Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor- (TGF), epidermalgrowth factor (EGF), cripto-1, amphireg-ulin and epidermal growthfactor receptor (EGFR ) was studied in 51 premenopausal humanovaries at various phases of the menstrual cycle. Localizationof mRNA for TGF and EGF was also studied by in-situ hybridization.Immunoreactive TGF was observed predominantly in theca cellsin 12 of 33 antral follicles in the follicular phase (6/14 dominantfollicles, and 6/19 non-dominant) but not in any of the 18 folliclesin the luteal phase or in primordial and pre-antral follicles.TGF immunoreactivity was present predominantly in the luteinizedgranulosa cells in 13 of 15 corpora lutea in the luteal phase,which are considered to be active in steroidogenesis, but notin any of the regressed corpora lutea. Accumulation of TGF mRNAhybridization signal was observed only in the theca cells inthe follicles and luteinized theca cells in the ovaries thatwere immunohistochemically positive for TGF. EGFR immunoreactivitywas detected in 24 of 33 antral follicles in the follicularphase and in two of 18 follicles in the luteal phase but notin any of the corpora lutea. Immunoreactive EGF, cripto-1 andamphiregulin or EGF mRNA was not detected in any follicles,corpora lutea, or the stroma cells examined. These results indicatethat, of the epidermal growth factors examined in this study,TGF is locally synthesized in normal cycling human ovaries andTGF may be synthesized in theca cells and act on the granulosacells in a paracrine fashion through the EGFR in ovarian follicles. EGF family/human/immunohistochemistry/in-situ hybridization/ovary     

An enzyme immunoassay for determining epidermal growth factor (EGF) in human serum samples using an ultramicroanalytical system

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39–2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4–7.3% and 0–5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96–104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine^® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.     

Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn(2+)-induced EGFR activation. Exposure to Zn(2+) induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn(2+) on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn(2+)-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn(2+)-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn(2+)-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn(2+)-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn(2+) induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.     

A new model system identifies epidermal growth factor receptor–human epidermal growth factor receptor 2 (HER2) and HER2–human epidermal growth factor receptor 3 heterodimers as potent inducers of oesophageal epithelial cell invasion

Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas show distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homodimers or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion during early carcinogenesis, remain unknown. Here, a new cellular model system for controlled activation of epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) and EGFR–HER2 or HER2–human epidermal growth factor receptor 3 (HER3) homodimers and heterodimers was studied in non‐neoplastic squamous oesophageal epithelial Het‐1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA/DmrA and DmrC), and transduced into Het‐1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR–HER2 and HER2–HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct downstream signalling pathways, such as phospholipase Cγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB dimers caused cell rounding and non‐apoptotic blebbing, specifically in EGFR–HER2 and HER2–HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2 dimer cells as compared with empty vector cells. In addition, HER2 dimer cells showed in increased cell invasion, reaching significance for induced HER2–HER3 heterodimers (P = 0.015). Importantly, in three‐dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2 homodimer cells were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of cytokeratin 7 (when HER2 homodimers were modelled) and p63 (when EGFR–HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homodimers and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three‐dimensional microenvironment, thereby functionally identifying ErbB homodimers and heterodimers as important drivers of oesophageal carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The epidermal growth factor receptor in human pancreatic cancer.

The epidermal growth factor receptor (EGFR) and its ligands are thought to be important in the control of proliferation of many epithelial systems, including the exocrine pancreas. Abnormalities in expression of two of the known ligands of the EGFR, transforming growth factor alpha and epidermal growth factor, occur frequently in ductal adenocarcinoma of the human pancreas. We have examined an archival series of cases of pancreatic pathology for expression of the EGFR using the anti-EGFR antiserum 12E and found that there is almost ubiquitous overexpression of EGFR in pancreatic cancer and in chronic pancreatitis. Southern blot analysis showed no evidence of amplification or rearrangement of the EGFR gene. We conclude that an autocrine loop involving the EGFR system may be involved in the genesis of both neoplasia and reactive hyperplasia of pancreatic ductal epithelium.     

