Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

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.     

Signaling within a caveolae-like membrane microdomain in human neuroblastoma cells in response to fibroblast growth factor

It is now clear that the plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these microcompartments are now recognized to be sites of localized signal transduction for several extracellular stimuli. At least two different types of microdomains can be identified, largely based on the presence or absence of the caveolin proteins. The generic name of caveolae-like domains is commonly used to refer to both domains indistinguishably. Although caveolin proteins were long thought to be absent from the brain, we have shown that the human neuroblastoma cell line LAN-1 expresses both caveolin-1 and caveolin-2. Basic fibroblast growth factor (FGF)-2 induced a specific signaling response within the caveolae-like domain of LAN-1 cells, characterized by the tyrosine phosphorylation of a 75-80-kDa protein. This protein present in the caveolae-like domains has properties suggesting that it is a member of the SNT family of adapter proteins. The signaling event originating in the caveolae-like domains in response to FGF-2 appeared to require the activation of at least Fyn and Lyn, two members of the Src family of tyrosine kinases. This work suggests that compartmentalized signaling within caveolae-like domains may create a level of specificity for certain growth factors such as FGF.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.     

Cloning,expression and purification of human epidermal growth factor using different expression systems

Epidermal growth factor (EGF) is a protein that belongs to the family of growth factors that bind the ErbB receptors, which play a prominent role in the development of carcinomas. We had demonstrated that potato carboxypeptidase inhibitor (PCI) acts as an EGF antagonist. Because of the low affinity of PCI for the epidermal growth factor receptor, it was decided to design EGF mutants with PCI abilities. In order to achieve this we have first cloned, expressed and purified the native protein, EGF. Different expression systems with different locations of the recombinant protein were designed and a purification protocol was designed with those which allowed expression of EGF. Finally, the sample needed folding. Differences in the amount of EGF obtained and its activity were observed depending on the expression system used.     

Expression of porcine epidermal growth factor in Pichia pastoris and its biology activity in early-weaned piglets

Early-weaned piglets often have abnormalities in intestinal morphology and function. Epidermal growth factor (EGF) is critical in the development and in the repair of the gastrointestinal tract in pigs. This study investigated the effects of dietary EGF supplementation on growth performance and small intestinal morphology of early-weaned piglets. The functional domain of porcine EGF (pEGF) was cloned after RT-PCR amplification. The recombinant protein was expression by the Pichia pastoris expression system and the construct pPIC9K-pEGF was transformed into host GS115. The secretary recombinant protein in the supernatants was analyzed by SDS-PAGE. The gel indicated that the extra band at 6 kDa in the transformant, which corresponds to the standard hEGF, were both reactive to anti-pEGF antibody by Western blotting. The expression level of pEGF in the culture supernatant was 870 microg/mL. An animal feeding test was conducted to identify the effects of pEGF supplementation on growth performance and the development of digestive tracts of 14-day weaned piglets. The dietary treatment was a corn-soybean meal basal diet either with or without 1.5 mg/kg recombinant pEGF from the transformant fermentative supernatant. Dietary treatments enhanced the daily gain during 0-7 days postweaning (p < 0.05), but did not affect the performance throughout the entire test period. Dietary supplemental pEGF significantly increased serum IgA levels on day 18 postweaning, and increased the mucosa IgA levels and crypt depth at jejunum on day 28 postweaning (p < 0.05). The experimental results showed that the recombinant pEGF could be secreted by P. pastoris. The trophic effects of pEGF on growth performance, immune response, and small intestine development were determined by feeding recombinant pEGF to early-weaned piglets.     

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of ¹²⁵I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two type of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

A quinazoline-derivative DOTA-type gallium(III) complex for targeting epidermal growth factor receptors: synthesis, characterisation and biological studies

The novel DOTA-like chelator 1,4,7,10-tetraazacyclododecane-1-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}propionamide-4,7,10-triacetic acid (H₃L) was synthesised by alkylation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate) with N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-bromopropionamide, followed by hydrolysis of the ester groups with trifluoracetic acid. H₃L has been fully characterised by multinuclear NMR spectroscopy, mass spectrometry and high-performance liquid chromatography (HPLC). Five protonation constants, log K _Hi , of H₃L were determined by potentiometry and UV–vis spectrophotometry and the values found are 10.47, 9.18, 5.24, 4.00 and 2.23. These methods, complemented with variable-pH ⁷¹Ga NMR studies, allowed us to ascertain the stability constant of the Ga(III) complex of L. GaL has a remarkably high thermodynamic stability constant (log K _ML = 24.5). The radioactive complex ⁶⁷GaL was prepared in high yield and high radiochemical purity. Its HPLC chromatogram is identical to that obtained for the GaL complex prepared at the macroscopic level. At pH 7.4, ⁶⁷GaL has an overall neutral charge, is highly hydrophilic (log D = −1.02 ± 0.03) and presents high in vitro stability in physiological media and in the presence of an excess of diethylenetriaminepentaethanoic acid . In vitro studies indicated that H₃L and GaL do not inhibit the cell growth of epidermal growth factor receptor expressing cell lines, such as A431 cervical carcinoma cells, a result which agrees with the very low cell internalisation found for ⁶⁷GaL in the same cell line. Biodistribution studies in mice indicated high in vivo stability for ⁶⁷GaL, a high total excretion rate and a relatively slow blood clearance, in full accordance with its hydrophilic character and the relatively important protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

