125I-labeled Epidermal Growth Factor Research Articles

Blocking EGFR in the liver improves the tumor-to-liver uptake ratio of radiolabeled EGF

Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumor-bearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of (125)I-labeled EGF. The anti-EGFR Affibody molecule (Z(EGFR:955))(2) was then assessed as a blocking agent of (111)In-labeled EGF in a dual isotope study (50, 100, and 200 microg, preadministered 30 or 60 min before (111)In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased (125)I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50 microg (Z(EGFR:955))(2) as a blocking agent 30 min before the (111)In-EGF decreased the uptake of (111)In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (Z(EGFR:955))(2) shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFR-expressing tumor diagnostics and imaging.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Phospholipase C-ϵ Augments Epidermal Growth Factor-dependent Cell Growth by Inhibiting Epidermal Growth Factor Receptor Down-regulation

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

Controlled-release of epidermal growth factor from cationized gelatin hydrogel enhances corneal epithelial wound healing

We designed a new ophthalmic drug-delivery system for epidermal growth factor (EGF) from the biodegradable hydrogel of cationized gelatin. We placed a cationized gelatin hydrogel (CGH) with incorporated 125I-labelled EGF in the conjunctival sac of mice and measured the residual radioactivity at different times to evaluate the in vivo profile of EGF release. Approximately 60–67% and 10–12% of EGF applied initially remained 1 and 7 days after application, respectively; whereas EGF delivered in topically applied solution or via EGF impregnation of soft contact lenses disappeared within the first day. We also placed CGH films with 5.0 μg of incorporated EGF on round corneal defects in rabbits to evaluate the healing process using image analysis software and to assess epithelial proliferation immunohistochemically by counting the number of Ki67-positive cells. The application of a CGH film with incorporated EGF resulted in a reduction in the epithelial defect in rabbit corneas accompanied by significantly enhanced epithelial proliferation compared with the reduction seen after the topical application of EGF solution or the placement of an EGF-free CGH film. The controlled release of EGF from a CGH placed over a corneal epithelial defect accelerated ocular surface wound healing.

Characterization of EGF coupling to aminated silicone rubber surfaces

Tethering of growth factors to biomaterial substrates via a polyethylene glycol (PEG) spacer has been established as a means of controlling dosage and conformation of the protein at the material surface, while retaining biological activity. However, the extent of modification through a comparison of bound versus unbound protein has not generally been characterized. In this work, covalent tethering of epidermal growth factor (EGF) to allylamine plasma modified polydimethylsiloxane (PDMS) substrates is characterized to determine the nature of the bound growth factor and to optimize the conditions for the reaction. Tethering is achieved via conjugation of EGF with homobifunctional N-hydroxysuccinimide (NHS) ester of PEG-butanoic acid (SBA2-PEG) in solution, followed by exposure of the pegylated EGF to the aminated surfaces (solution first reaction). SDS-PAGE analysis indicates that a low ratio of EGF:PEG is required to maximize the yield of the EGF-PEG reaction; a relatively short reaction time is needed to limit hydrolysis of the NHS ester. With increasing amounts of PEG and a higher reaction time, a higher fraction of the EGF can be covalently tethered to the surfaces, as shown by binding of 125I-labeled EGF and subsequent washing with sodium dodecyl sulfate (SDS) to remove adsorbed protein. However, even under the optimal reaction conditions established by the SDS-PAGE analysis, higher molecular weight EGF-PEG complexes are observed by SDS-PAGE and matrix-assisted laser desorption/ionization (MALDI). The presence of these complexes, as well as unreacted growth factor, can lead to a surface of heterogeneous composition. While these surfaces were found to have biological activity, stimulating the adhesion and growth of corneal epithelial cells versus PDMS controls, further optimization of reaction conditions, including the use of a homobifunctional PEG linker and possibly separation of reaction species are required to achieve a uniformly active and well-defined biomaterial surface.

