Establishment of epithelial cell lines from adult mouse regenerating liver

A simple technique for developing epithelial cell lines from regenerating mouse liver has been described. Twenty-one epithelial cell lines have been developed and can be divided into four groups according to their morphology. All these near diploid cell lines have the capacity to metabolize diverse classes of chemical carcinogens (3-methylcholanthrene, 2-acetylaminofluorene, dimethylnitrosamine and aflatoxin B1) to cytotoxic metabolites. It is not yet possible to determine which ones of these cell lines originated from hepatocytes. Studies are in progress to further characterize and to use these cell lines as lethally irradiated feeder layers for cell-mediated activation of various classes of chemical carcinogens and mutagens with C3H/10T1/2 mouse embryo fibroblasts as indicator cells.     

Lectins as markers of rumen epithelial cell differentiation

Summary Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B₄ (Bandeiraea simplicifolia, isolectin B₄)) were explored for use as differentiation markers of rumen epithelial cellsin vivo andin vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA),N-acetylglucosamine (WGA) or forN-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B₄ and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.     

Nonrandom chromosome changes in nine rat liver epithelial cell lines

Using G-banding technique, the karyotypes of nine liver epithelial cell lines from Wistar rats cultivated under standard conditions were studied in early successive passages. Preferential involvement of chromosome n∘ 1 abnormalities including numerical and structural changes appeared at about the 14th passage. These apparently nonrandom chromosome changes have been compared with those found in neoplastic cell lines.     

Progression of spontaneous malignant transformation of epithelial rat liver cell lines

Cultured epithelial cell lines from normal rat livers were shown to undergo gradual transformation and malignancy which increased with time. Morphological changes appeared both before and after cells had attained a malignant state, as detected by agar tests. The progression of the degree of malignancy was determined by the morphological appearance of the cells, the increase in the number and size of cell colonies in soft agar, the expression of gamma glutamyl transferase (GT) and the shortening of the latency period necessary for tumor formation after transplantation to syngeneic rats of cells from sequential passages.     

Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines.

The progeny of single teratocarcinoma cells will give rise to several different cell types in vitro, and the latter were shown to be functionally differentiated by biochemical criteria. In all these studies, cloned lines of mouse teratocarcinoma cells were assayed during the course of differentiation for some biochemical products characteristic of the tissues formed. The carcinoembryonic protein, alpha-foetoprotein, was not synthesized by undifferentiated embryonal carcinoma (EC) cells, but was synthesized in increasing amounts during their differentiation to endoderm-type cells in suspension culture. alpha-Foetoprotein was shown to be a product of endoderm cells, but not all endoderm cells synthesized this protein. During the course of further differentiation when EC cells or aggregates were grown in tissue-culture dishes, other biochemical products appeared. In cultures containing predominantly nerve-type cells, there was a 30-fold increase in the specific activity of acetylcholinesterase, with concomitant appearance of the aldolase isoenzyme characteristic of mouse brain. In some cultures, a small amount of muscle-type cell formation was marked by the appearance of the MB isoenzyme of creatine phosphokinase. Generally, biochemical differentiation was immature.     

Amino acid transport in established adult rat liver epithelial cell lines

The capacities of Na⁺-dependent transport of -aminoisobutyrate, glutamine and glutamate in four established and three transformed rat liver epithelial cell lines were found to be considerably higher than those of isolated and cultured hepatocytes. At least for transport systems A and G^– this seemed to be due to elevated values of V _max , whereas the values for K _m were quite comparable to those of hepatocytes. In contrast to hepatocytes, however, no significant hormonal stimulation of amino acid uptake could be detected in the cell lines.Each normal cell line expressed a distinct pattern of transport capacities with respect to the three systems measured and this was not altered by chemical transformation of the lines. The individual patterns of the lines showed no similarity to presumptive patterns of subpopulations of liver parenchymal cells. In particular, there was no evidence for a direct relationship of one of the cell lines with a small subpopulation of parenchymal cells located adjacent to hepatic venules as revealed by additional measurements of glutamine synthetase, a marker enzyme for this particular subpopulation.It is concluded that established rat liver epithelial cell lines express features characteristic of normal hepatocytes with respect to amino acid transport, but have developed a distinct phenotype adapted to a rapid, hormone-independent growth in vitro. Alteration of their phenotype by transformation is not coupled with a further increase in amino acid transport capacity.Abbreviations AIB -aminoisobutyrate - LPC liver parenchymal cells - N-Methyl-AIB N-methyl--aminoisobutyrate - TAT tyrosine aminotransferase     

Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.     

Role of stromal tenascin-C in mouse prostatic development and epithelial cell differentiation

Deregulation of epithelial-stromal interactions is considered to play a critical role in the initiation and promotion of benign prostatic hyperplasia (BPH) and prostate carcinoma (PCa). Expression of tenascin-C (TN-C), an extracellular matrix (ECM) glycoprotein, is reportedly higher in BPH and PCa as compared with normal prostate. Remodeling of the ECM alters the homeostatic balance between epithelium and stroma, resulting in physiological changes in cellular functions. To investigate the role of TN-C in prostatic development and differentiation, we evaluated the morphological phenotype of TN-C knockout (KO) mouse prostate (ventral: VP, dorsolateral: DLP, and anterior: AP) and examined tissue recombinants composed of adult mouse DLP epithelium and fetal TN-C KO urogenital sinus mesenchyme (UGM). Histological analysis showed epithelial cell clusters protruding into the ductal lumens in TN-C KO AP and DLP. Interestingly, binucleated cells appeared in epithelium of TN-C KO DLP at 8 weeks. Simultaneously, androgen receptor (AR)-positive cells were decreased in TN-C KO epithelia. Similar to the TN-C KO phenotype, protruded epithelial clusters, binucleated cells, and AR-negative nuclei were induced in DLP epithelium by recombining with TN-C KO UGM. Our results suggest that stromal TN-C might be involved in maintaining epithelial cytodifferentiation, morphogenesis, and androgen receptor expression of normal prostate glands in adult mice.     

Ontogeny of pulmonary alveolar epithelial markers of differentiation

We studied differentiation of the pulmonary epithelium in the periphery of fetal rat lung in vivo and in vitro by comparing the ontogeny of cell-surface glycoconjugates with that of surfactant phospholipids. Apical surface binding of the lectin Maclura pomifera agglutinin (MPA) and expression of a 200-kDa MPA-binding glycoprotein (MPA-gp200) was evident at 20 days gestation in type 2 cells, but did not correlate with ultrastructural features of type 2 cell differentiation. Epithelial cells isolated from peripheral lung of 18-day gestation fetal rats displayed hormone-sensitive surfactant synthesis prior to the hormone-insensitive expression of MPA-gp200. Expression of MPA-gp200 occurred in association with the appearance of many new apical surface proteins suggesting a hormone-independent process of polar membrane differentiation. Thus membrane and secretory differentiation are discordant and can be dissociated. In vivo binding of Ricinus communis 1 agglutinin (RCA1), an apical marker of the differentiated alveolar type 1 cell occurred in undifferentiated peripheral lung epithelial cells as early as 18 days gestation, disappeared from differentiating type 2 cells and appeared in differentiated type 1 cells. Both undifferentiated fetal epithelial cells at 18 days gestation and fully differentiated type 1 cells express multiple glycoproteins with terminal beta-linked galactose residues which bind RCA1. Some of these RCA1-binding glycoproteins appear to be similar. These observations suggest that alveolar epithelial type 1 cells may derive directly from undifferentiated peripheral lung epithelial cells as well as from fully differentiated type 2 cells. In addition, terminal differentiation of fetal lung peripheral epithelium into type 1 and type 2 cells may involve repression as well as induction of differentiation-related genes.     