肺腺癌细胞中EGFR蛋白的表达与细胞内胶原化的相关性研究

目的探讨表皮生长因子受体（epidermal growth factor receptor,EGFR）在肺腺癌细胞中的表达及与细胞发生胶原化的相关性。方法从胸水中提取肺腺癌细胞为研究对象,以32例良性胸水中的增生上皮细胞、炎性细胞为对照,采用免疫细胞化学方法检测细胞中EGFR、E钙粘素蛋白、Vimentin、TTF-1和胶原蛋白亚型I的表达。Masson染色方法检测胶原纤维表达。结果78例胸水标本中,EGFR在肺腺癌细胞中的阳性率为79．5％,胶原蛋白亚型I为32．1％,Masson染色的阳性率为70．5％,明显高于对照组且EGFR和Masson染色的阳性表达结果的相关性具有统计学意义（P〈0．01）。结论EG—FR在肺腺癌细胞中阳性表达,可能与细胞内基质胶原蛋白形成有关。     

Identification and expression of epidermal growth factor gene in mouse testis

INTRODUCTIONEpidermalgrowthfactor(EGF)wasinitiallyisolatedandpurifiedfromthesubmaxillarygland(SMG)ofmalemouse[1].Itisapolypeptidecomposedof53aminoacidresidues[2].Itinfluencescellproliferationanddifferentiationandmodulatesthegrowthanddevelopmentofmammalianorgans[3--7].AnoteworthyfindingisthatextirpationofmouseSMGresultsinamarkedreductionofserumEGFconcentrationassociatedwithanimpairedspermatogenesis[3].ThisfindingsuggeststhatEGFmayregulatespermproductionanddifferentiation.Inhumantest…     

Dexamethasone acts as a negative regulator of epidermal growth factor receptor synthesis in fetal rat lung cells

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung cells is decreased by approximately 50% following 24-h dexamethasone treatment. Ligand binding assays identified an average of 30,000 receptors per cell in untreated FRL cells, while analysis of dexamethasone treated cells showed a decrease to about 16,000 receptors per cell. No substantial changes in receptor affinities were detected. Immunoprecipitation of 35S-methionine-labeled EGF receptor protein demonstrated a 50% decrease in total EGF receptor protein after 24-h dexamethasone treatment. Brief pulse labeling with 35S-methionine showed that the reduction in total EGF receptor protein content was due to a decrease in EGF receptor synthesis. Receptor synthesis declined about 25% after 1 h of dexamethasone treatment and at 3 h, EGF receptor synthesis was maximally decreased to nearly 50% that of cells not exposed to dexamethasone. Dexamethasone treatment was also effective in reducing EGF receptor synthesis in cells pretreated with retinoic acid, an agent which enhances receptor synthesis. These data are the first to document a dexamethasone-induced decrease in EGF receptor synthesis. Furthermore, these findings may provide a plausible mechanism by which dexamethasone could regulate EGF responsiveness.     

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 μM) (p < 0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p < 0.01). However, treatment with GM3 (1 μM) resulted in decreased ALP activation (p < 0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.     

Nordihydroguaiaretic acid inhibits insulin-like growth factor signaling, growth, and survival in human neuroblastoma cells

Neuroblastoma is a common pediatric malignancy that metastasizes to the liver, bone, and other organs. Children with metastatic disease have a less than 50% chance of survival with current treatments. Insulin-like growth factors (IGFs) stimulate neuroblastoma growth, survival, and motility, and are expressed by neuroblastoma cells and the tissues they invade. Thus, therapies that disrupt the effects of IGFs on neuroblastoma tumorigenesis may slow disease progression. We show that NVP-AEW541, a specific inhibitor of the IGF-I receptor (IGF-IR), potently inhibits neuroblastoma growth in vitro. Nordihydroguaiaretic acid (NDGA), a phenolic compound isolated from the creosote bush (Larrea divaricata), has anti-tumor properties against a number of malignancies, has been shown to inhibit the phosphorylation and activation of the IGF-IR in breast cancer cells, and is currently in Phase I trials for prostate cancer. In the present study in neuroblastoma, NDGA inhibits IGF-I-mediated activation of the IGF-IR and disrupts activation of ERK and Akt signaling pathways induced by IGF-I. NDGA inhibits growth of neuroblastoma cells and induces apoptosis at higher doses, causing IGF-I-resistant activation of caspase-3 and a large increase in the fraction of sub-G0 cells. In addition, NDGA inhibits the growth of xenografted human neuroblastoma tumors in nude mice. These results indicate that NDGA may be useful in the treatment of neuroblastoma and may function in part via disruption of IGF-IR signaling.