Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line

The MDA 468 human breast carcinoma cell line was examined for changes in epidermal growth factor (EGF) receptor synthesis and degradation under the influence of EGF. This cell line was used because it overexpresses the EGF receptor such that each cell has 10(6) receptors, but unlike the well-studied A431 cell, its receptor gene is amplified but is not rearranged. On exposure to EGF, total cellular receptor protein, measured by immunoprecipitation with monoclonal antibody B1D8, is reduced. The half-life of receptor metabolically labeled with L-[35S]methionine is 24 h in the absence of EGF and is reduced to 12 h in the presence of 10(-9) M EGF. To measure the effect of EGF on synthesis of the receptor, pulse labeling conditions were selected in which the rate of synthesis of the receptor precursor were followed. EGF had no significant effect on the rate of general protein synthesis in these cells, yet stimulated the synthesis of the EGF receptor 1.8-fold over the unstimulated rate. This increase in receptor precursor synthesis showed time and dose dependence. Stimulation could be detected after 3 h exposure to EGF with a maximum at 6-8 h. A concentration of 10(-11) M EGF gave detectable stimulation with maximal stimulation occurring at 10(-9) M. Longer times and higher concentrations gave submaximal stimulation. A similar dose-response relationship was observed when the rate of mature 170-kDa receptor protein synthesis was measured. These studies demonstrate that EGF stimulates the synthesis of it own receptor. Downregulation of the receptor by EGF results from an increased rate of receptor degradation and not decreased synthesis.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.     

Epidermal growth factor receptor expression in human gliomas

Summary The expression of epidermal growth factor receptor (EGFR) was determined in cryosections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and two monoclonal antibodies (mAb) against EGFR. All gliomas were found to express EGFR when examined with B-EGF, whereas 33 expressed EGFR when examined with the two mAbs. The highly malignant gliomas (glioblastomas and anaplastic astrocytomas) had a more heterogeneous staining pattern and a larger proportion of tumour cells staining strongly with B-EGF than did the low-grade gliomas (astrocytomas, oligodendrogliomas, mixed gliomas, and ependymomas). This indicates that high-grade gliomas contain more tumour cells rich in EGFR than do the low-grade gliomas. Reactive astrocytes, ependymal cells, and many types of nerve cells (cerebral cortical pyramidal cells, pyramidal and granular hippocampal cells, Purkinje cells, cerebellar granular cells and neurons in the molecular layer of the cerebellum) expressed EGFR, whereas small neurons and normal glial cells were not found to express EGFR.     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.     

EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line

The effects of epidermal growth factor(EGF) on intracellular calcium ([Ca²⁺]_i)responses to the muscarinic agonist carbachol were studied in a humansalivary cell line (HSY). Carbachol (10⁴ M)-stimulated[Ca²⁺]_i mobilization was inhibited by 40%after 48-h treatment with 5 × 10¹⁰ M EGF. EGF alsoreduced carbachol-induced [Ca²⁺]_i inCa²⁺-free medium and Ca²⁺ influx followingrepletion of extracellular Ca²⁺. UnderCa²⁺-free conditions, thapsigargin, an inhibitor ofCa²⁺ uptake to internal stores, induced similar[Ca²⁺]_i signals in control and EGF-treatedcells, indicating that internal Ca²⁺ stores were unaffectedby EGF; however, in cells exposed to thapsigargin, Ca²⁺influx following Ca²⁺ repletion was reduced by EGF.Muscarinic receptor density, assessed by binding of the muscarinicreceptor antagonistL-[benzilic-4,4'-³HCN]quinuclidinyl benzilate([³H]QNB), was decreased by 20% after EGF treatment.Inhibition of the carbachol response by EGF was not altered by phorbolester-induced downregulation of protein kinase C (PKC) but was enhancedupon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of thecarbachol response by EGF were both blocked by the MAP kinase pathwayinhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca²⁺ release from internal stores andalso exerts a direct inhibitory action on Ca²⁺ influx. Adecline in muscarinic receptor density may contribute to EGF inhibitionof carbachol responsiveness. The inhibitory effect of EGF is mediatedby the MAP kinase pathway and is potentiated by a distinct modulatorycascade involving activation of PKC. EGF may play a physiological rolein regulating muscarinic receptor-stimulated salivary secretion.

     

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.     

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.     

Epidermal growth factor receptor signaling activates orthodenticle expression during Drosophila head development

The relation between signal transduction pathways and the genes that specify regional identity remains poorly understood. We investigated the interaction between the epidermal growth factor receptor (EGFR) pathway and the homeobox gene orthodenticle (otd), which specifies cell fate during head development. Previous studies of head formation in Drosophila melanogaster demonstrated that reducing either EGFR signaling or otd expression in the imaginal primordium of the dorsal head capsule eliminates the ocelli and other dorsal head structures. Here, we show that blocking EGFR signaling reduces otd expression and that activating EGFR signaling outside this primordium induces ectopic otd expression. We also demonstrate that loss of EGFR can be rescued by constitutive otd expression. Our results indicate that otd is a downstream target of the EGFR pathway during head development.     

Epidermal growth factor receptors in the human glioblastoma cell line SF268 differ from those in epidermoid carcinoma cell line A431

Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF.     

Epidermal growth factor receptor signal transactivation

Cross-communication between heterologous signaling systems and the epidermal growth factor receptor (EGFR) has been shown to be critical for a variety of biological responses: EGFR transactivation when G-protein-coupled receptors (GPCRs) are stimulated represents the paradigm of an interreceptor network that is dependent on G-proteins, kinases, metalloproteases, and growth factor precursors. Investigating the mechanism of this process will help expand our knowledge of physiological regulatory mechanisms and diverse pathophysiological disorders.     

Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.