Unlabeled Epidermal Growth Factor Research Articles

Accumulation of 111In-Labelled EGF-Au-PEG Nanoparticles in EGFR-Positive Tumours is Enhanced by Coadministration of Targeting Ligand.

The successful use of targeted radionuclide therapy in the treatment of solid tumours may be limited by radioresistance, which necessitates delivery of a high dose of radioactivity. Nanoparticle (NP)-based delivery systems possess a large surface area for attachment of radioisotopes and so offer a solution to this challenge. However, tumour uptake may be limited by rapid hepatic clearance of NP via the mononuclear phagocyte system. Liver uptake is further compounded when epidermal growth factor (EGF) is used as a targeting ligand, as EGF-tagged NP bind the EGF receptor (EGFR), which is expressed to a moderate extent by hepatocytes. This report describes an indium-111 (111In)-labelled PEGylated EGF-tagged gold (Au) NP (111In-EGF-Au-PEG) and an effective strategy of coadministration of targeting ligand to address these issues. Direct attachment of EGF to the surface of Au NP did not compromise surface coating with long-chain PEG. In vitro experiments showed that 111In-EGF-Au-PEG targets EGFR-positive cancer cells (MDA-MB-468): >11% of radioactivity was internalised after incubation for 4 h. In in vivo studies accumulation of NP was observed in MDA-MB-468 xenografts and tumour uptake was enhanced by the coadministration of 15 µg of the unlabelled targeting ligand, EGF, to block hepatic EGFR. Uptake was 3.9% versus 2.8% injected dose/g (%ID/g) of tumour tissue with and without unlabelled EGF, respectively. Coadministration of EGF reduced liver uptake by 25.95% to 7.56 %ID/g. This suggests that the coadministration of unlabelled targeting ligand with radiolabelled PEGylated NP offers a promising strategy for targeting EGFR-positive cancer and for minimising liver uptake.

PET imaging of EGF receptors using [18F]FBEM-EGF in a head and neck squamous cell carcinoma model

To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF). Cys-tagged EGF (cEGF) was labeled with (18)F by coupling the free thiol group of the Cys tag with N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide ([(18)F]FBEM) to form [(18)F]FBEM-cEGF. Cell uptake, internalization and efflux of [(18)F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results. The radiolabeling yield for [(18)F]FBEM-cEGF was over 60%, based on starting [(18)F]FBEM. [(18)F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 ± 0.59 %ID/g at 30 min after injection. Significantly higher tumor uptake of [(18)F]FBEM-cEGF (5.99 ± 1.61%ID/g at 30 min after injection, p < 0.01) and tumor/nontumor ratio were achieved by coinjection of 50 μg of unlabeled EGF. Decreased liver uptake of [(18)F]FBEM-cEGF was observed when unlabeled EGF was coadministered. With optimized liver blocking, [(18)F]FBEM-cEGF has the potential to be used in a noninvasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity.

Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR). Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48,000 synthetic compounds. The competition between fluorescent EGF and unlabeled EGF reveals the IC(50) of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK). This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.

Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.

