Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro

An organotypic, tridimensional cell culture, also called a raft system, was used to study the influence of fibroblasts on epithelial carcinogenesis in a cell line derived from laryngeal squamous cell carcinoma and harboring a mutated p53. Differences between the effects of normal fibroblasts and those of tumor-derived fibroblasts were compared by means of fibroblasts taken from the normal skin and from the tumor of a cancer patient and cultivated with epithelial carcinoma cells in an organotypic culture. To study cell contact-mediated changes, the fibroblasts were either simply embedded in collagen matrix or additionally brought into direct contact with epithelial cells. Control epithelial cells were cultivated without any fibroblasts in an organotypic model. A protein panel [p53, p21, PCNA, bcl-2, Ki67, total cytokeratin (CK), CK 8, CK 10, CK 17, CK 18, CK 19, vimentin] involved in cell cycling and epithelial differentiation was assessed immunocytochemically in all organotypic cultures with fibroblasts, in tumor cells cultivated as a monolayer, and in the original tumor sample. The most dysplastic phenotype was obtained when tumor-derived fibroblasts were used in direct contact with epithelial cells, whereas the most benign phenotype was seen when skin fibroblasts had no contact with them. The intensive staining seen for p53 can be explained by p53 mutations also reflecting the weak expression of p21 and abundant expression of PCNA. The intensive Ki67 staining seen in all sections paralleled that of PCNA and marked active cellular proliferation. The CK staining pattern seen in cultured epithelia toward embryonic CKs, CK 8 and CK 18, suggested a simple epithelial phenotype. CK 19 was found only in the epithelium where no direct contacts had occurred. Vimentin expression increased when the raft epithelium was shifting toward a more benign phenotype. The results stress the importance of the origin of fibroblasts as well as the role of direct cellular contacts in modifying the epithelial phenotype even when the epithelial cells are malignant.     

Identification of epithelial cells and fibroblasts in hypophysis intermediate lobe cultures by scanning electron microscopy

Cell cultures of the rat pituitary intermediate lobe grown for eight days were studied in the scanning electron microscope. The epithelial cells and fibroblasts could be differentiated by the characteristic structures of the cell surface and the cell association features, The epithelial cells were characterized by blebs and rugae, microvilli, and occasionally by some cilia. The surface of the fibroblasts was smooth or bore microvilli. Scanning electron microscopy may provide special information for the characterization of endocrine cell cultures.     

The mitogenic effect of KGF and the expression of its cell surface receptor on cultured normal and malignant human oral keratinocytes and on contiguous fibroblasts

This study examined the mitogenic response to keratinocyte growth factor (KGF) of normal and tumour-derived human oral keratinocytes in which the degree of cellular differentiation was known and in contiguous fibroblast cultures derived from the malignant epithelial cultures. Keratinocytes, but not fibroblasts, were stimulated by KGF, thereby demonstrating epithelial target cell specificity of the ligand. KGF-induced stimulation of the tumour-derived keratinocytes cultured in the absence of the 3T3 fibroblast support broadly correlated with the degree of cellular differentiation; well-differentiated keratinocytes were stimulated more by KGF than their less differentiated counterparts. Malignant oral keratinocytes expressed KGF cell surface receptors (KD 451-709 pM; receptors/cell 2306-13645), but KGF receptor mRNA did not correlate with either KGF-induced mitogenesis or the degree of epithelial cell differentiation. When the tumour-derived keratinocytes were cultured in the presence of 3T3 fibroblasts, the mitogenic response to KGF was comparable to normal epithelial cells. The results suggest that KGF-mediated growth stimulation may not be significant in providing a selective advantage for the growth of malignant keratinocytes.     

Toxicity of formaldehyde to human oral fibroblasts and epithelial cells: influences of culture conditions and role of thiol status