Chimeric receptor analyses of the interactions of the ectodomains of ErbB‐1 with epidermal growth factor and of those of ErbB‐4 with neuregulin

A series of chimeric receptors was generated between the epidermal growth factor (EGF) receptor, ErbB-1, and its homologue, ErbB-4, to investigate the roles of the extracellular domains (I-IV) in the ligand specificities. As compared with ErbB-1 and the chimeras with both domains I and III of ErbB-1, the chimeras with only one of these domains exhibited reduced binding of 125I-labeled EGF. Particularly, the contribution of domain III was appreciably larger than that of domain I of ErbB-1 in 125I-labeled EGF binding. Nevertheless, the chimeras with domain III of ErbB-1 and domain I of ErbB-4 were prevented from binding to 125I-labeled EGF competitively by the ErbB-4 ligand, neuregulin (NRG). On the other hand, NRG did not compete with 125I-labeled EGF for binding to the chimeras with the ErbB-1 domain I and the ErbB-4 domain III. Therefore, NRG binding to ErbB-4 depends much more on domain I than on domain III. With respect to autophosphorylation and subsequent ERK activation, EGF activated the chimeras with either domain I or III of ErbB-1. In contrast, NRG activated the chimeras with the ErbB-4 domain I and the ErbB-1 domain III, but not those with the ErbB-1 domain I and the ErbB-4 domain III. Therefore, the relative contributions between domains I and III of ErbB-4 in the NRG signaling are different from those of ErbB-1 in the EGF signaling.

Apical and basolateral EGF receptors regulate gastric mucosal paracellular permeability.

Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.

Ganglioside Modulates Ligand Binding to the Epidermal Growth Factor Receptor

Whereas previous investigations have shown that pharmacologic addition of gangliosides inhibits keratinocyte proliferation by downregulating epidermal growth factor receptor phosphorylation, the underlying biochemical basis and physiologic relevance are unknown. Using Scatchard and displacement plots, we have shown that supplemental purified gangliosides decrease the binding of (125)I-labeled epidermal growth factor to keratinocyte-derived SCC12 cells. Conversely, SCC12 cells transfected with sialidase and thus depleted of gangliosides show increased ligand binding to the epidermal growth factor receptor, which is consistent with their increased proliferation in response to epidermal growth factor and transforming growth factor-alpha, and increased phosphorylation of the epidermal growth factor receptor, and downstream signal transduction pathway components. The mechanism of the altered binding appears to involve primarily decreased numbers of available receptors within the intact membrane, but not altered receptor protein expression. These studies provide evidence that the effect of gangliosides on keratinocyte proliferation results, at least in part, from the direct binding of ganglioside to the receptor and disruption of the receptor-ligand interaction. Manipulation of membrane ganglioside content may be a powerful new means to alter epidermal growth factor receptor-dependent cell proliferation.

Zonal differences in effects of HGF/SF and EGF on DNA synthesis in hepatocytes under fed or starved conditions.

Zonal differences of DNA synthesis in hepatocytes induced by hepatocyte growth factor and/or scatter factor (HGF/SF) and epidermal growth factor (EGF) were investigated using male Wistar rats under fed or starved conditions. Overall, DNA synthesis was greater in fed rats than in starved rats. The predominance of EGF in periportal hepatocytes (PPH) on zonal DNA synthesis was reversed by starved conditions, but the predominance of HGF/SF on zonal DNA synthesis in perivenous hepatocytes (PVH) was not influenced by nutritional conditions. 125I-labeled EGF and 125I-labeled HGF/SF-receptor binding studies revealed no significant difference between PPH and PVH in starved or fed rats. To investigate the mechanism of the signal transduction pathway, we used genistein, an inhibitor of tyrosine kinase. Genistein had different effects on zonal difference in EGF and HGF/SF. In EGF, 1 microgram/ml genistein abolished zonal differences, but in HGF/SF 1 microgram/ml genistein did not abolish zonal differences. These data suggest that, in contrast to HGF/SF, zonal difference of DNA synthesis by EGF was dependent on nutritional conditions and DNA synthesis induced by HGF/SF and EGF might be related to tyrosine kinase, but the influence of tyrosine kinase on DNA synthesis was different between HGF/SF and EGF.