ERBB4/HER4在子痫前期胎盘中的表达

目的探讨子痫前期患者胎盘滋养细胞人表皮生长因子受体4(ERBB4/HER4)的表达及意义。方法采用RT-PCR法检测35例子痫前期患者(子痫前期组)胎盘ERBB4的表达及其与胎盘重量及新生儿出生体重的关系,并与20例正常妊娠妇女(正常妊娠组)比较。结果在子痫前期患者胎盘中,ERBB4 mRNA表达明显高于正常妊娠胎盘,两者有显著性差异(P<0.05)。结论子痫前期患者胎盘ERBB4表达显著增高,与子痫前期的发病密切相关。     

EGFR单克隆抗体抗结肠癌作用的实验研究

目的:观察表皮生长因子受体单克隆抗体(EGFRMcAb)对结肠癌的作用。方法:用不同剂量的EGFRMcAb处理LST174结肠癌细胞系, 采用细胞计数、生长曲线测定及MTT法测定体外培养细胞的生长和增殖抑制率。结果:可见一定程度的增殖抑制并呈剂量依赖性。抗-EGFR抗体组细胞数明显低于对照组(P<0.01).不同浓度之间比较:当EGFRMcAb为0.625mL/L时细胞计数相对高, 是对照组的61.3%;当EGFRMcAb为2.5mL/L时细胞计数最低, 是对照组的33.8%.EGFRMcAb组细胞生长受到抑制, 细胞生长曲线较对照组速度减慢。MTT值显示实验组细胞增殖能力低于对照组, 抑制率为42.3%(P<0.01).结论:EGFRMcAb可抑制人结肠癌LST174细胞生长, 具有一定的抗结肠癌作用。     

Expression of epidermal growth factor receptor and transferrin receptor by human trophoblast populations

Epidermal growth factor (EGF) has several roles, including stimulation of cell division and differentiation. EGF receptor (EGFR) has been localized to villous syncytiotrophoblast, but expression by other human trophoblast populations has not been reported. EGFR expression was examined in normal and pathological placental tissues using a streptavidin-biotin-peroxidase technique; results were compared with expression of transferrin receptor (Tf-R) in similar tissues. EGFR was detected on villous syncytiotrophoblast in early and term pregnancy with labelling of the apical membrane, focal cytoplasmic reactivity, and patchy labelling of the trophoblast basement membrane. In contrast with other reports, EGFR was also consistently localized to villous cytotrophoblast, chorion laeve, and extravillous trophoblast populations in maternal uterine tissues. Maternal decidua showed diffuse labelling of stromal cells, particularly in the superficial zones. The reaction pattern in ectopic tubal pregnancy was similar to that in early intrauterine pregnancy. In molar pregnancy, EGFR was detected on villous syncytiotrophoblast and cytotrophoblast. In contrast, in normal, ectopic, and molar pregnancies labelling for Tf-R was confined to syncytiotrophoblast and to the proximal portions of the cytotrophoblast columns. Expression of EGFR by all trophoblast cells may represent a mechanism of placental growth and proliferation control. EGFR may also be involved with establishment of differentiated trophoblast functions including hormone secretion.     

胎肺发育分化中表皮生长因子受体的表达和作用

目的　观察表皮生长因子受体 (EGFR)在小鼠胎肺发育过程的表达特征 ,研究表皮生长因子 (EGF)和转化生长因子 α(TGF α)通过与EGFR的作用 ,对胎肺形态发生和肺泡上皮成熟分化的作用。方法　昆明小鼠 ,13- 19天胎肺组织、生后1、7、14、30天及成鼠肺组织共 12组 ,常规石蜡切片 ,采用免疫组化SP两步法检测EGFR的表达。结果　胎肺假腺期 ,EGFR的表达主要定位于支气管呼吸道上皮 ,小管期EGFR阳性反应达到最高峰。在原始肺泡期 ,阳性反应主要在肺泡上皮细胞。出生后 ,支气管上皮细胞EGFR表达重新呈阳性表达。结论　在胎肺发育的不同时期 ,EGFR在上皮细胞的定位有迁移 ,免疫组化反应强弱也有差异 ,说明EGFR在胎肺不同发育阶段发挥不同的功能 ,它不仅参与支气管树的形成 ,还对呼吸道上皮细胞和肺泡上皮细胞的成熟分化有重要调节作用。     

Subcellular localization of epidermal growth factor receptor in human submandibular gland

The subcellular distribution of the epidermal growth factor receptor (EGFr) was demonstrated in the normal human submandibular gland by means of immunogold cytochemistry. EGFr labelling appeared in both acinar and ductal cells, where strong immunoreactivity was associated with a tubulovesicular system near the basolateral surfaces. In addition, groups of reactive vesicles were highlighted among secretory granules of both serous and mucous cells and at the apex of ductal cells. Basolateral vesicles were interpreted as being a result of EGFr internalization after activation by an exogenous ligand, although the functional meaning of those located apically remains unclear.     