Retrograde Transport of Filopodial erbB1

Lidke et al. used epidermal growth factor (EGF) conjugated to fluorescent quantum dots (EGF-QDs) to track individual ligand-bound erbB1 receptors and investigate in detail the retrograde transport on filopodia of activated erbB1 receptors (a process this group had recently reported). Using confocal microscopy to compare the localization of EGF-QD and activated erbB1 receptors (visualized through immunofluorescence detection of erbB1 phosphorylated on tyrosine-1068) on the filopodia of cultured human A431 cells, the authors found a direct correlation between EGF-QD binding and erbB1 activation. Pharmacological analysis indicated that the retrograde transport on filopodia of EGF-QD-erbB1 complexes depended on both activation of the erbB1 receptor tyrosine kinase and an intact actin cytoskeleton. Moreover, a comparison of EGF-QD-erbB1 transport and actin flow in the filopodia of HeLa cells that expressed enhanced green fluorescent protein (EGFP)-labeled actin indicated that both moved at the same rate. Receptor endocytosis occurred on the cell body, following retrograde transport, and not on the filopodia themselves. Isolated EGF-QD-erbB1 complexes obtained in the presence of very low concentrations of EGF-QD showed only diffusive movement; the addition of higher concentrations of unlabeled EGF under these conditions initiated retrograde transport, which suggests that monomeric EGF-QD-erbB1 complexes did not undergo retrograde transport. Visualization of low concentrations of EGF-QD of two different colors revealed that a switch in EGF-QD-erbB1 movement from diffusion to transport followed merging of the two colors, again suggesting that transport depended on EGF-erbB1 dimerization (or oligomerization). Thus, the authors suggest that filopodia may act as a sensory system to detect signaling molecules far from the cell body. D. S. Lidke, K. A. Lidke, B. Rieger, T. M. Jovin, D. J. Arndt-Jovin, Reaching out for signals: Filopodia sense EGF and respond by directed retrograde transport of activated receptors. J. Cell Biol. 170 , 619-626 (2005). [Abstract] [Full Text]

Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor

The purpose of this study was to analyse whether non-radiolabelled epidermal growth factor (EGF) can modify the cellular uptake of 125I when delivered as [125I]trastuzumab. 125I was used as a marker for the diagnostically and therapeutically more interesting isotopes 123I (SPECT), 124I (PET) and 131I (therapy). The cell-associated radioactivity was measured in squamous carcinoma A431 cells following addition of [125I]trastuzumab. Different concentrations of [125I]trastuzumab and unlabelled EGF were used, and the total, membrane-bound and internalised radioactivity was measured. We also analysed how EGF and trastuzumab affected the cell growth. It was generally found that the cellular 125I uptake was decreased by the addition of EGF when [125I]trastuzumab was added for short incubation times. However, if the incubation times were longer, EGF increased the 125I uptake. This shift came earlier when higher [125I]trastuzumab concentrations were applied. The addition of EGF also influenced cell proliferation, and concentrations above 10 ng/ml reduced cell growth by approximately 20% after 24 h of incubation. By adding unlabelled EGF, it was possible to modify the cellular uptake of [125I]trastuzumab. This points towards new approaches for the modification of radionuclide uptake in EGFR- and HER2-positive tumours.

Diffusion of epidermal growth factor in rat brain extracellular space measured by integrative optical imaging.

Epidermal growth factor (EGF) stimulates proliferation, process outgrowth, and survival in the CNS. Understanding the actions of EGF necessitates characterizing its distribution in brain tissue following drug delivery or release from cellular sources. We used the integrative optical imaging (IOI) method to measure diffusion of fluorescently labeled EGF (6,600 Mr; 4 microg/ml) in the presence of excess unlabeled EGF (90 microg/ml) to compete off specific receptor binding and reveal the "true" EGF diffusion coefficient following injection in rat brain slices (400 microm). The effective diffusion coefficient was 5.18 +/- 0.16 x 10(-7) (SE) cm2/s (n = 22) in rat somatosensory cortex and the free diffusion coefficient, determined in dilute agarose gel, was 16.6 +/- 0.12 x 10(-7) cm2/s (n = 27). Tortuosity (lambda), a parameter representing the hindrance imposed on EGF by the convoluted brain extracellular space (ECS), was 1.8, the lowest yet measured by IOI for a protein in brain. Control experiments with fluorescent dextran of similar molecular weight and tetramethylammonium confirmed EGF did not affect local ECS structure. We conclude that transport of smaller growth factors such as EGF through brain ECS is less hindered than that of larger proteins (>10,000 Mr, e.g., nerve growth factor) where typically lambda > 2.1. Modeling was used to predict that low lambda will allow EGF sources in the brain to be further from target cells and still elicit a biological response. High lambda values for larger growth factors imply more constrained local biological effects than with smaller proteins such as EGF.

177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma.

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.