An Enhancement of Uterine Epidermal Growth Factor Receptor Associated with Embryo Implantation in Rats.

The present study was designed to investigate the physiological significance of epidermal growth factor (EGF) receptors expressed on rat uterine epithelial cells during embryo implantation. The number of uterine EGF receptor sites, as determined with 125I-labelled EGFas a ligand, progressively increased from the 3rd to the 7th day of pregnancy. Analysis of the EGF receptor by Scatchard transformation of EGF-binding data and affinity labelling with 125I-labelled EGF indicated that EGF receptor proteins were noticeably expressed during postimplantation. Determination for EGF receptor sites was further carried out in the uterine tissues of rats, which were superovulating after transplantation of pituitary tissue into a capsule of kidney or subcutaneous injection of 50 IU pregnant mare serum gonadotropin (PMSG) and 50 IU human chorionic gonadotropin (hCG). The transplantation caused an increase in the number of implantation sites (24.5 ± 3.9, n=4) as compared to control rats (14.0 ± 0.6, n=7). Nevertheless, PMSG- and hCG-primed rats totally failed to initiate the implantation process, whereas the number of EGF receptor sites significantly increased during preimplantation (3rd day). In pituitary-transplanted rats on the 7th day, the number of EGF receptor sites was increased as observed in control rats (30.0 ± 0.5 fmol/mg proteins). In PMSG-and hCG-primed rats, however, the EGF receptor was noticeably reduced (7.3 ± 0.4 fmol/mg proteins). These results strongly suggest that functional EGF receptor proteins were noticeably expressed during postimplantation and that its drastic enhancement depends upon the interaction of blastocysts with uterine epithelial cells.

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.

Humanization of a mouse monoclonal antibody that blocks the epidermal growth factor receptor: recovery of antagonistic activity

Background: Antibody humanization by transplanting the complementarity determining regions (CDRs) of a murine antibody to a human framework aims to reduce the response of the human immune system against a foreign molecule. Frequently, however, some murine amino acids from the framework have to be retained to recover binding affinity. Objectives: To redesign R3, a mouse monoclonal antibody (mAb) that binds the human epidermal growth factor (EGF)-receptor and inhibits the binding of EGF, to be a human IgGl. Study design: The light and heavy chains of REI and Eu, respectively, were selected as human immunoglobulin (lg) frameworks for CDR-grafting based on their high homology with the corresponding sequences of murine R3. Molecular modeling was used to analyze the possible effects of mutating murine residues that underlie the CDRs. Results: CDR-grafting dramatically reduced the binding capability of the antibody. Molecular modeling suggested that two amino acids (Thr 76 and Thr 93), among five immunoglobulin heavy chain variable region (VH) residues underlying the CDRs, were critical for antigen binding. The five residues were mutated back to the original murine amino acids in different combinations contained in six variants of humanized antibodies. In agreement with molecular modeling analysis, the variant in which three murine residues were retained (Ser 75, Thr 76 and Thr 93) exhibited a similar capacity to inhibit the binding of 125I-labeled EGF to its receptor as compared with the original antibody. This humanized antibody was at least 2-fold less immunogenic in African Green monkeys than the chimeric antibody. Conclusions: Only very few mutations in the frameworks may be necessary to recover the binding capability of a humanized antibody. Molecular modeling can serve as a powerful tool to identify residues critical for binding.

