Balb-C 3T3 Fibroblasts Research Articles

Antiproliferative effects and insulin-like growth factor-I expression in Balb-C 3T3 fibroblasts after treatment with somatostatin and gonadotropin-releasing hormone analogs

The present study was aimed to compare antiproliferative effects of somatostatin (SS) and gonadotropin-releasing hormone analogs (GnRHa) on a fibroblast cell line. Proliferation index, cell count, viability of the cells and insulin-like growth factor-I (IGF-I) immunoreactivity were determined after treatment with either SS (100 microM/ml), GnRHa (35 nM/ml) or SS and GnRHa of Balb-C 3T3 mouse fibroblasts. It was found that the proliferation index, cell count, viability and IGF-I immunoreactivity were not affected by GnRHa treatment as compared with no treatment (p > 0.05). Application of SS to the fibroblasts resulted in a significant reduction in proliferation index, cell count, and IGF-I immunoreactivity as compared with GnRHa treatment and no treatment, but it had no effect on cell viability. The labelling index in SS-treated cells was significantly reduced as compared with combined treatment with SS and GnRHa. In conclusion, a direct effect of GnRHa on fibroblast cells in culture could not be demonstrated. SS had direct inhibitory effects on cell proliferation possibly via inhibition of IGF-I effects without affecting cell viability.

Arachidonic acid mediates calcium influx induced by basic fibroblast growth factor in Balb-c 3T3 fibroblasts

Basic fibroblast growth factor (bFGF), a peptide acting as a mitogen in different cell types, is able to induce a long lasting non capacitative calcium influx from the extracellular medium in Balb-c 3T3 mouse fibroblasts. This effect is mediated by the tyrosine kinase activity of bFGF receptors and the opening of voltage independent, agonist activated calcium channels. In this paper we investigate the signal transduction steps involved in this process using single cell calcium fluorimetry and electrophysiological techniques. One of the pathways initiated by the binding of growth factors to their tyrosine kinase receptors is the activation of cytosolic phospholipase A 2 (cPLA 2) and the release of arachidonic acid (AA) from the plasma membrane with the subsequent production of eicosanoids. We show here that, in our preparation, this pathway is involved in the opening of the bFGF-activated calcium permeable channels, through the activation of mitogen activated protein kinase (MAPK) and cPLA 2. Evidence for direct involvement of AA is given by the finding that: (i) bFGF induces AA release from Balb-c 3T3 cells; (ii) blockers of AA metabolism are not effective; and (iii) the application of either arachidonic acid or its non metabolizable analogue 5,8,11,14-eicosatetraynoic acid (ETYA) reproduces the responses described for bFGF. Finally, single channel analysis indicates that bFGF, AA and ETYA can activate the same calcium permeable channel.

TTGase/G αh protein expression inhibits adenylate cyclase activity in Balb-C 3T3 fibroblasts membranes

Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn 2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.

Cell-biomaterial interactions: role of transglutaminase enzyme

Cell interaction(s) with biomaterial undergo a multistep-step paradigm of adhesion, contact, attachment, and spreading. However, the molecular mechanism(s) underlying the cell anchorage on a material surface is not yet well understood. Transglutaminases, a family of calcium-dependent enzymes, has been implicated in the interactions between cells and extracellular matrix. To investigate the functions of this enzyme in cell-material interactions, Balb-C 3T3 fibroblasts were cultured, stably expressing high levels of tissue transglutaminase (TGase), on dishes precoated with thin layers of the hydrophilic hydrogel poly (2-hydroxyethyl methacrylate) (polyHEMA). Results suggest that in the presence of a relatively high poly (HEMA) dilution, the transfected cells showed a more complex morphologic pattern, characterized by many spread and flattened cells, in comparison with a control culture. These studies support the hypothesis that the overexpression of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min elevation of intracellular free calcium concentration ([Ca 2+] i). Both the sustained [Ca 2+] i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73–82], could be abolished either by chelation of extracellular calcium with EGTA or by SKF 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SKF 96365 was applied from the 4 th to the 8 th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G 2/M phase. Both growth inhibition and G 2/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.

Sustained calcium influx activated by basic fibroblast growth factor in Balb‐c 3T3 fibroblasts.

1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long-lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF-activated ionic events are compatible with their involvement in the control of cell proliferation.

Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts

In quiescent Balb-c mouse 3T3 fibroblasts, the application of whole or dialyzed 10% foetal calf serum elicits a biphasic electrical response, consisting of a transient outward current, flowing through Ca 2+-activated K + channels, followed by an inward one, lasting up to 15 min. On the basis of experiments with ion substitutions and blockers, the inward current can be attributed to the opening of cationic channels permeable to Na + and Ca 2+ ions. This current could mediate the calcium influx involved in the sustained elevation of [Ca 2+] that has been observed in many preparations in response to mitogen stimulation and that is involved in triggering cell proliferation.

Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion.

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

Rapid modulation of a 64 K Dalton fibroblast protein: a PDGF mediated early cellular event

The results presented here reveal a novel platelet derived growth factor (PDGF) mediated early cellular event. Treatment of growth arrested Balb c3T3 fibroblasts with PDGF induces a specific and rapid modulation of a 64,000 Dalton (64 KD) protein preexisting in quiescent cells. The kinetics of 64 KD protein modulation indicate that, temporally, this PDGF mediated step lies between the membrane associated immediate events such as receptor autophosphorylation or ion mobilization and the earliest known transcriptional event, the activation of the proto-oncogene c-fos.

Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro.

We studied the in vitro secretion of macrophage-derived growth factor (MDGF) activity by peritoneal macrophages from fertile and infertile women. Peritoneal fluid was obtained from 55 women undergoing laparoscopy for evaluation and treatment of infertility or for tubal sterilization. Isolated macrophages were plated in tissue culture wells and incubated in Dulbecco's Modified Eagle's Medium plus 0.2% lactalbumin hydrolyzate at 37 C for 24 h. Medium MDGF activity was assayed by determining the ability of medium to stimulate [3H] thymidine incorporation in BALB-c 3T3 fibroblasts. Macrophages from 23 women released significant MDGF activity in vitro; the release was linear for up to 72 h. Among the 55 women, macrophages from 10 of 36 (28%) women with normal pelvic anatomy or tubal occlusion/pelvic adhesions released significant MDGF activity. In contrast, macrophages from 13 of 19 (68%) women with endometriosis, a significantly higher proportion (P less than 0.02), released MDGF. The finding that endometriosis is associated with in vivo primed peritoneal macrophages that produce MDGF in vitro may help to explain the proliferation or maintenance of endometrial tissue in the peritoneal cavity.

Inhibition of early platelet-derived growth factor responses in [formula omitted] cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium ([Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALBc-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect of PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALBc-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.

Molecular and developmental biology aspects of fibroblast growth factor.

Basic and acidic fibroblast growth factors (FGFs) are two closely related peptides of Mr =16000 that act as potent mitogens and differentiation factors or a wide variety of mesoderm and neuroectoderm derived cells. Basic FGF (bFGF pI 9.6) was first identified by its ability to cause the proliferation and phenotypic transformation of BALB-C 3T3 fibroblasts (Gospodarowicz,1974;Gospodarowicz and Moran,1974), while acidic FGF (aFGF, pI 5.6) was first identified by its ability to cause the proliferation and delayed differentiation of myoblasts (Gospodarowicz et al.,1975). It was later rediscovered on the basis of its ability to stimulate endothelial cell proliferation (Lemmon et al.,1982; Maciag et al.,1978).

Insulin and epidermal growth factor stimulate poly ADP-ribosylation

Treatment of contact-inhibited BALBc 3T3 fibroblasts with insulin or epidermal growth factor elicited a considerable increase in the capacity of poly (ADP-ribose) synthesis. Stimulation of the poly ADP-ribosylation was observed after long-term hormone treatment (18 hours) but not after short-term treatment (1 hour) and it appeared to be correlated with the cell's entry into DNA synthesis. Several cellular components were found to be enhanced in poly ADP-ribosylation. The data suggest a possible role for poly ADP-ribosylation in mitogenic action of polypeptide hormones and growth factors.

Production of macrophage-dependent fibroblast-stimulating activity (M-FSA) by murine macrophages: Effects on BALBc 3T3 fibroblasts

Macrophages cultured in vitro in medium containing platelet-poor plasma serum (PPPS) produce a substance (or substances) that stimulates the proliferation of fibroblasts in vitro. Two alternative assays have been used to study the production of this “macrophage-dependent fibroblast-stimulating activity” (M-FSA) by mouse peritoneal macrophages. The first determined increase in cell numbers induced in cultures of quiescent 3T3 fibroblasts (Growth Curve Assay); the second determined the incorporation of [ 3H]thymidine into the DNA of quiescent 3T3 cells induced by various culture media. Medium containing 5% blood serum (5% BS) resulted in higher final cell numbers (5-fold), and greater incorporation of [ 3H]thymidine into DNA than did medium containing 5% platelet-poor plasma serum (PPPS). Macrophages cultured in PPPS produced two different operationally defined activities. The first, M-FSA, resulted in greater final cell numbers, and was non-dialysable. The second reduced the incorporation of [ 3H]thymidine into DNA, and was found to be dialysable. Maximum levels of M-FSA were produced at a concentration of approx. 5 × 10 6 macrophages/10 ml culture medium containing 5 % PPPS. Successive 18 h cultures of macrophages in fresh changes of medium containing PPPS resulted in equivalent levels of M-FSA in each successive batch of medium. The presence of PPPS in the culture medium appeared to be an absolute requirement for the production of M-FSA. Culture in the absence of serum, in the presence of BSA, FCS, or of RBS did not result in the production of M-FSA. The dialysable factor, on the other hand, was produced non-specifically, in media containing either PPPS, FCS, RBS, BSA, or no serum. The level of production increased with increasing macrophage concentrations. At a concentration of 100 × 10 6 macrophages/10 ml medium, the stimulatory effect of M-FSA in the [ 3H]thymidine incorporation assay was completely masked. The dialysable substance did not affect the proliferation of 3T3 cells. Only the incorporation of [ 3H]thymidine into DNA was affected. It is suggested that M-FSA could be produced by macrophages, indirectly, by activation or de-inhibition of inactive (or inhibitory) substances present specifically in PPPS.