A STOPPED-FLOW FLUORESCENCE ANISOTROPY METHOD FOR MEASURING HORMONE BINDING AND DISSOCIATION KINETICS WITH CELL-SURFACE RECEPTORS IN LIVING CELLS

ABSTRACTWe have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1–15 nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.

Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy.

We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace Rh-EGF binding, demonstrating no cross-reaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K(ass) of 1.5 x 10(9) M(-1). The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k(diss) = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.

Epidermal Growth Factor Radiopharmaceuticals: 111In Chelation, Conjugation to a Blood-Brain Barrier Delivery Vector via a Biotin-Polyethylene Linker, Pharacokinetics, and in Vivo Imaging of Experimental Brain Tumors

Epidermal growth factor (EGF) is a potential peptide radiopharmaceutical for detection of brain tumors, because many human gliomas overexpress the EGF receptor (EGFR). The transport of EGF to the brain, however, is restricted by the blood-brain barrier (BBB). The purpose of the present study was to develop a vector-mediated brain delivery system for radiolabeled EGF. Human EGF was monobiotinylated with NHS-PEG3400-biotin, where NHS is N-hydroxysuccinimide and PEG3400 is poly(ethylene glycol) of 3400 Da molecular mass. EGF-PEG3400-biotin was radiolabeled with either 125I or 111In through the metal chelator, diethylenetriaminepentaacetic acid (DTPA). The radiolabeled EGF was then conjugated to a BBB delivery vector comprised of a complex of the OX26 monoclonal antibody (MAb) to the rat transferrin receptor, which was coupled to streptavidin (SA). Following intravenous injection in rats, the 125I conjugate was rapidly degraded in vivo, while the 111In conjugate was metabolically stable. The brain delivery of [111In]DTPA-EGF-PEG3400-biotin was enabled by conjugation with OX26/SA and was optimized by co-injection of unlabeled EGF to saturate EGF receptors in the liver. The specific binding of the [111In]DTPA-EGF-PEG3400-biotin conjugated to OX26/SA to the EGF receptor was confirmed in C6 rat glioma cells, which had been transfected with a gene encoding for the human EGF receptor under the regulation of a dexamethasone-inducible promoter. In vivo studies of C6-EGFR experimental tumors in Fischer 344 rats demonstrated successful brain imaging only when the peptide radiopharmaceutical was conjugated to the BBB delivery system, although the C6-EGFR tumors did not express EGFR in vivo. In conclusion, these studies describe the molecular formulation of a peptide radiopharmaceutical that can be used for imaging brain tumors behind the BBB.

Bovine Milk Inhibits Proteolytic Degradation of Epidermal Growth Factor in Human Gastric and Duodenal Lumen

R. K. Rao, R. D. Baker and S. S. Baker. Bovine milk inhibits proteolytic degradation of epidermal growth factor in human gastric and duodenal lumen. Peptides 19(3) 495–504, 1998.—Degradation of epidermal growth factor (EGF) in human gastric and duodenal lumen was analyzed by incubating 125I-labeled or unlabeled human recombinant EGF with human gastric or duodenal luminal fluids in vitro. Degradation of EGF was assessed by measuring the generation of acid soluble radioactivity or by reversed-phase high-performance liquid chromatography (HPLC). Incubation with gastric luminal fluids resulted in a time- and dose-dependent degradation of labeled and unlabeled EGF at pH 2.5 but not at pH 7.5. Duodenal luminal fluids, on the other hand, degraded EGF at pH 7.5 but not at pH 2.5. The rate of degradation of unlabeled EGF in gastric luminal fluids was nearly 12-fold higher than the rate of degradation of labeled EGF, whereas only a slight difference in rates of degradation of labeled and unlabeled EGF was observed in duodenal luminal fluids. High-performance liquid chromatography analysis detected three major degradation products that eluted with retention time of 17.5 min, 20.0 min, and 22.5 min that was associated with a reduction of intact EGF (retention time 23.5 min). Defatted and decaseinated supernatant of bovine milk effectively inhibited the degradation of EGF in both gastric and duodenal luminal fluids. Dietary derived protease inhibitors, such as soya bean trypsin inhibitor, lima bean trypsin inhibitor, egg white protease inhibitor, and Bowman–Birk protease inhibitor prevented EGF degradation in duodenal luminal fluids but failed to inhibit EGF degradation in gastric luminal fluids. These results suggest that bovine milk may contain specific inhibitors that protect EGF from proteolytic degradation in human gastric lumen.