同一患者から樹立した３種の口腔癌細胞株とその性状

In the present study, we used a modified primary culture method to establish human cancer cell lines derived from the primary lesion and lymphnode metastases in a patient with oral cancer.Three novel cell lines were established: MO-T from a primary squamous cell carcinoma of the tongue, and MO-N 1 and MO-N 2 from neck lymphnode metastases. These cell lines have been cultured for more than 1 year and are in good condition. The population doubling time of MO-T, MO-N 1, and MO-N 2 are approximately 48.0, 46.5, and 36.0 hours, respectively. The cell lines were implanted in nude mice, and the resulting tumors histopathologically resembled the original lesions. Epidermal growth factor (EGF) binding assay was performed with the use of 125I-labeled human EGF, and the number of cell surface receptors was estimated by Scatchard plot. The number of epidermal growth factor receptors (EGFR) of MOT, MO-N 1, and MO-N 2 was 7.80×105, 1.01×106, and 7.60×105 per cell, respectively. These cells expressed both high-and low-affinity receptors. We also investigated whether or not these cells carry an amplified gene for EGFR by Southern blot analysis using its complimentary DNA, pE 7 insert, as a probe. None of the cell lines shbwed distinct evidence of an amplified gene for EGFR. Moreover, we examined mRNA expression of EGFR by Northern blot analysis. The mRNA levels differed among the three cell lines.These findings suggest that the three cell lines derived from a single patient are useful for studying the biological features and the mechanism of invasion and metastasis of oral cancer.

Saturation of the Endocytic Pathway for the Transferrin Receptor Does Not Affect the Endocytosis of the Epidermal Growth Factor Receptor

Cell-surface receptors that undergo clathrin-mediated endocytosis contain short amino acid sequences in their cytoplasmic domain that serve as internalization signals. Interactions between these sequences and components of the endocytic machinery should become limiting upon overexpression of the constitutively recycling transferrin receptor (TfR). A tetracycline-responsive system was used to induce overexpression of the TfR up to 20-fold in HeLa cells. Internalization assays indicate the rate of 125I-transferrin uptake per surface TfR is reduced by a factor of 4 in induced cells. Consistent with endocytosis being the rate-limiting step, TfRs shift from an endosomal to more of a plasma membrane distribution with TfR overexpression. The clathrin-associated protein AP-2 has been proposed to interact directly with the cytoplasmic domain of many receptors, yet no changes in the amount or distribution of AP-2 were detected in induced cells. The internalization rate for the epidermal growth factor receptor was also measured, with or without induction of TfR expression. Even though endocytosis of the TfR is saturated in induced cells, 125I-labeled epidermal growth factor continues to be internalized at a rate identical to that seen in uninduced cells. We propose that there are different limiting steps for the endocytosis of these two receptors.

An enhanced and sensitive autocrine stimulation by transforming growth factor-alpha is acquired in the brain metastatic variant of a human non-small-cell lung cancer cell line.

Transforming growth factor-alpha (TGF-alpha)-mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line.

High molecular mass forms of epidermal growth factor in pig uterine secretions.

Accumulating evidence suggests that uterine luminal fluids contain a variety of polypeptide growth factors and cytokines that, it is speculated, have roles in the development, growth and differentiation of the uterus and, during pregnancy, in the growth and survival of the embryo. Although epidermal growth factor (EGF) has previously been identified by radioimmunoassay and immunohistochemistry in the pig uterus, there have been no detailed studies of the secreted EGF protein. EGF was therefore purified from uterine flushings and uterine fluids of nonpregnant pigs of mixed breed using a variety of ion-exchange chromatography steps. Uterine flushings and fluids contained an anionic factor(s) that at 4 degrees C competed with 125I-labelled mouse EGF for binding to EGF receptors on an endometrial carcinoma cell line and stimulated DNA synthesis in Balb/c mouse 3T3 fibroblasts. As analysed by gel filtration, uterine fluids contained a 3-6 kDa factor that stimulated 3T3 cell DNA synthesis and was a competitor of cellular 125I-labelled EGF binding. Gel filtration further revealed that uterine flushings and fluids contained, respectively, 45 kDa and 40-70 kDa moieties that were mitogenic and that bound to the EGF receptor. SDS-PAGE and western blotting using an antiserum specific for pig EGF revealed immunoreactive forms of EGF of approximately 25 kDa in partially purified uterine flushings. It is concluded that uterine secretory EGF occurs, at least in part, as high molecular mass proteins. The ability of these high molecular mass EGFs to bind to and activate the EGF receptor suggests that they may be authentic ligands for the EGF receptor in utero.