The toxicity of formaldehyde, a monomer released from certain polymeric dental materials, was studied in cultured human oral fibroblasts and epithelial cells. The influences of growth conditions were evaluated for both cell types, as well as the role of the internal and external thiol states. A one-hour exposure to formaldehyde decreased the colony-forming efficiency (CFE) of both cell types in a concentration-dependent manner, although the toxicity varied up to 100-fold with the conditions. Clearly, the presence of serum and the thiol cysteine counteracted the toxicity in fibroblasts. Similarly, pituitary extract and cysteine, or a mixture of amino acids and ethanolamines, counteracted the formaldehyde toxicity in serum-free cultures of epithelial cells. In contrast, a growth-promoting surface matrix of fibronectin and collagen did not influence the formaldehyde toxicity, as shown by both the CFE assay and a dye reduction assay. Further, a short-term change to the various growth media per se with or without the supplements serum or cysteine did not significantly alter the CFE. Analysis of the thiol state demonstrated significant differences between epithelial cells and fibroblasts, i.e., comparatively lower cellular levels of the free low-molecular-weight thiols glutathione and cysteine in fibroblasts. This result correlated to significantly higher formaldehyde toxicity in the fibroblasts than in the epithelial cells. Taken together, the results indicated the cytoprotective function of both intracellular and extracellular thiols toward formaldehyde, as well as the usefulness of thiol-free and chemically defined conditions for toxicity assessments in oral epithelial cells and fibroblasts. We conclude that the combined use of a controlled external milieu and the presumed target cell type may be advantageous in evaluations of oral toxicity mechanisms or the toxic potency of dental materials, particularly those which, like formaldehyde, may react with thiols or amines.     

Colon carcinoma cells blocked in polarization exhibit increased expression of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is an oncofetal protein whose regulation is poorly understood, although CEA is commonly expressed on many carcinoma cell types and enhances experimental metastases. The abundance of membrane-associated CEA was increased 3-fold when HD6 colon carcinoma cells were prevented from polarizing by culture for 3 days in low calcium medium. Polarization is an early event in HD6 cell differentiation, with the polarized cells forming a tight, laterally adherent monolayer by culture in normal calcium medium. Lateral adherence can occur because 3 days of culture in normal calcium medium increases expression of calcium-dependent intercellular adhesion proteins: a 35-fold increase in membrane abundance of LCAM and a 16-fold increase in membrane abundance of the desmosomal protein desmoglein I. Polarized HD6 cells exhibit low levels of CEA only at their apical luminal surface. Rounded, unpolarized HD6 cells do not exhibit increases in either LCAM or desmoglein I membrane expression, but express increased levels of CEA molecules throughout their cell surface, where they act as intercellular adhesion molecules, allowing unpolarized cells to form random cell to cell contacts. Cells cultured in low calcium medium form calcium-independent cell aggregates whose formation can be blocked by Fab' fragments of anti-CEA monoclonal antibody col-1. The familiar pattern of random, multilayered associations of tumor cells both in vitro and in xenographs in vivo may be due to intercellular adhesion mediated by CEA which is up-regulated and expressed throughout the cell surface of unpolarized tumor cells.     

Harvest and storage of adult human retinal pigment epithelial sheets

PURPOSE: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells. METHODS: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination. RESULTS: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro. CONCLUSION: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.     

Cytoskeletal reorganization by soluble Wnt-3a protein signalling

BACKGROUND: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles. RESULTS: We prepared mouse L cells transfected with Wnt-3a cDNA, and found that their beta-catenin protein level was up-regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the beta-catenin level of the latter was also increased. Approximately 50% of the Wnt-3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt-3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt-3a had any effect on cellular behaviour in vitro. When the CM containing Wnt-3a (W3a-CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin-microfilamental system was re-organized by the W3a-CM treatment. It induced a directional alignment of actin stress fibres and other actin-associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re-distributed to the leading edges of the cells, co-localizing with F-actin, in the presence of Wnt-3a. CONCLUSION: Our findings suggest that Wnt-3a protein, in the soluble form, can act to re-organize cytoskeletal structures.     

Case report: right-sided periadrenal metastasis supplied by the hepatic artery. Clue to the genesis of pedunculated hepatocellular carcinoma

To demonstrate that cells both perceive and respond to external force, a strain/relaxation regimen was applied to normal human fetal and aged dermal fibroblasts cultured as monolayers on flexible membranes. The precisely controlled protocol of stretch (20% elongation of the culture membrane) at 6.67 cycles/min caused a progressive change in the monolayers, such that the original randomly distributed pattern of cells became a symmetric, radial distribution as the cell bodies aligned parallel to the applied force. High cell density interfered with the success of re-alignment in the fetal cell cultures observed, which may reflect a preference in this cell strain for cell-cell over cell-matrix contacts. The chronologically aged cells observed did not demonstrate this feature, aligning efficiently at all seeding densities examined. The role of microfilaments in force perception and transmission was investigated through the addition of cytochalasin D in graded doses. Both intercellular interactions and cytoskeletal integrity mediate the morphological response to mechanical strain.     