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.

Epidermal growth factor binding sites in the mouse exocrine and endocrine pancreas shown by in vivo quantitative microautoradiography and confocal laser scanning microscopy

Microautoradiography at 3, 6 and 15 min after intravenous injection of 125I-EGF was used to investigate the distribution of epidermal growth factor (EGF) binding sites in the pancreas of normal male mice. The autoradiographs were observed by confocal laser microscopy, which allows the quantification of silver grains. The results demonstrated that both endocrine and exocrine pancreatic cells exhibited substantial specific binding of 125I-EGF. The highest level of EGF binding was found in the duct cells of the exocrine pancreas followed by the acinar cells. The cells of the islets of Langerhans also showed substantial specific binding of 125I-EGF though the binding level was lower than that of the exocrine pancreas. In the control experiments, mice were injected with 125I-EGF and various amounts of unlabeled EGF.

Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Aims: Mitogenic effect and cellular handling of epidermal growth factor (EGF) were analyzed in primary cultured rat hepatocytes at several cell densities. Methods/Results: DNA synthesis, assessed by the incorporation of 125I-deoxyuridine, was accelerated by EGF at a low cell density while that stimulated by EGF was relatively low at the highest cell density, suggesting a cell density-dependent regulation of mitrogenic response to EGF. An equilibrium binding study of 125-I-EGF in the presence of various concentrations of unlabeled EGF at 0°C revealed that the dissociation constant (K d) was 0.47–0.88 nM while the specific binding capacity (n) was 86–96 fmol/mg protein at each cell density. No significant difference was observed in the time profiles of the surface-bound, internalized, and degradation products of 125I-EGF, assessed per mg protein, between different cell densities. Based on a kinetic analysis of the time-profiles, the internalization rate constant and the degradation rate constant were found to be independent of cell density. Conclusions: These results indicate that the cellular binding and disposition of EGF are not regulated by cell density, and that the cell density-dependence of the mitogenic effect cannot be attributed to differences in the affinity or capacity of the EGF receptor, internalization, or degradation of EGF. We speculate that the cell density-dependent mitogenic response may be accounted for by the difference in other factors such as the signal transduction processes induced by the receptor binding of EGF, or the translocation of a small fraction of the total EGF to hepatocyte nuclei.

Epidermal growth factor-binding protein in Mycobacterium avium and Mycobacterium tuberculosis: a possible role in the mechanism of infection.

Epidermal growth factor (EGF) is a potent mitogen for a variety of eukaryotic cells. EGF is found in a number of tissues and is prevalent in necrotic tissues and granulomata. The biological effect of EGF on mammalian cells is initiated by the binding to a specific receptor. Both Mycobacterium avium and Mycobacterium tuberculosis cause lung infections and localized or disseminated disease in both patients without AIDS and those with AIDS. Histopathologic studies show necrosis in the lung, liver, and splenic tissues of patients with disseminated mycobacterial infection. In the course of experiments to examine the effect of growth factors on macrophages, it was observed that M. avium and M. tuberculosis but not Mycobacterium smegmatis cultured in the presence of 5, 50, or 500 ng of EGF per ml grew significantly faster than mycobacteria cultured in the absence of EGF. 125I-EGF was found to bind to M. avium and M. tuberculosis, and the binding was competitively inhibited by unlabeled EGF. A receptor for EGF was identified on mycobacteria. Incubation of mycobacteria with EGF prior to infection of macrophage monolayers resulted in faster bacterial growth within macrophages compared with that of mycobacteria not incubated with EGF. EGF-binding protein was cloned and expressed in Escherichia coli, and subsequently the protein was purified and the N-terminal amino acids were sequenced. These results suggest that EGF is a growth factor for pathogenic mycobacteria in granulomatous tissues and within macrophages and might enhance growth rates of both intracellular and extracellular mycobacteria in the site of infection.