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.

Nitric oxide: a modulator for the epidermal growth factor receptor expression in developing ovarian granulosa cells.

the present study was designed to assess the involvement of nitric oxide (NO) in the regulation of the epidermal growth factor (EGF) receptor during development of rat granulosa cells, which were prepared by puncturing ovaries of diethylstilbestrol-treated rats. The immature cells were cultured for 48 h with follicle-stimulating hormone (FSH) to be transformed into mature cells. A marked accumulation of guanosine 3',5' -cyclic monophosphate (cGMP) was observed during development. The accumulation of cGMP, but not of adenosine 3',5'-cyclic monophosphate (cAMP), was suppressed by specific inhibitors of NO synthase, L-NG-monomethyl-L-arginine (L-NMMA) and L-N(G)-nitro-L-arginine, and a selective inhibitor of the inducible NO synthase, aminoguanidine. The L-NMMA-induced suppression was partially reversed by addition of L-arginine to cultures but not D-arginine, indicating that NO formation is inhibited by competing with analogues of L-arginine for NO synthase. Treatment with 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide , an antagonist of NO, caused suppression in the increase of EGF binding sites, whereas exposure of the cells to sodium nitroprusside (SNP), an NO donor, caused elevation in EGF binding sites, with increasing extra- and intracellular cGMP levels. Analysis of the EGF receptor by affinity labeling with 125I-labeled EGF revealed that the intensity of the cross-linked receptor molecular with a molecular mass of 180 kDa was increased by exposure to SNP. The facilitatory effect of SNP on the EGF receptor was observed when the cAMP-dependent pathway was fully activated by FSH. However, the NO effect may be mediated by a cGMP-independent pathway, as 8-bromo-cGMP did not mimic the action of SNP. These results indicate that the L-arginine-NO system may contribute to the regulation of EGF receptor expression in developing granulosa cells stimulated by FSH.

Production of Heparin-Binding Epidermal Growth Factor–like Growth Factor (HB-EGF) at Sites of Thermal Injury in Pediatric Patients

Fluids that accumulate at wound sites may be an important reservoir of growth factors that promote the normal wound healing response. The presence of heparin-binding growth factors was studied in burn wound fluid (BWF) from 45 pediatric patients who had sustained partial thickness burns. One of the growth factors present was similar to platelet-derived growth factor (PDGF) based on its heparin affinity, inhibition of bioactivity by a PDGF antiserum, and detection in a PDGF-AB enzyme-linked immunosorbent assay. A second growth factor was identified as heparin-binding epidermal growth factor-like growth factor (HB-EGF) based on its heparin affinity, competition with 125I-labeled epidermal growth factor (EGF) for EGF receptor binding, and recognition in biological assays and Western blots by two HB-EGF antisera. Amino acid sequence analysis of one form of this second growth factor verified its identity as an N-terminally truncated form of HB-EGF. Immunohistochemical analysis of partial thickness burns demonstrated the presence of HB-EGF in the advancing epithelial margin, islands of regenerating epithelium within the burn wound, and in the duct and proximal tubules of eccrine sweat glands. HB-EGF in the surface epithelium of burn wounds was uniformally distributed, whereas it was restricted to the basal epithelium in nonburned skin. These data support a role for PDGF and HB-EGF in burn wound healing and suggest that the response to injury includes deposition of HB-EGF and PDGF into blister fluid and a redistribution of HB-EGF in the surface epithelium near the wound site.