E-cadherin engagement stimulates proliferation via Rac1

E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit epithelial cell proliferation in different settings.     

The neuropeptide calcitonin gene-related peptide differently modulates proliferation and differentiation of smooth muscle cells in culture depending on the cell type

Calcitonin gene-related peptide (CGRP) is a neuropeptide present around vasculature very early during development, when smooth muscle cells (SMC) are still proliferating and not yet totally differentiated. We investigated the effects of CGRP on proliferation and differentiation of SMC in culture; 10(-7) M CGRP added in the medium of cultured smooth muscle cells every 2 days did not significantly changed cells growth rate in 1% FCS. At the opposite, this treatment modulated proliferation of cells grown in 10% FCS medium. Two distinct populations of SMC with different growth rates were obtained from our primary cultures. SMC which proliferated slowly in the presence of 10% fetal calf serum (FCS) had growth rates positively influenced by CGRP. The quantity of alpha-smooth actin expressed by these cells was not influenced by the peptide. On the contrary, SMC which proliferated more rapidly in 10% FCS medium had growth rate inhibited by CGRP. In these cells, CGRP significantly reduced the amount of expressed alpha-smooth actin, an index of SMC differentiation. In both cases, the peptide significantly increased the level of mRNA for all the actin genes. In the light of this dual role of CGRP, it can be presumed that this peptide controls smooth muscle cells proliferation and differentiation in vivo and could thus regulate the homeostasis of the vessel wall.     

Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications

Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.     

Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A

Mesenchyme cells derived from limb buds of day 10 mouse embryos were plated out at confluent and sub-confluent cell densities. Cells in confluent cultures multiplied and differentiated into chondrocytes. The addition of vitamin A to the culture medium inhibited both cell proliferation and chondrogenesis. However, cytosine arabinoside, which also inhibited growth, did not block chondrogenesis. This indicates that the inhibition of growth in the vitamin A-treated cultures did not necessarily contribute to the inhibition of chondrogenesis. Cells in sub-confluent cultures multiplied but did not differentiate into chondrocytes. In contrast to confluent cultures, vitamin A did not inhibit growth in sub-confluent cultures. This observation suggests that vitamin A may inhibit growth by causing contact inhibition.     

Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Immobilized glycolipids from human diploid fibroblasts inhibit DNA synthesis of cultured human fibroblasts

Several previous studies have shown that glycolipids isolated from plasma membranes of cultured cells and added to cells in culture inhibit the growth rate in a concentration-dependent fashion. In order to investigate the possible involvement of glycolipids in the growth regulation of normal cells by cell-cell contacts, we tested the effect of immobilized glycolipids, isolated from human fibroblasts, on the DNA synthesis of freshly seeded fibroblasts. Gangliosides inhibited DNA synthesis to a great extent, whereas neutral glycolipids had only a minor effect. The degree of inhibition of DNA synthesis by immobilized gangliosides depended both on the cell density of the cultures from which the gangliosides were isolated and on the pretreatment of the immobilized gangliosides: Preincubation with DMEM without FCS of immobilized gangliosides, isolated from confluent cultures, resulted in a 75% inhibition of growth rate of embryonal human lung fibroblasts (FH109) cultured on immobilized gangliosides. Under the same conditions, gangliosides from sparse cultures reduced the growth rate by about 30%. On the other hand, the degree of inhibition exerted by immobilized gangliosides isolated from confluent cultures was found to be greatly reduced by preincubation with DMEM with FCS, whereas the slight inhibition of growth rate, exerted by gangliosides from sparse cultures, was found to be reversed into a slight stimulation of growth rate after preincubation with complete medium. Concomitantly with the reduction of the inhibition of DNA synthesis, it was found that the complete medium, used for preincubation of the gangliosides, was no longer able to support DNA synthesis to the same extent as untreated complete medium. The data suggest that gangliosides bind growth-supporting factors of the serum, gangliosides isolated from sparse cultures being more potent in the binding of these molecules than gangliosides isolated from dense cultures.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Contact-dependent regulation of growth of diploid human fibroblasts is dependent upon the presence of terminal galactose residues on plasma membrane glycoproteins

The growth of diploid human fibroblasts has previously been shown to be regulated mainly by the extent of cell-cell contacts [R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361], these contacts being effective only when terminal, beta-glycosidically linked galactose residues were present on plasma membrane glycoproteins. These studies, in which a high cell density in sparse cell cultures has been mimicked by the addition of immobilized plasma membrane glycoproteins, have been further extended to investigate the role of terminal galactose residues directly in cell cultures. The studies presented herein show that (i) culturing human fibroblasts in the presence of beta-galactosidase resulted in an approximately twofold higher saturation density, as well as a twofold higher proliferation rate at high cell densities when compared to the rates found in control cultures. (ii) The presence of alpha-lactalbumin in the culture medium, which acts as a modifier of the activity of galactosyltransferase, had the same effect as beta-galactosidase. (iii) Addition of the lectin I from Bandeiraea simplicifolia (BS I), which is specific for terminal galactose residues, resulted in an increase in the proliferation rate of cell cultures at high cell densities, while the proliferation was not affected at low cell densities. These data show that the presence of terminal, beta-glycosidically linked galactose is vital for the efficient growth control of normal cells.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or alpha-difluoromethylornithine

We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and alpha-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 microM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 x 10(5)), medium (5 x 10(5)), or high (2 x 10(6)) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Chondrogenic differentiation of human mesenchymal stem cells within an alginate layer culture system