Effects of titanium on transcriptional and post-transcriptional regulation of fibronectin in human fibroblasts

The effects of commercially pure titanium (Ti) on the regulation of fibronectin gene expression and synthesis were investigated in early-passage human gingival fibroblasts. The fibroblasts were cultured on 50 nm Ti-coated silicon wafers treated with radio-frequency glow discharge prior to use and on Falcon tissue culture plastic (TCP) dishes as a control. Northern hybridization analysis revealed that fibroblasts cultured on Ti reduced the fibronectin mRNA level by 58% at 16 h, but increased it by 2.6-fold at 90 h, although the cell numbers and house-keeping gene GAPD mRNA levels on these two surfaces were essentially the same. The amount of total RNA was slightly less on the Ti surface. While the total [35S]methionine incorporation was essentially unaltered, the amount of [35S]methionine-labeled fibronectin was significantly increased in cells cultured on a Ti surface in early cultures but decreased in the late cultures. The apparent discrepancy between the increased fibronectin mRNA levels and decreased translation could be explained by a 30% reduction in fibronectin mRNA half life in cells cultured on Ti. The distribution of fibronectin between the medium and the cell layer also was altered on Ti surfaces, with a approximately 100-fold increase of fibronectin assembled in extracellular matrix at 16 h, but a 36% reduction at 90 h. In contrast, the amount of fibronectin recovered in the medium was essentially unchanged. The total amount of protein assembled into the extracellular matrix by cells on Ti increased 2.1-fold at 16 h but decreased by 19% in 90-h cultures. These significant changes in fibronectin gene activity and gene product distribution by cells cultured on Ti surfaces demonstrate that the surface chemistry of biomaterials can selectively regulate the cellular behavior at the molecular level and, conversely, that molecular biological techniques provide sensitive indicators of the molecular biocompatibility of implant materials.     

Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro

The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10(5) per cm2 once cultures reached steady state, (2) most cells entered the G0/G1 phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.     

Partially purified soy hydrolysates retard proliferation and inhibit bacterial translocation in cultured C2BBe cells

Hydrolyzed soybean isolates SP-A and SP-B (Abbott Laboratories, OH), developed for use in enteral nutritional products, were tested in cultures of C2BBe cells, a colonic adenocarcinoma cell line with enterocytic differentiation, to evaluate effects on cell growth, maturation and ability to resist infection by enteric bacteria. SP-A delayed formation of confluent monolayers by 10 d compared with cells cultured without SP-A. SP-A also caused a retardation in the development of intercellular tight junctions as measured by transmonolayer electrical resistance (TER). SP-B had no effect on cell proliferation or TER of intestinal cell cultures. SP-A and SP-B enhanced the development of the brush border enzymes alkaline phosphatase and isomaltase over a 28 d period. By these criteria, SP-A and SP-B appear to affect intestinal epithelial cell development in culture. When C2BBe monolayers were exposed to the enteric bacteria, Salmonella typhimurium or Salmonella typhi, an inhibition of the passage of S. typhi was seen in cultures with SP-A and SP-B. No effect on the passage of S. typhimurium was seen with either soy isolate. Partially purified soy isolates therefore impart resistance to selected enteroinvasive bacteria. Addition of soy hydrolysates to the media of cultured intestinal cells may serve as a rapid and economical screening mechanism for preclinical trials that would test the therapeutic benefits of soybean isolates.     

Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)-80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.     

Autocrine regulation of protein secretion in mouse mammary epithelial cells

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.     

Evidence for an impaired ability to determine semantic relations in Alzheimer''s disease patients

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.     

Failure of complex supplementation of minimal cultures to elicit a shift-up response in Pseudomonas putida

Primary cell cultures are established from 8-day quail embryo livers. During the first three days the culture is made up of areas of epithelial-like cells and scattered fibroblasts. The cytoplasm of the epithelial cells shows a high glycogen content as detected by the PAS reaction controlled with salivary amylase digestion. During the following days an important increase in the number of fibroblastic cells is observed. After 6-7 days of cultivation, the epithelial cells have disappeared and the culture is entirely fibroblastic. PAS technique does not show any trace of glycogen in these cultures which have been prolonged up to 45 days. Six-to 45-day primary cultures entirely made up of fibroblasts were associated with hepatic or pulmonary mesenchyme in organotypic culture for 3-4 days. In some cases the explant was first cultivated in vitro for 2 days and then grafted into a 5-day-old chick embryo on the chorioallantoic membrane for 6 days. In the secondary cultures hepatocytes showing an epithelial arrangement and a high glycogen content were observed. It appears from this observation that some of the primary culture fibroblasts are in fact dedifferentiated parenchymal cells. Such a dedifferentiation is a reversible phenomenon since the cells retain the ability to express their initial determination if they are placed in convenient environmental conditions. The role of the specific tissular arrangement in the stability of the differentiated state is discussed.     

Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis. In the early stages of fetal rat development, the liver is mainly a hematopoietic organ and hepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarce and are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin is found in some cells. Intercellular contacts between epithelial and mesenchymal cells are more numerous than between epithelial cells. Later in development, hepatocytes are arranged in a "muralium duplex" architecture (two-cell-thick sheets). Contacts between hepatocytes become more numerous and bile canaliculi become well developed. The density of cytokeratin filaments increases and appears to be very high near the bile canaliculi. In adult liver, hepatocytes are arranged in a "muralium simplex" architecture. Cytokeratin filaments show a symmetrical distribution in relation to the nuclear region. The highest density of filaments is found near the cytoplasmic membrane. Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentiation were investigated in a pilot study using computerized image analysis. We found significant differences between the filament networks in fetal and adult hepatocytes.     

Effects of keratinocyte-secreted soluble factors on spreading, number of dendrites and cell-cell contacts of human epidermal melanocytes and dermal fibroblasts: a quantitative analysis

We previously demonstrated that spreading and clustering of in vitro cultured human keratinocytes are autocrine-induced phenomena, mediated by keratinocyte-secreted soluble factor. In this paper, the effects of this factor on spreading, number of dendrites and cell-cell contacts of the two cellular components of skin, melanocytes and fibroblasts have been studied 24 h after plating cells on uncoated plastic surfaces in MCDB153 serum-free medium or in the same medium conditioned by keratinocytes (KCM). Spreading of melanocytes present in the epidermal cell population remained constant at increasing cell density, while that of keratinocytes showed a statistically significant increase. Moreover, time-course experiments showed that the rate of spreading was faster for melanocytes. At increasing epidermal cell density, a statistically significant increase in number of dendrites and cell-cell contacts of melanocytes was observed. Similar results were obtained when melanocytes were plated both in coculture with keratinocytes (as epidermal cell cultures) or as a pure cell population in keratinocyte conditioned medium (KCM), suggesting that the observed phenomena are due to keratinocyte-secreted soluble factors and not to direct keratinocyte-melanocyte interactions. The addition of nerve growth factor (NGF) to fresh medium or addition of an inactivating anti-NGF monoclonal antibody (alpha D11) to KCM did not affect the number of dendrites or cell-cell contacts of melanocytes. Keratinocyte-secreted soluble factor(s) present in KCM also dramatically influenced morphology and cell-cell contacts of human dermal fibroblasts.     

The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

PURPOSE: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-beta-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. METHODS: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes in E. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. RESULTS: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. CONCLUSION: The results of the "high-density" assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the "low-density" assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.     

1,25-dihydroxyvitamin D3 stimulates the assembly of adherens junctions in keratinocytes: involvement of protein kinase C

Signaling via intercellular junctions plays an important role in the regulation of growth and differentiation of epithelial cells. Loss of cell-cell contacts has been implicated in carcinogenesis, tumor progression, and metastasis. Here, we investigated whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was able to stimulate the assembly of adherens junctions and/or desmosomes in cultured human keratinocytes. After 4-day incubation, 1,25-(OH)2D3 caused assembly of adherens junctions, but not desmosomes. The adherens junctions were identified upon known ultrastructural criteria and evidence of the translocation of specific junctional proteins (E-cadherin, P-cadherin, alpha-catenin, and vinculin) to the cell-cell borders. The presence of alpha-catenin and vinculin at cell-cell borders indicated that the adherens junctions were functional. This was further supported by showing that anti E-cadherin antibody inhibited the 1,25-(OH)2D3-induced keratinocyte stratification. A relation between protein kinase C and adherens junction regulation was noticed. 1,25-(OH)2D3-dependent formation of junctions was blocked by the inhibitors of protein kinase C, bisindolylmaleimide and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and treatment of keratinocytes with 1,25-(OH)2D3 caused a rapid activation of protein kinase C and its translocation to the membranes. Formation of intercellular contacts may be an important mechanism of 1,25-(OH)2D3 action in hyperproliferative and neoplastic diseases.