Investigating Epidermal Growth Factor Receptor Trafficking with High-Resolution Enzymatic Protein-Tagging and Transmission Electron Microscopy.

Enzymatic ascorbate peroxidase (APEX) tagging allows for high-resolution, three-dimensional protein distribution analyses in cells and tissues. This chapter describes the application of APEX-tagging to visualize the trafficking of the epidermal growth factor receptor (EGFR) during epidermal growth factor-mediated receptor activation. Here, we describe the preparation of cells, methods to validate the stimulation of the EGFR, and visualization of the APEX-resolved distribution of the EGFR in the transmission electron microscope.

Synthesis and biological evaluation of EGFR binding peptides for near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Crosstalk between KDEL receptor and EGF receptor mediates cell proliferation and migration via STAT3 signaling.

Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands.

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants-R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low-affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called "high"-affinity biological responses arise from activation by isolated receptor dimers, whereas "low"-affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Epidermal growth factor receptors, testosterone levels and parotid gland changes in rats infected with Trypanosoma cruzi

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Signaling pathway in human airway mucous hypersecretion / 基础医学与临床

Objective To explore the upstream signal transduction pathway of neutrophil elastase(NE)-induced mucin(MUC)5AC gene expression.Methods A549 cells were either incubated with NE alone or with DMTU,aprotinin or AG1478.The level of MUC5AC mRNA was measured with RT-PCR.The activation of epidermal growth factor receptor(EGFR) and signaling were assessed by measuring the release of epidermal growth factor(EGF) and the phosphorylation of EGFR.Results NE increased MUC5AC gene expression accompanied by an increase of EGF and phosphorylated EGFR.DMTU,aprotinin and AG1478 significantly inhibited MUC5AC gene expression.DMTU and aprotinin significantly decreased the level of EGF and phosphorylated EGFR.ConclusionNE can induce MUC5AC gene expression via EGFR signalling in A549 cells.The upper stream involves oxidants,activation of tissue kallikrein and EGF.

Epidermal Growth Factor Receptor and c-erbB-2 Expressions in Biliary Tract Cancers

PURPOSE: The expressions of epidermal growth factor receptor (EGFR) and c-erbB-2 have been considered to be implicated in the genesis and progression of cholangiocarcinomas. However, their clinical roles and pathological characteristics remain uninvestigated. The purpose of this study was to assess the expressions of EGFR and c-erbB-2, and to identify their clinical and pathological significances in biliary tract cancers. METHODS: One hundred and fourteen samples were obtained from surgically resected biliary tract cancers (72 extrahepatic bile duct cancers, 33 gallbladder cancers, and 9 intrahepatic bile duct cancers). Expressions of EGFR and c-erbB-2 were examined by immunohistochemical staining. Expression rates were analyzed according to the location of the tumor, histologic differentiation, depth of invasion, lymph node metastasis, lymphovascular invasion, recurrence, and survival rate. RESULTS: The expression rate of EGFR was 10.7% in biliary tract cancers. EGFR expression was more often observed in moderately- or poorly-differentiated carcinomas than in well-differentiated carcinomas (p=0.0252). No correlations were observed with age, gender, location of tumor, depth of invasion, lymph node metastasis, lymphovascular invasion, recurrence rate, or survival rate. c-erbB-2 was expressed in 4.5% of biliary tract cancers. c-erbB-2 expression had no significant relationships with clinical and pathological prognostic factors. CONCLUSION: EGFR expression can be used restrictively as a prognostic indicator of biliary tract cancers. c-erbB-2 expression in biliary tract cancers is very low, and does not show any prognostic significance.