Human mesenchymal stem cells (hMSCs) derived from bone marrow have the capacity to differentiate along a number of connective tissue pathways and are an attractive source of chondrocyte precursor cells. When these cells are cultured in a three-dimensional format in the presence of transforming growth factor-beta, they undergo characteristic morphological changes concurrent with deposition of cartilaginous extracellular matrix (ECM). In this study, factors influencing hMSC chondrogenesis were investigated using an alginate layer culture system. Application of this system resulted in a more homogeneous and rapid synthesis of cartilaginous ECM than did micromass cultures and presented a more functional format than did alginate bead cultures. Differentiation was found to be dependent on initial cell seeding density and was interrelated to cellular proliferation. Maximal glycosaminoglycan (GAG) synthesis defined an optimal hMSC seeding density for chondrogenesis at 25 x 10(6) cells/ml. Inclusion of hyaluronan in the alginate layer at the initiation of cultures enhanced chondrogenic differentiation in a dose-dependent manner, with maximal effect seen at 100 microg/ml. Hyaluronan increased GAG synthesis at early time points, with greater effect seen at lower cell densities, signifying cell-cell contact involvement. This culture system offers additional opportunities for elucidating conditions influencing chondrogenesis and for modeling cartilage homeostasis or osteoarthritic changes.     

Technological problems in cultivation of plant cells at high density. Reprinted from Biotechnology and Bioengineering, Vol. XXII, No. 6, Pages 1203-1218 (1981)

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: effects of seeding density

Autologous cell transplantation is a promising approach for cartilage repair, but the expansion of chondrocytes in a monolayer, a common approach to amplifying the cell number, inevitably leads to cell de-differentiation. To explore whether porous alginate sponges could be utilized for chondrocyte expansion and investigate the effects of seeding densities, the porcine chondrocytes were seeded to porous alginate sponges at low (5 x 10(5) cells per 40 sponges), medium (5 x 10(6) cells per 40 sponges), or high (2 x 10(7) cells per 40 sponges) density. After 4-week perfusion culture, all three groups resulted in chondrocyte proliferation, maintenance of chondrocytic gene (collagen II, Sox 9 and aggrecan) expression, and formation of cell clusters resembling cartilaginous tissues. The higher the seeding density, the higher the final cell density and GAGs production and, accordingly, the larger the cell clusters. Strikingly, the cumulative expansion ratios achieved by the low-density group ( approximately 150-fold) significantly exceeded those achieved by the medium (approximately 21-fold) and high (approximately 4.7-fold) density groups, as well as those achieved using other scaffolds. In conclusion, seeding chondrocytes to the alginate sponges at a low density, combined with perfusion culture, represents a drastic improvement in expanding autologous chondrocytes.     

Bioenergetics and end-product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation q(s)(1 - Y(x) (c))G(ATP) = (mu/Y(ATP) (max)) + m, with q(s) the specific rate of xylose consumption (moles xylose/hour . g cells), Y(x) (c) the carbon based cell yield (g cell carbon/g substrate carbon), G(ATP) the ATP gain (moles ATP produces/mol substrate catabolized), mu the specific growth rate (1/h), Y(ATP) (max) the ATP-based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour . g cells). Y(ATP) (max) was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour . g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased G(ATP) by initiating production of the secondary metabolites, propanediol, and in some cases, D-lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen-limited continuous free-cell cultures maintained a constant cell yield, whereas phosphate-limited continuous free-cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus-limited continuous free-cell cultures exhibited an altered G(ATP) in response to mineral nutrient limitation, and neither produced secondary metabolites. (c) 1993 John Wiley & Sons, Inc.     

Xeno-free proliferation of human bone marrow mesenchymal stem cells

The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.     

Heterotypic and homotypic cellular interactions influencing the growth and differentiation of bipotential oligodendrocyte-type-2 astrocyte progenitors in culture

Cell populations highly enriched in oligodendrocyte-type-2 astrocyte (O-2A) progenitors (so defined by their ability to bind the monoclonal antibodies LB1 and O4, and by the lack of expression of the differentiated glial markers galactocerebroside and glial fibrillary acidic protein (GFAP) were obtained from rat mixed cortical glial cultures. The O-2A progenitors were grown at low density (2 X 10(4) cells/cm2) in BME + 10% fetal calf serum (FCS) on a poly-L-lysine (PLL) substrate (controls) or on a substrate of purified type-1 astrocytes (AS) killed by air drying (K-AS), in order to analyze the effects of the interaction between the two cell types on the growth and differentiation of the immature O-2A cells, independently of the mitogenic soluble factors (e.g., platelet-derived growth factor; see Raff, 1989, Science 243, 1450-1455) secreted by type-1 AS. While on PLL most of the progenitors differentiated into GFAP+ type-2 AS within 1 week, on K-AS they largely differentiated into GalC+ oligodendrocytes (OL). On the latter substrate, however, the precursors achieved a higher density, due to higher proliferative activity. The additional observation, that when immature O-2A cells were seeded at high density (greater than 5 X 10(4) cells/cm2) on PLL their differentiation into OL was much more pronounced than in cultures of lower density, indicates that there is a close correlation between the density of immature O-2A cells and lineage decision, and that the increased OL differentiation of the immature O-2A cells on K-AS is at least partly related to the higher density achieved by the cells on this substrate. The enhanced proliferation of immature O-2A cells on K-AS did not appear to be related to platelet-derived growth factor or fibroblast growth factor remaining attached to the substrate, nor to known components of the extracellular matrix (ECM), such as heparan sulfate, chondroitin sulfate, laminin, or fibronectin, but was probably due to other components of a polypeptide nature present in the ECM produced by type-1 AS. A cell-free ECM was in fact almost as mitogenic as the K-AS substrate, and the mitogenic activities of both K-AS and AS-ECM were similarly inhibited by a set of enzymatic (pronase, trypsin) and physicochemical (heat, pH) treatments.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

Switching from differentiation to growth in hepatocytes: control by extracellular matrix.

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell-cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading.     

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.