Selenium retention and inhibition of cell growth in mouse mammary epithelial cell lines in vitro

The steady state levels of growth inhibitory doses of inorganic selenium were examined in five different mammary epithelial cell lines: MOD, COMMA-D, COMMA-F, COMMA-T, and YN-4. The retention of selenium was monitored using a radioactive isotope,⁷⁵Se. Growth inhibition correlated with high levels of selenium in the cell. Generally, the retention of intracellular selenium was not dependent upon cell density, cell number, net growth rate, or tumorigenicity of the mammary cell lines. One cell line, COMMA-D, exhibited an unique response wherein the amount of selenium retained was low and the growth inhibitory effects of selenium were negligible when the cells were exposed to selenium at low density. However, at high cell densities, the COMMA-D cells responded like the other four cell lines. The growth inhibitory effect of selenium was reversible; upon removal of selenium from the medium, cells start synthesizing DNA within 24h. The retention of selenium was influenced by constituents in the growth medium. In particular, cysteine, but not methionine, purines, or pyrimidines altered selenium retention and counteracted the growth inhibitory effects of selenium. These results indicated that the mammary cell lines, particulary COMMA-D and MOD are good model systems to examine the uptake, retention, localization, and function of inorganic selenium under conditions where it acts as a growth inhibitory agent.     

Membrane differentiation markers of airway epithelial secretory cells

We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.     

Partial transformation of mouse fibroblastic and epithelial cell lines with the v-myc oncogene

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v-myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic.     

Regulation of vascular endothelial growth factor production in mouse thymic epithelial cell lines

Vascular endothelial growth factor (VEGF) in adults is synthesized in small amounts by thymic epithelial cells and is important for the maintenance of vascular homeostasis. However, its role dramatically increases during the process of thymic reconstitution after damage caused by radiation, chemo-, or hormonotherapy. The aim of the study was to evaluate the influence of different factors on VEGF production by mouse thymic epithelial cells in vitro. As a model, two cell lines were used: cortical cTEC1-2 and medullar mTEC3-10 cells. These cells were characterized by their ability to synthesize VEGF mRNA and VEGF protein, as well as by the presence of VEGF receptors. No VEGFR1 or VEGFR2 mRNA expression was observed in these cells, while NRP-1 mRNA was expressed at a low level. An ELISA test showed that cTEC1-2 cells produced VEGF about 30 times more than mTEC3-10 cells. These cell lines, when exposed to cytokines, hormonal factors, or thymocytes, responded differently. Keratinocyte growth factor (KGF) enhanced VEGF mRNA expression, as well as VEGF protein production, in medullar cells, but down-regulated VEGF mRNA synthesis in cortical cells. Dexamethasone suppressed the levels of VEGF mRNA expression and its protein production in cortical cells, while in medullar cells only VEGF production was reduced. Introduction of IL-7, IL-1β, or murine thymocytes increased, while administration of semaphorin-3A, SDF-1α, or ACTH decreased, VEGF production by cortical epithelial cells with no influence on medullar cells. We suggest that our data, obtained in vitro, can serve for further development of special strategies directed for regulation of VEGF synthesis in the thymic epithelial cells in vivo.     

Alkaline phosphatase isozymes as possible markers of differentiation in human testicular teratocarcinoma cell lines

The alkaline phosphatase (ALP) activity in eight independent cell lines derived from human testicular germ cell tumors was characterized. Seven out of eight of the lines had high ALP levels, and most of the activity in each case was of the liver/bone/kidney ALP type, as judged by thermostability, inhibition, and electrophoretic studies. Low levels of a heat stable, placental-like ALP were also present in these seven lines; only a small subpopulation of the cells of each line reacted strongly with an anti-placental ALP monoclonal antibody. The heat-stable, placental-like isozyme characteristic of these lines differed from the normal placental ALP in its inhibition profile. Thus it is possible that a subpopulation of the cells in these lines expresses a new embryonic ALP form.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers

Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor alpha-subunit and the muscular chloride channel CIC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.