ANTIPROLIFERATIVE ACTIVITY OF HUMAN IFN-γ-EGF3 FUSION PROTEIN ARE RELATED TO ITS EGF RECEPTOR COMPETITION / 药物分析学报

The relationship between antiproliferative effect of human IFN-γ-EGF3 fusion protein and the influence of EGF receptor binding activity has been studied on A431 cell line. Antiproliferative activity of human IFN-γ-EGF3 was higher than that of its parent IFN-γ. In the 125 I-EGF receptor competition experiment, the inhibition of EGF receptor binding capacity on the target cells was observed in the treatments of human IFN-γ or IFN-γ-EGF3, but the later was more significant. Our data suggests that the antiproliferative effects by IFN-γ and its fusion protein are closely related to their EGF receptor competitions.

Expression,Purification of Fusion Protein TGF?-PE40 and the Cytotoxicity of TGF?-PE40 on Tumor Cells / 中国肿瘤生物治疗杂志

Objective: To express and purify transforming growth factor ?(TGF?)-pseudomonas exotoxin 40 and investigate its cytotoxic effect on cancer cells overexpressing epidermal growth factor (EGF) receptor. Methods: Recombi nant plasmid pV28 was constructed by inserting the gene coding TGF?-PE40 into the vector pET28a Expression of fusion protein was conducted using the host BL21. Production of the recombinant protein was induced by IPTG, following extraction and purification of inclusion bodies with His-tag purification system. Cell viability assay (by MTT) was performed to determine the cytotoxic effect of TGF?-PE40 on cancer cells ( A431 and SK-OV3). Results: Recombinant plasmid pV28, which expresses TGF?-PE40, was constructed successfully. Purity of TGF?-PE40 was about 98% after a purification procedure using His-tag column. Cytotoxic experiment showed that at a concentration of 0. 86?0. 07 UUUUg/ml, TGF?-PE40 could reduce 50% viability of A431, which has high expression of EGFR. Whereas the IC50 for ovarian cancer cell SK OV3, which expresses less EGFR, was 6.37?2.18 ?g/ml. There was a significant difference between these two groups (P

An Immunohistochemical Study of Molluscum Contagiosum / 대한피부과학회지

BACKGROUND: Molluscum contagiosum is a common viral infect oudisease of the skin and mucous membrane that is caused by a molluscum contagiosum virus(MCV; which belongs to the poxviridae family. One of the characteristic histopathologic findings is an epidermal hyperplasia Porter and Archard reported that this phenomenon might be explained by a virus induced epidermal growth factor (EGF) like polypeptide. There was a report that epidermal prolifeation in viral infection might be modulated by other factors than the virus itself such as local immune response. OBJECTIVE: The purpose of this study was to examine the expression pattern of epidermal growth factor receptor and other immunocompetent cells by immunohistochemical stainings. METHOD : We performed iinmunoperoxidase staining on the 11 slaecmens of formalin-fixed, paraffin-embedded molluscum lesions and 15 specimens of snap frozen mollucum lesions with nine primary antibodies(EGFR, factor XIIIa, CDla, S-100 protein, MAC 387, HLA-IR, CD4, CDS, L26) RESULTS: EGF receptors were strongly expressed in lesional MCV ifect,ed keratinocytes. The number of CDla and factor XIIIa positive dermal dendritic cells were sigtly increased. In inflamed lesions, CD4 and HLA-DR expressions were increased in the dermis and per lesional epidermis. CONCLUSION: This study shows that 1) increased EGFR expression is of MCV infected keratinocytes may be related to the pathogenesis of epidermal hyperplasia. 2) helper T lyrnphocytes may operate in inflamed molluscum lesions.

In vitro Inhibitory Effect on Growth of Human Glioma Cells with p21 Gene Mediated by a Novel Gene Delivery System / 中国肿瘤生物治疗杂志

Objective: To investigate the effects of novel targeted non-viral vector in gene therapy of human glioma. Methods: The EGF-R targeting gene delivery system GE7 was constructed. Human Glioma cell line U251 was transfected in vitro with ?－gal as reportor gene and p21 as therapeutic gene using this gene delivery system. By means of the assay of ?-galactosidase staining, Western blotting, in situ end labeling apoptosis cells and DNA ladder, the transferring of exogenous genes and the apoptosis of the tumor cells were examined.Results: It was showed that gene transfer efficiency is over 80%. When transfected with p21 gene, the growth of cells was inhibited significantly, and the apoptosis was detected in the transfected cell by the methods of in situ end labeling and DNA ladder. Conclusion: The GE7 gene delivery system has the ability to transfer exogenous gene to tumor cells and the expression of the therapeutic gene can inhibit the growth of the cells.

