Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig.

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.     

Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors alpha and beta 1.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.     

Alteration in growth, cell morphology, and cytoskeletal structures of KB cells induced by epidermal growth factor and transforming growth factor-beta

Long-term biological effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) were examined with human epidermoid carcinoma KB cells. EGF inhibited the growth of KB cells in both serum-containing and serum-free synthetic media by reducing the growth rate and by lowering the saturation density. The cells cultured with EGF showed relatively high motility and grew dispersely as single cells, whereas the cells cultured in the absence of EGF grew in clusters. Although TGF-beta itself did not inhibit the growth of KB cells, it augmented the growth inhibition by EGF. TGF-beta also affected the cell morphology. In the presence of TGF-beta, the cells became flattened and actin stress fibers were well developed compared to those cultured in its absence. The effects of EGF on growth, cell motility, and cell morphology were reversible. Tyrosine phosphorylation of EGF receptors was continuously observed for at least 50 h in the presence of EGF. TGF-beta did not increase the phosphorylation induced by EGF. These results suggested that signals continuously transmitted through EGF receptors caused the changes in cell growth and morphology and that TGF-beta did not act on the cells by modulating binding of EGF to its receptors or activation of the receptor kinase. In contrast to EGF and TGF-beta, neither insulin nor IGF-I affected cell morphology or growth, although KB cells express their receptors and the receptor kinases were also continuously activated during exposure of the cells to insulin or IGF-I.     

Growth regulation of ovarian cancer cells by epidermal growth factor and tranforming growth factors α and β1

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGT, TGFα and TGFβ1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGFα stimulated cell growth and DNA synthesis in OVCAR-3 cell, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGFβ1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These efefects on growth factors are dose dependent. The interaction of TGFβ1 and EGF/TGFα was antagonistic in OVCAR-3 cells. In contrast, EGF/TGFα and TGFβ1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2–3 fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGFα, TGFβ1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine can paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for futher study into the mechanism of growth regulation by growth factors in these cell lines.     

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

A Mathematical Model for the Effects of HER2 Overexpression on Cell Proliferation in Breast Cancer

We present a mathematical model to study the effects of HER2 over-expression on cell proliferation in breast cancer. The model illustrates the proliferative behavior of cells as a function of HER2 and EGFR receptors numbers, and the growth factor EGF. This mathematical model comprises kinetic equations describing the cell surface binding of EGF growth factor to EGFR and HER2 receptors, coupled to a model for the dependence of cell proliferation rate on growth factor receptors binding. The simulation results from this model predict: (1) a growth advantage associated with excess HER2 receptors; (2) that HER2-over-expression is an insufficient parameter to predict the proliferation response of cancer cells to epidermal growth factors; and (3) the EGFR receptor expression level in HER2-over-expressing cells plays a key role in mediating the proliferation response to receptor-ligand signaling. This mathematical model also elucidates the interaction and roles of other model parameters in determining cell proliferation rate of HER2-over-expressing cells.     

Down-modulation of EGF receptors in cells transformed by the src oncogene

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.     

人肝癌细胞表皮生长因子受体以及佛波酯对它的调度

Using radioligand binding assay, the presence of epidermal growth factor (EGF) receptors in cells of two human liver cancer cell lines, BEL-7402 and SMMC-7721, was demonstrated. The ligand binding data were analyzed by a computer program. The dissociation constants (KD) of the ligand-receptor binding complex at equilibrium for 7402 and 7721 cells were 1.2 nM and 0.8 nM respectively, and their number of EGF receptors per cell were 6.2 x 10(4) and 2.5 x 10(4) respectively. After the treatment of cells with phorbol 12-myristate 13-acetate (PMA), no change either in the affinity or in the number of EGF receptors was found in 7721 cells. However, in the case of 7402 cells, while the number of receptors, like 7721 cells, remained unchanged, the affinity of EGF receptors displayed a time dependent modulation after PMA treatment. It dropped within the first hour to a KD value of 3.0 nM and then gradually returned to the normal control value at 48 hours or even slightly higher than normal (0.95 nM) at 96 hours of treatment. The modulation or down-regulation of EGF receptors by PMA in 7402 cells was paralleled by the simultaneous inhibition of DNA synthesis in these cells as evidenced from their reduction of 3H-TdR uptake. It is not clear what is the basis for the differences found between 7402 cells and 7721 cells in their number of EGF receptors per cell and their responsiveness to PMA treatment. It might be related to their difference in autocrine secretion of alpha-transforming growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

Epidermal growth factor: structure, location, phosphorylation and regulation of its receptor

Epidermal growth factor (EGF) is a Mr 6045 polypeptide first characterized for its ability to stimulate mitogenesis in epidermal and epithelial cells. The first step in the action of the growth factor is its binding to specific, high affinity membrane receptors. These receptors have been studied in a number of tissues and cell culture lines. The level of EGF receptors is modulated by many agents. EGF down-regulates its receptor. In addition, the number of EGF receptors is decreased by other growth factors (platelet-derived growth factor; transforming growth factor), by many tumor promoters and by viral transformation. Several hormones also can regulate EGF binding in its target tissues.     

Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

Inhibition of epidermal growth factor-stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level

The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EGF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialoglycopeptide was a very potent inhibitor of EGF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11-1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Transfer of functional EGF receptors to an IL3-dependent cell line

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.     

Characteristics of specific binding of epidermal growth factor (EGF) on human tumor cell lines

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.