Glycoconjugate and adhesion molecule changes during the cell cycle in a human embryonic epithelial cell line

The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell cycle phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G₁ than S and G₂ phases; in the S and particularly in G₂ phases the cytoplasm glycoconjugates are rearranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor β₁ integrin are well expressed in G₁, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G₂ than in G₁; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle.     

Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent α-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 ± 0.12 mM and a Vmax of 3.24 ± 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 ± 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference. © 1995 Wiley-Liss, Inc.     

Characterization of JOK-1, a human gastric epithelial cell line

Summary Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line “JOK-1” These cells express conventional, epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherins, β-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epitheliallike barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line “MDCK” also expresses serveral epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherin, β-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.     

Characterization of a white bass (Morone chrysops) embryonic cell line with epithelial features

The genus Morone is an important one for U.S. aquaculture, but there has been no available cell line from this genus. We report here a cell line (the WBE line) derived from white bass embryos that has been grown for more than 80 passages over 21 mo in Dulbecco modified Eagle medium supplemented with fetal bovine serum. The WBE line showed epithelial features with positive immunohistochemical staining for cytokeratin and intercellular junctions when observed by electron microscopy. The doubling time, transformation characteristics, response to cryopreservation, and karyotype were determined. The WBE line was also tested by polymerase chain reaction to verify the species of origin and to screen for mycoplasma infection. This epithelial cell line may prove useful for the study of host-pathogen interactions that occur at the epithelial surface in this commercially important fish species.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Growth hormone kinetics during adaptation to a hyperosmotic environment in rainbow trout

Summary To clarify the role of growth hormone (GH) in salmonids during seawater (SW) adaptation, we examined GH kinetics in chronically cannulated rainbow trout, weighing about 1 kg. When trout were transferred from fresh water (FW) to 75% SW, plasma chloride concentration was normalized within 1 week. Plasma GH concentration increased significantly 2 days after transfer and decreased to the initial level thereafter. Metabolic clearance rate (MCR) and secretion rate (SR) of GH were calculated from the plasma levels of GH measured by radioimmunoassay after intra-arterial injection of recombinant chum salmon GH. 4 days after transfer to 75% SW, both MCR and SR increased to levels five times higher than those in FW, and returned to the FW levels after 3–4 weeks. In rainbow trout GH seems to be involved in the development of hypoosmoregulatory mechanisms, especially during the early phase of adaptation.     

Calcium adaptation to sodium pump inhibition in a human megakaryocytic cell line

The unique characteristics of the platelet Na/Ca exchanger, i.e., its dependence on both transmembrane Na and K gradients, render it highly sensitive to Na pump inhibition. In this project, we observed that the human megakaryocytic cell line CHRF-288 expresses both the ₁- and ₃-isoforms of the Na-K-ATPase. Inhibition of the Na pump increased the RNA and protein expressions of sarco(endo)plasmic reticulum Ca-ATPase 2b, cytosolic Na and Ca, and the freely exchangeable Ca in the endoplasmic reticulum. These changes occurred in concert with diminished store-operated Ca entry and an increase in the maximal activity of the Na/Ca exchanger. Inhibition of the Na pump by ouabain was more effective in inducing these changes than diminishing medium K. Collectively, these observations point to an integrative effort to counteract the impact of Na pump inhibition by Ca sequestration into the endoplasmic reticulum, diminished Ca entry, and increased activity of the Na/Ca exchanger. The implications of these findings in platelet biology are discussed. platelets; thrombosis; potassium; stroke     

High-yield and high-degree purification of human alpha-fetoprotein produced by adaptation of the human hepatoma cell line Hep G2 in a serum-free medium

The human hepatoma cell line Hep G2 secretes both albumin and alpha-fetoprotein when grown in the presence of serum. The present report describes how adaptation to growth in serum-free medium results in a progressive switch in the expression of the two proteins; i.e., alpha-fetoprotein becomes the main protein secreted while albumin production is greatly reduced. The culture supernatant obtained, being very enriched in the protein, allows the development of a purification procedure by preparative electrophoresis. By this procedure it is possible to easily obtain large amounts of alpha-fetoprotein from a constant and unlimited source. The availability of these protein preparations should improve the reproducibility and the quality of standardization in clinical immunoassays for alpha-fetoprotein and should permit a more accurate study of the structure and biological functions of the protein.     

Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.     

Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.     

Comparison of an animal product free medium and normal growth supplement on the growth and barrier integrity of a human corneal epithelial cell line

With the development of defined media for general and specific use with cell cultures, and concern over the use of human cells and over potential prion infections associated with growth factor extracts such as bovine pituitary extract, an animal product-free medium has become available. The basic keratinocyte defined medium can be used with a choice of animal product-containing or animal product-free supplements. Human corneal epithelia cell lines were cultured in the media with these two types of supplement, and compared in terms of their growth rates, their capacity to form tight barriers, and calcium regulation of the location of a junction-associated protein, zonula occludins-1 (ZO-1). The growth rates were not different in the two media, as long as the recommended coating was applied to the culture flask for the animal product-free medium. The barrier function was equally effective for confluent cultures seeded at the same densities. A calcium concentration of 100 microM or above resulted in ZO-1 localisation at the cell membrane in either medium. Hence, cultures in the media are comparable, when the coating is employed. Further experiments are being conducted to establish the comparability of responses to chronic treatment with surfactants.     

Characterization and antigen-presenting function of a murine thyroid-derived epithelial cell line

We have developed and evaluated the antigen-presenting function of a murine thyroid-derived epithelial cell line M.5 in order to further investigate the possible role of the thyroid follicular epithelium in the inductive phase of autoimmune thyroiditis. M.5 cells did not express class II major histocompatibility complex encoded (Ia) antigens constitutively, but these could be readily induced with interferon-gamma. We found that Ia expressing M.5 cells were ineffective in stimulating T cell proliferation when tested in a 4-day primary mixed leukocyte reaction (MLR). However, significant T cell stimulation was obtained when phorbol myristate acetate (PMA) was added either to the M.5-T cell co-cultures, or for a brief period to the M.5 cells prior to adding the responder T cells. Cytofluorographic analysis of M.5 cells disclosed that PMA did not significantly alter the expression of Ia antigens. Additional experiments indicated that interleukin 1 (IL-1) was unlikely to represent the co-stimulatory factor generated by PMA. This was based on the observations that M.5 cells failed to secrete significant IL-1 either spontaneously, or in the presence of various stimuli, and that murine recombinant IL-1 failed to substitute for PMA in the activation of T cells. The nature of the co-stimulatory signal is as yet unknown. We conclude from these experiments that a pure population of thyroid-derived epithelial cells may be able to function, under the described circumstances, as antigen presenting cells.     

Proliferation of human embryonic corneal stromal cells in a serum-free medium

Substantial multiplication of stromal cells from human embryonic corneas has been obtained in a basal medium MCDB 104 supplemented with 25 ng EGF/ml, 10 micrograms insulin/ml, 20 micrograms transferrin/ml, 25 ng MSA/ml, 500 micrograms ovalbumin/ml, 50 micrograms LDL/ml, 50 micrograms HDL/ml and 10(-6) M hydrocortisone. Even though the growth rate appears to be similar to that in 10% serum, the cells cease proliferating at a lower density.     

Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).     

A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium

Summary A new, nontumorigenic human breast epithelial cell line, HMT-3522, has been established from fibrocystic breast tissue. Cells were explanted and propagated in chemically defined medium including insulin, transferrin, epidermal growth factor, hydrocortisone, estradiol, prolactin, and Na-selenite. The epithelial nature of the cell line was established by immunocytochemical detection of cytokeratins. Moreover, electronmicroscopy revealed monolayers of polarized cells connected by desmosomes and provided with apical microvilli. Milk fat globule membrene antigen, specific for the apical membrane domain of normal, luminal breast epithelial cells, was expressed only in confluent cultures where some cells overlaid others, indicating “stem cell”-like properties. After 25 to 30 passages, the cells are diploid with a few marker chromosomes and loss of chromosomes in the D-group. The cells are nontumorigenic in athymic mice; they lack estrogen receptors, and estradiol does not stimulate growth. The HMT-3522 cell line may represent a useful model for the study of brest cell differentiation and carcinogenesis in vitro. This work was supported by a grant from the Danish Cancer Society.