Immunohistochemical Study of TGFalpha , EGF and EGF Receptor on the Epithelial Tumors of the Skin / 대한피부과학회지

BACKGROUND: Several reports have demonstrated that TGFalpha and EGF are mitogenic for keratinocytes. Whenther its expression on epithelial tumors is a marker of malignancy or signifies an important step in the development of neoplasia is poorly understood. EGF receptors are also present in normal epidermis and epithelial tumors but their physiological roles are not yet understood. OBJECTIVE: Our purpose was to examine the staining patterns of TGFalpha, EGF and EGF receptors on the npithelial tumors of the skin, and to investigate kinetics of expression of EGF receptors. METHODS: We performed immunoperoxidase staining(ABC technique) with monoclonal anti-TGFalpha antibody, polyclonal anti-EGF antibody and polyclonal anti-EGF receptor antibody on the formalinfixed, paraffin-embedded tissue specimens of benign, premalignant and malignant skin tumors. RESULTS: The density of the expression of TGFalpha and EGF was not correlated with the degree of the malignancy of the epithelial tumors and is neither constant in any kind of the tumors. However the infiltrative type of basal cell carcinoma(BCC) is stronger that its solid type on the expression of TGFalpha and EGF. All benign tumors demonstrated a diffuse pattern within tumor lobules. pression of TGFalpha and EGF. All benign tumors demonstrated a diffuse pattern within tumor lobules. Focal TGFalpha immunostaining was seen in three of 10 squamous cell carcinomas(SCC) and four of 10 BCCs. TGFalpha immunostaining was absent from the outermost one to two layers of tumor lobules of all keratoacanthomas. The specimens which increased the expression of TGFalpha and EGF tended to decrease the expression of EGF receptor. CONCLUSION: These data suggest that the density of immunohistochemical expression of TGFalpha and EGF may be not dependent on the differentiation of tumor cells, and the pattern of immunohistochemical expression of TGFalpha can differentiate SCC from benign tumors such as keratoacanthoma. FGF receptor may be occupied by both of TGFalpha and EGF. With the receptors being occupied, a down regulation of the receptors may occur which results in decreased EGF receptor expression.

Effect of epidermal growth factor on growth of human renal cell carcinoma and transitional cell carcinoma cell lines / 대한비뇨기과학회지

We evaluated the relationship between content of epidermal growth factor receptor (EGFR) and responsiveness to epidermal growth factor (EGF) in human renal cell carcinoma (RCC) and transitional cell carcinoma (TCC) cell lines. EGFR was measured by radioisotope ligand binding assay using I-EGF. Three renal cell carcinoma cell lines (Caki-1. Caki-2, A-498) and 4 transitional cell carcinoma cell lines (T-24. J-82. HT-1197. HT-1376) were used. Growth of cell lines was tested in serum free RPMI 1640 media with or without various concentration of EGF by MTT assay. The content of EGFR was far higher in HT-1197than the other cell lines. EGFR contents were higher in RCC than in TCC cell lines except HT-1197. All RCC and TCC cell lines did not grow in serum free RPMI 1640 media. In EGF supplemented condition, HT-1197 and 3 RCC cell lines which showed high content of EGFR were stimulated to grow in a dose related fashion, but T-24. J-82 and HT-1376 cell lines which showed low content of EGFR were not. Growth rates of RCC and TCC cell lines were closely related with the content of EGFR under influence of EGF.