Reactivity of epidermal growth factor receptor monoclonal antibodies with human uterine tissues

Two monoclonal antibodies (29.1 and 528), which were raised against the epidermal growth factor receptor, were used to evaluate expression of epidermal growth factor receptor in frozen uterine tissue with immunohistochemical techniques. Monoclonal antibody 29.1 stained only vascular endothelium and glandular epithelium in patients who were blood type A. This staining pattern is consistent with the previously reported blood type A specificity of this antibody. Monoclonal antibody 528, which recognizes a peptide determinant is thought to be specific for the epidermal growth factor receptor, stained both endometrial glands and endometrial stromal cells heavily. Faint staining was also seen in myometrium in most cases. This marked difference in expression of epidermal growth factor receptor between endometrium and myometrium contrasts with results of prior biochemical studies in which tissue homogenates were used. No variation in intensity of staining was seen between proliferative and secretory endometrium with the use of either antibody.     

Immunohistochemical localization of the epidermal growth factor receptor in normal human tissues

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used to localize this protein in selected normal human tissues. Two patterns of reactivity were recognized: strong linear or granular cell surface staining, and granular cytoplasmic staining. In one tissue, the endometrium, a change in the reaction pattern associated with changes in hormonal stimulation was observed. In some tissues such as epididymis and skin, the antibody showed surface reactivity with cells considered to represent part of the proliferating cell compartment, whereas in liver, pancreas, and prostate, all cells were reactive with the antibody, though the predominant reactivity was localized in the cytoplasm. The differential distribution of the epidermal growth factor receptor to specific cell types and cellular compartments may signify adaptations that permit growth factor responsiveness in a milieu of available ligand.     

表皮生长因子受体在肝细胞癌及胆管细胞癌中的表达

目的：比较表皮生长因子受体（ＥＧＦＲ） 在肝细胞癌及胆管细胞癌中表达的差异。方法：采用免疫组化及原位杂交方法检测９２ 例肝细胞癌及５０ 例胆管细胞癌组织中ＥＧＦＲ蛋白及ｍ ＲＮＡ的表达。结果：９２ 例肝细胞癌标本中,４２ 例ＥＧＦＲ 蛋白或ｍＲＮＡ阳性,占４５－７％ 。５０ 例胆管细胞癌中,３６ 例ＥＧＦＲ蛋白或ｍＲＮＡ 阳性,占７２－０ ％ 。结论：与肝细胞癌相比,胆管细胞癌的发生、发展与ＥＧＦＲ 表达增高的关系更加密切。     

Analysis of epidermal growth factor receptor and activated epidermal growth factor receptor expression in pituitary adenomas and carcinomas.

Epidermal growth factor receptor plays an important role in the pathogenesis of many malignancies. Various growth factors, including epidermal growth factor receptor, have been shown to influence pituitary tumor growth and differentiation. To analyze the role of epidermal growth factor receptor in pituitary tumor development, we examined normal pituitaries (n=8), pituitary adenomas (n=158), and pituitary carcinomas (n=7) for expression of epidermal growth factor receptor protein and messenger RNA using tissue microarrays and RT-PCR. We also examined (a) the expression of phospho-epidermal growth factor receptor, the activated form of epidermal growth factor receptor, in pituitary tumors and normal pituitaries by immunohistochemistry and (b) the effects on epidermal growth factor receptor expression of treating pituitary cells (HP75 cell line) with epidermal growth factor. Epidermal growth factor receptor and the phosphorylated variant expression were present in normal pituitary cells. Epidermal growth factor receptor messenger RNA was also detected in normal pituitaries, pituitary adenomas, and carcinomas by in situ hybridization and RT-PCR. Most pituitary adenomas showed expression of epidermal growth factor receptor and the phosphorylated variant. Nonfunctional adenomas showed higher levels of expression of epidermal growth factor receptor (76 vs 34%) and of phospho-epidermal growth factor receptor (26 vs 8%) as compared to functional adenomas. Five of seven pituitary carcinomas showed strong expression of both epidermal growth factor receptor and phospho-epidermal growth factor receptor. When a human pituitary cell line (HP75) was cultured in the presence of epidermal growth factor receptor, there was an increase in the levels of both epidermal growth factor receptor and phospho-epidermal growth factor receptor after 5 h of treatment, thus confirming that epidermal growth factor receptor signaling was active in pituitary tumors. These results indicate that activated epidermal growth factor receptor is expressed in pituitary adenomas and carcinomas. Higher levels in pituitary carcinomas suggest a role in pituitary tumor progression.     