Regulation of epidermal growth factor receptor synthesis by ovarian steroids in human endometrial cells in culture.

The aim of this study was to investigate the effect of oestradiol and progesterone on epidermal growth factor (EGF) binding in human endometrial glands and stromal cells in culture. Monolayers of isolated glands or stromal cells were cultured for 6 days in the presence or absence of steroids which were replenished daily. Binding of 125I-labelled EGF was measured in the presence and absence of unlabelled EGF. Over a 6 day period, oestradiol caused a dose-dependent increase in the number of EGF receptors in stromal cells, with a maximum effect of 150% control at a concentration of 10 nmol l-1. The effect of progesterone was also dose dependent and reached a maximum of 170% control at 100 nmol progesterone l-1. Oestradiol and progesterone in combination caused a greater increase in EGF receptor concentration than did either steroid alone (control, 100 +/- 11%; oestradiol, 144 +/- 11% control; progesterone, 200 +/- 20% control; oestradiol and progesterone, 288 +/- 6% control). Steroid treatment did not alter the affinity of the receptor for EGF. The stage of the cycle of the tissue did not affect the response to steroids. The effect of oestradiol was inhibited by the anti-oestrogen ICI182780, and that of progesterone by the anti-progestin RU486. In endometrial glands, neither oestradiol nor progesterone affected the number of EGF receptors, but the two steroids in combination induced an increase of 140 +/- 23% control where control was 100 +/- 15%. These data demonstrate that oestradiol and progesterone increase the number of EGF receptors in vitro, and suggest that EGF is involved in mediating the actions of these steroids on the processes of proliferation and differentiation in the human endometrium.

Identification of an EGF/TGF-alpha receptor in primary cultures of guinea pig gastric mucous epithelial cells.

Binding and localization of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) were assessed using in vitro primary cultures of guinea pig gastric mucous epithelial cells (GMEC). GMEC were isolated and cultured in six-well plates with Dulbecco's modified Eagle's medium + 10% serum and then changed to serum-free medium for 24 h for binding studies. The binding time course of 125I-labeled EGF and 125I-TGF-alpha in GMEC cultures at 4 degrees C was saturable, reaching a plateau within 4-6 h. Competition-binding curves revealed that the amount of unlabeled EGF and TGF-alpha to reduce 125I-EGF binding by 50% was 0.35 and 0.23 nM, respectively. The amount of unlabeled EGF and TGF-alpha to decrease 125I-TGF-alpha binding by 50% was 0.30 and 0.21 nM, respectively. A Scatchard analysis of the data disclosed that a single class of high-affinity binding sites (dissociation constant = 0.24 nM) was present. The maximal binding capacity was approximately 20 fmol/10(6) cells or approximately 12,000 receptors per cell. The binding of 125I-EGF and 125I-TFG-alpha to GMEC cultures was maximal between pH 7.0 and 8.5. No specific binding of EGF or TGF-alpha could be detected below pH 5.0. The half-maximal pH dissociation value for EGF and TGF-alpha was pH 5.89 and pH 6.83, respectively. We found no difference in the final amounts of membrane-bound or internalized 125I-EGF and 125I-TGF-alpha. However, there was a significant difference (P < 0.05) at 5-30 min in the rate of dissociated and internalized 125I-EGF- and 125I-TGF-alpha. Immunofluorescence microscopy of GMEC cultures for EGF/TGF-alpha receptors showed increased fluorescence at the leading edges and around the perimeter of cells. Detection of an EGF/TGF-alpha receptor was also confirmed by Western blotting. Our findings demonstrate that guinea pig GMEC possess a specific EGF/TGF-alpha receptor, which further supports a physiological role for EFG and TFG-alpha as mitogens in these cells.

Binding of epidermal growth factor and transforming growth factor-α in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.