Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.     

Secretory cell types and cell proliferation of human bronchial epithelial cells in an organ-culture system

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3β-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3β-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3β-HSD.     

Establishment and characterization of a human papillary thyroid carcinoma cell line with oxyphilic differentiation (ONCO-DG 1).

In the present study the establishment and characterization of a new oxyphilic papillary thyroid carcinoma cell line--ONCO-DG1- is given. With immunohistological, histochemical and flow cytometric methods, ONCO-DG 1 cells revealed features of epithelial differentiation. Furthermore the cells formed von Kossa-positive deposits resembling psammoma bodies in monolayer and spheroid culture until late passages. The tumor cell line is now in the 40th subculture. Because of the ability to form multicellular tumor spheroids (MCTS), this cell line is a good model for examining the interaction between thyroid tumor cells and confluent human endothelial cells on extracellular matrix in vitro. It is also suitable for xenotransplantation studies, because it is tumorigenic in NMRI nude mice in vivo.     

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.     

Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line

The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl- and K+ channels. We now describe the molecular nature of the K(+) channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o-. These cells, under isotonic conditions, present a K+ current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+ channels.     

Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95).

The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Establishment of a human fetal cardiac myocyte cell line

Summary Human cardiac myocytes undergo degeneration, cytolysis, and necrosis in a number of clinical disease conditions such as myocarditis, dilated cardiomyopathy, and during episodes of cardiac allograft rejection. The precise cellular, biochemical, and molecular mechanisms that lead to such abnormalities in myocytes have been difficult to investigate because at present it is not possible to obtain and maintain viable cell cultures of human adult cardiac myocytes in vitro. However, human fetal cardiac myocytes are relatively easy to maintain and culture in vitro, but their limited availability and growth, variability from one preparation to another, and varying degrees of contamination with endothelial and epithelial cell types have made it difficult to obtain reliable data on the effect of cardiotropic viruses and cardiotoxic drugs on such myocytes. These thoughts prompted us to attempt to derive a cell line of human cardiac origin. Highly enriched human fetal cardiac myocytes were transfected with the plasmids pSV2Neo and pRSVTAg and gave rise to a cell line (W1) which has been maintained in culture for 1 yr. Morphologic and phenotypic analyses of W1 cells by flow microfluorometry and immunoperoxidase techniques indicate that the W1 cell line shares many properties of human fetal cardiac myocytes, but appears not to react with specific antibodies known to react with markers unique to human endothelial, epithelial, skeletal muscle, and dendritic cells. These preliminary data suggest that the W1 cells may provide a unique source of an established cell line that shares many properties ascribed to human cardiac myocytes. This study was supported by grant 1RO1-25566-03 from the National Institutes of Health, Bethesda, MD, to A. Ahmed-Ansari and by a Grant-in-Aid from the American Heart Association, Georgia Affiliate, to Nicolas Neckelmann.     

A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.     

Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95)

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate–dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner. Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion. Treatment with chlorate, an inhibitor of A ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JARRL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate–containing proteoglycans participated in cell-cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Effects of PUVA on a human skin epithelial cell line

An established human epithelial cell line was exposed to photoactivated 8-methoxy psoralen (PUVA) during exponential growth. Effects of PUVA treatment on cell growth were measured by cell kinetic methods (counting of cell numbers, flow cytometric measurements (FCM) of DNA and calculations of labelling indices (LI)). Doses of 8-methoxy psoralen and UVA were comparable to those used in patients. The cell number in PUVA treated cultures remained almost constant, and very few mitoses were seen for 144 h. About 9 h after PUVA, both FCM and LI showed an increase in the fraction of cells in S-phase, reaching a maximum of 85-90% after 24 h. DNA synthesis took place at a low rate in these cells. FCM showed an increasing fraction of polyploid cells after PUVA treatment. The possibility that inhibition of cell proliferation is one of the main effects of PUVA, is discussed.     

Regulation of growth and differentiation by vitamin a in a cloned fetal lung epithelial cell line cultured on collagen gell in hormone-supplemented medium

Summary Proliferative and differentiative responses to various doses of vitamin A (VA) were studied in the predifferentiated cells of a fetal Syrian hamster pulmonary epithelial line (M3E3/C3), which were cultured on a collagen gel in a hormone-supplemented medium. These predifferentiated cells possessed well-developed endoplasmic reticulum (ER) and Golgi apparatus. At VA doses higher than 8 μg/ml, periodic acid Schiff and slightly alcian blue positive mucuslike granules were produced, which were also detectable electron microscopically. These mucuslike products were rich in sialic acid and resembled quite well those from primary cultures of tracheal epithelial cells of Syrian hamster sucklings when analyzed by column chromatography on various types of gel. At all VA doses studied (2.4, 8, 24 μg/ml), cells grew exponentially with an average population doubling time of around 74 h, whereas in the absence of VA they had a linear growth rate and a population doubling time of 158 h between Days 4 and 11. The uptake of [³H]glucosamine into the whole cell homogenates showed a peak at Day 8, irrespective of VA doses (0 to 24 μg/ml), and at the highest VA dose (24 μg/ml) it exceeded by twofold the control (0 μg/ml) level. At the same time, [¹⁴C]thymidine demonstrated a high peak of uptake on Day 8 at 8 and 24 μg/ml VA. There was virtually no difference between 0 and 2.4 μg/ml VA, with both doses yielding much lower peaks. Based on the results currently presented and previously reported, three successive stages were hypothesized for the mucous differentiation processes in M3E3/C3. The process from the first undifferentiated stage to the second predifferentiated stage with well-developed ER and Golgi apparatus requires both collagen gels and hormones. Differentiationn from the second stage to the third secretory stage with mucous granules is stimulated by VA. These observations indicate that the cell line M3E3/C3 could provide a new system for investigating the mechanisms of mucus differentiation by VA. This study was partly supported by a grant for Humanisierung des Arbeitslebens from Bundesministerium für Forschung und Technologie