No association between epidermal growth factor and epidermal growth factor receptor polymorphisms and nasopharyngeal carcinoma

Numerous candidate genes have been proposed as susceptibility factors for the development of nasopharyngeal carcinoma (NPC). Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) interaction plays a pivotal role in cell proliferation, differentiation, and tumourigenesis of epithelial tissues. To our knowledge, however, no study has examined the relationship between the EGF/EGFR and NPC. The aim of this study is to investigate the potential association between single-nucleotide polymorphisms of EGF +61 G/A and EGFR +2073 A/T and NPC. A total of 173 patients with NPC and 206 age- and sex-matched controls were the participants. Genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism strategy and DNA sequencing. There were no significant differences in the genotype and allele frequencies of EGF +61 G/A and EGFR +2073 A/T polymorphisms between the group of patients with NPC and the control group in a Chinese population (for EGF +61 G/A: OR=1.29, 95% CI: 0.95-1.74; for EGFR +2073 A/T: OR=0.91, 95% CI: 0.67-1.23). Further studies are still needed to explore the complicated interaction between environmental factors and EGF +61 G/A and EGFR +2073 A/T polymorphisms in the risk of NPC, particularly in ethnically different populations.     

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P < 0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.     

Endocrinology and paracrinology: Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor- (TGF), epidermalgrowth factor (EGF), cripto-1, amphiregulin and epidermal growthfactor receptor (EGFR ) was studied in 51 premenopausal humanovaries at various phases of the menstrual cycle. Localizationof mRNA for TGF and EGF was also studied by in-situ hybridization.Immunoreactive TGF was observed predominantly in theca cellsin 12 of 33 antral follicles in the follicular phase (6/14 dominantfollicles, and 6/19 non-dominant) but not in any of the 18 folliclesin the luteal phase or in primordial and pre-antral follicles.TGF immunoreactivity was present predominantly in the luteinizedgranulosa cells in 13 of 15 corpora lutea in the luteal phase,which are considered to be active in steroidogenesis, but notin any of the regressed corpora lutea. Accumulation of TGF mRNAhybridization signal was observed only in the theca cells inthe follicles and luteinized theca cells in the ovaries thatwere immunohistochemically positive for TGF. EGFR immunoreactivitywas detected in 24 of 33 antral follicles in the follicularphase and in two of 18 follicles in the luteal phase but notin any of the corpora lutea. Immunoreactive EGF, cripto-1 andamphiregulin or EGF mRNA was not detected in any follicles,corpora lutea, or the stroma cells examined. These results indicatethat, of the epidermal growth factors examined in this study,TGF is locally synthesized in normal cycling human ovaries andTGF may be synthesized in theca cells and act on the granulosacells in a paracrine fashion through the EGFR in ovarian follicles.     

Expression of epidermal growth factor receptor in human meningiomas and meningeal tissue.

The aim of this study was to examine meningeal tissue under normal, reactive, and neoplastic conditions for expression of epidermal growth factor receptor (EGFR) using an improved histochemical method, namely biotinylated epidermal growth factor. EGFR was found in all the examined meningiomas (12 benign and three anaplastic) and in neonatal rat meninges, whereas normal and injured adult human and rat meninges did not exhibit detectable EGFR. These observations indicate that EGFR is involved in the development of meningeal tissue. Further, EGFR is abnormally expressed in meningeal tumours, indicating a role of EGFR in the neoplastic process of these tumours. The regular expression of EGFR in human meningiomas suggests EGFR as a tumour marker for this tumour type.