion.

We describe morphologic and biochemical changes in the colonic epithelial HCT-116 cell line following depletion of glucose from the culture medium. Cultured cells under permissive differentiation conditions (inosine-supplemented glucose-free medium) exhibited, after confluence, an enterocytic differentiation, in contrast to cells grown under standard culture conditions, where they remain in an undifferentiated state. The differentiated phenotype was characterized by the presence of a monolayer of polarized cells displaying an apical tight junction, and by the presence of alkaline phosphatase, a well known brush border marker. We demonstrated that the formed tight junctions were functional using the following criteria: a) labeling of the junctions with antibodies recognizing the tight juntion proteins occludin and ZO-1, as observed by immunofluorescence and immunoblotting analysis; b) characteristic organization of the tight junction strands, as observed in freeze-fracture replicas; c) increase ofthe transepithelial resistance across the monolayer; d) not permeation of the ruthenium red stain across the tight junction, and e) presence of the hyperphosphorylated form of occludin.     

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.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Morphological and functional polarity of an epithelial thyroid cell line

The thyroid epithelial cell line FRT in monolayer culture appeared to be strongly polarized by morphological criteria. Cells were connected by tight junctions, exposed microvilli toward the culture medium and formed domes at confluency. FRT cells were infected with vesicular stomatitis virus (VSV) and Sindbis virus and the budding polarity was examined 8 and 16 h after infection, respectively. VSV budding occurred preferentially from the basolateral domain of plasma membrane, while Sindbis virus budding was mostly apical. The distribution of VSV and Sindbis virus glycoproteins, as determined by the immuno-gold technique, correlated well with the budding polarity. Polarized budding was not observed in isolated cells in suspension.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.     

Formation of the apical pole of epithelial (Madin-Darby canine kidney) cells: polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions

The time course of development of polarity of an apical (184-kD) and a basolateral (63-kD) plasma membrane protein of Madin-Darby canine kidney cells was followed using semiquantitative immunofluorescence on semithin (approximately 0.5-micron) frozen sections and monoclonal antibody probes. The 184-kD protein became highly polarized to the apical pole within the initial 24 h both in normal medium and in 1-5 microM Ca2+, which results in well-spread, dome-shaped cells, lacking tight junctions and other lateral membrane interactions. In contrast, the basolateral 63-kD membrane protein developed full polarity only after incubation in normal Ca2+ concentrations for greater than 72 h, a time much longer than that required for the formation of tight junctions (approximately 18 h) and failed to polarize in 1-5 microM Ca2+. These results demonstrate that intradomain restriction mechanisms independent of tight junctions, such as self-aggregation or specific interactions with the submembrane cytoskeleton, participate in the regionalization of at least some epithelial plasma membrane proteins. The full operation of these mechanisms depends on the presence of normal cell-cell interactions in the case of the basolateral 63-kD antigen but not in the case of the apical 184-kD protein.     

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.     

Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH- sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti- ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)