Dissociation of uridine and (86Rb+) uptake from stimulation of DNA synthesis in Swiss 3T3 cells

The importance of the stimulation of uridine and (⁸⁶Rb⁺) uptake to the stimulation of DNA synthesis was investigated using three defined growth factors, EGF,FGF and PGF_2α′ and three hormones, hydrocortisone, insulin and PGE_1′, which do not stimulate proliferation of Swiss 3T3 cells but modify the response to these growth factors. Uridine uptake is stimulated by insulin, but not by PGF_2α′ indicating that its activation is neither sufficient nor necessary for stimulation of cell proliferation. (⁸⁶Rb⁺) uptake was stimulated by each growth factor tested, but also by insulin and PGE₁. Modifying effects of insulin, hydrocortisone or PGE₁ in combination with growth factors on the level of DNA synthesis were not reflected in changes in stimulation of these uptake systems. We conclude that these events are regulated separately and are not tightly coupled to the initiation of DNA synthesis.     

Colchicine enhances the effect of growth factors on the initiation of DNA synthesis in sparse and confluent Swiss 3T3 cells

We have studied the effect of colchicine on the initiation of DNA synthesis stimulated by serum or by growth factors such as EGF, FGF or PGF_2α in sparse and confluent resting Swiss 3T3 cells. Colchicine in combination with FGF or with low concentrations of serum is more effective in stimulating DNA synthesis than with EGF or PGF_2α. Only in the presence of insulin do these latter two growth factors give with colchicine a response comparable to that of FGF with colchicine. This phenomenon was observed both in sparse and in confluent resting cells. We conclude that the synergistic effect of colchicine with growth factors or with serum does not depend on the cellular saturation density at which Swiss 3T3 cells become quiescent.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.     

Endothelin-I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolal ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [¹²⁵I]ET-1 was achieved at concentrations greater than 100 pM. The K_d and B_max values for [¹²⁵I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [¹²⁵I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (K_i = 0.047 nM) than ET-3 (K_i = 10.8 nM). No specific binding of [¹²⁵I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ET_A receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley-Liss, Inc.     

Reduction of the calcium requirement of normal human epithelial cells by EGF

The influence of epidermal growth factor (EGF) on the Ca²⁺ requirement of normal (NP-2s) and neoplastic human epithelial (PC-3) cells was studied using a clonal growth assay. The interaction of Ca²⁺ and EGF was investigated by kinetic analysis of dose-response experiments in which the Ca²⁺ or EGF concentration necessary for half-maximal growth, K_m were determined. The normal epithelial cells required 80 times more calcium than did the cancer cells. In the presence of EGF, the Ca²⁺ requirement of both cell types was virtually identical. EGF did not affect the growth rate of the cancer line. The interaction between Ca²⁺and EGF was found to be unidirectional since EGF reduced the K_m^Ca2+ 120-fold, whereas Ca²⁺ had no effect on the EGF dose-response.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Cytosine arabinoside increases the binding of125I-labelled epidermal growth factor and125I-transferrin and enhances the in vitro targeting of human tumour cells with anti-(growth factor receptor) mAb

We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with aK _d of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with aK _d of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [³H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [³H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.This work has been supported by the Italian Association for Cancer Research (A.I.R.C.) and by the National Council of Research (C.N.R.) of Italy, contract 92.02274.PF39     

Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of ¹²⁵I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the ¹²⁵I-EGF – EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K_D≈200 pM on SKBR3 cells to K_D≈8 nM on A431 cells. The ¹²⁵I-EGF – EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the ¹²⁵I-EGF - EGFR affinity, in particular when the cells are starved. The ¹²⁵I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.     

Distinctive effects of hydrocortisone on the modulation of EGF binding and cell growth in hela cells grown in defined medium

Hydrocortisone modulates the binding capacity of HeLa cells for ¹²⁵I-labeled epidermal growth factor (EGF). A twofold increase in ¹²⁵I-labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 × 10^?8?1 × 10^?6 M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum-supplemented or completely defined, serum-free, hormone-supplemented medium. Scatchard analysis of the binding data indicates that the number of ¹²⁵I-EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum-free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 × 10^?9 M, while a concentration higher than 5 × 10^?8 M is required to affect the binding capacity for ¹²⁵I-EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum-supplemented medium is similar to that observed in the serum-free cultures, hydrocortisone is growth-inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10⁻⁹ M) with baFGF (40 μg/ml) and EGF(0.01 μg/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E₂ release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network. © 1996 Wiley-Liss, Inc.     

Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Effect of serum and growth factors on heat sensitivity in Swiss mouse 3T3 cells

Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46°C for 20 min by changing their flat, well-extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37°C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F_2α (PGF_2α) markedly increased the heat resistance to a 46°C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2–3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF_2α in these cells) was added alone or in combination with either EGF or PGF_2α, it had no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2–4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46°C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells.     

The stimulation of the initiation of DNA synthesis by fibroblast growth factor in swiss 3T3 cells: Interactions with hormones during the pre-replicative phase

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13–15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E₁ (PGE₁) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF. These results show that the stimulation of growth by FGF follows the same basic pattern as was previously shown with Prostaglandin F_2α (PGF_2α). However, since hydrocortisone inhibits stimulation by PGF_2α when added during the first 4 hr of the lag phase, there are clearly differences in some events stimulated by the two growth factors.     

Modulation of EGF binding and action by succinylated concanavalin A in fibroblast cell cultures

The role of the binding of succinylated concanavalin A to tissue culture cells in influencing epidermal growth factor (EGF)-mediated cell proliferation has been studied. Succinylated concanavalin A dramatically reduces the stimulation of 3T6 cells by EGF in Dulbecco's modified Eagle's medium (DME) containing insulin and vitamin B₁₂ as additional growth factors, but no serum. Furthermore, binding studies using ¹²⁵I-labeled EGF have shown that the binding of EGF to the cell surface is reduced upon addition of succinylated concanavalin A.     

The effect of 1.25 dihydroxyvitamin D3 on binding and internalization of epidermal growth factor in cultured cells : Studies on BT-20 cells using quantitative electron microscope autoradiography

The biological effects of 1.25(OH)₂D₃ on epidermal growth factor receptor (EGF-R) and on EGF internalization were examined in human mammary carcinoma BT-20 cells. In this cell line, with known amplification of the epidermal growth factor receptor gene, EGF was not stimulatory for growth. Biological assay and quantitative EM autoradiography combined with iodinated ligand binding to specific receptors demonstrated that the number of binding sites per unit of length of plasma membrane was 2.48-fold higher in treated than in control cells. ¹²⁵I-EGF was progressively internalized in a time- and temperature-dependent manner after selective association with the membrane-coated pits. No modification of the time course of ¹²⁵I-EGF internalization was noted in the control and in the treated cells, but a different distribution of the labeling in the subcellular compartment was observed in treated cells. In 1.25(OH)₂D₃-treated batches, the grain density remained low in the receptosomes throughout the experiment, whereas it was high and occurred early in the lysosomes. On the other hand, in control cells, the grain density of the receptosomes was high, whereas it occurred late and was relatively low in the lysosomes. These data suggest that 1.25(OH)₂D₃ is a regulator of EGF-R level in BT-20 cell line, but it cannot be affirmed whether this effect is direct or mediated by other parameters.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria.

Murine epidermal growth factor (EGF) stimulated the production of prostaglandin E₂ (PGE₂) and bone resorption in neonatal mouse calvaria in organ culture. The effect of EGF on bone resorption occurred at low concentrations of the polypeptide (half-max stimulation = 0.4 ng/ml, 6.6 × 10^?11 M). All concentrations of EGF which stimulated resorption also stimulated the production of PGE₂ by bone; concentrations of EGF which did not stimulate resorption did not enhance PGE₂ production. EGF-induced formation of PGE₂ and bone resorption were inhibited completely by indomethacin (200 ng/ml) and hydrocortisone (3 × 10^?6 M). Indomethacin did not inhibit the bone resorption-stimulating activity of exogenous PGE₂. The time courses of action of EGF, parathyroid hormone and exogenous PGE₂ on bone resorption were similar. Brief exposure (15 or 60 min) to EGF (10 ng/ml) did not cause bone resorption or an increase in PGE₂ accumulation in a subsequent 48-h incubation in the absence of EGF. High concentrations (30 to 100 ng/ml) of bovine fibroblast growth factor (FGF) also stimulated the production of PGE₂ and bone resorption. We conclude that concentrations of EGF equal to or less than those present in mouse plasma stimulate the resorption of mouse bone in organ culture by a mechanism that involves the enhanced local production of PGE₂.