Pretranslational enhancement of epidermal growth factor receptor by direct effect of testosterone in mouse liver

The effects of testosterone on the levels of epidermal growth factor receptor (EGFR) in mouse liver were studied. Orchiectomy resulted in 39% and 41% reductions in the levels of EGFR mRNA and EGF binding, respectively, within 2 weeks. Treatment of orchiectomized mice with a sc injection of testosterone propionate (TP; 100 micrograms/mouse.day) for 1 week restored these values to normal male levels. The hepatic levels of EGFR mRNA and EGF binding in females were 37% and 36% of those in males, respectively, and were not affected by ovariectomy, whereas treatment of females with TP (100 micrograms/mouse.day) increased EGFR to normal male levels within 1 week. On the other hand, neither orchiectomy nor androgen treatment affected levels of mRNAs for EGFR in the kidney or mRNAs for the structural protein beta-actin in the liver. To examine whether testosterone directly increased EGFR levels in the liver, TP (1.0 mg) pellets were implanted into the spleen of orchiectomized mice, so that testosterone released from the pellets reached the liver through the portal vein but did not enter the systemic circulation due to rapid clearance by the liver. The heptic levels of EGFR mRNA and EGF binding in orchiectomized mice were restored to normal male levels by intrasplenic implantation of TP (1.0 mg) pellets. This treatment also increased the hepatic levels of EGFR mRNA and EGF binding in female mice by 61% and 68%, respectively. In addition, sialoadenectomy, which reduced plasma EGF, as well as EGF antiserum treatment did not affect the androgen-dependent increase in EGFR levels in the liver, suggesting that endogenous EGF is not involved in the androgenic regulation of hepatic EGFR levels. These results suggest that hepatic EGFR levels are at least in part regulated at a pretranslational level by direct effects of androgens on the liver.     

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor alpha and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor alpha release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.     

The epidermal growth factor receptor family

The epidermal growth factor receptor family consists of four receptor genes and at least 11 ligands, several of which are produced in different protein forms. They create an interacting system that has the ability to receive and process information that results in multiple outputs. The family has an important role in directing and coordinating many normal processes, including growth and development, normal tissue turnover and wound healing. Its members are also aberrantly activated by overexpression or mutation in many common human tumour types and as such have been the target for anticancer drug development.     

Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver.

We have purified the epidermal growth factor (EGF) receptor/protein kinase from the livers of normal mice by affinity chromatography. The biochemical properties of the liver receptor are very similar to those of the EGF receptor previously prepared from the human tumor cell line A-431 [Cohen, S., Ushiro, H., Stoscheck, C. & Chinkers, M. (1982) J. Biol. Chem. 257, 1523-1531]. The liver receptor for EGF is a glycoprotein of Mr 170,000. It binds 125I-labeled EGF and possesses an EGF-stimulable protein kinase activity specific for tyrosine residues. Both autophosphorylation and kinase activity toward exogenous substrates are demonstrable. The EGF receptor purified from normal mouse liver is antigenically related to the receptor purified from human A-431 cells.     

表皮生长因子及其受体在糜烂胃黏膜中的表达

目的:观察胃窦黏膜糜烂区与糜烂旁胃黏膜、慢性萎缩性胃炎中表皮生长因子(epidermal growth factor,EGF)及其受体(epidermal growth factor receptor,EGFR)的表达,探讨其在胃黏膜损伤修复中的意义.方法:选择经胃镜及病理确诊的慢性萎缩性胃炎伴胃窦黏膜糜烂患者50例,距糜烂区3cm处40例,无糜烂慢性萎缩性胃炎40例,采用免疫组织化学染色法测EGF及EGFR的表达.结果:胃窦黏膜糜烂区EGF、EGFR阳性表达率分别为40%和30%,明显高于糜烂旁胃黏膜15%和10%及无糜烂慢性萎缩性胃炎20%和12.5%的阳性表达率(P<0.05),有统计学意义,无糜烂慢性萎缩性胃炎组略高于糜烂旁胃黏膜组,但无统计学差异.结论:EGF、EGFR在胃黏膜损伤后高表达,对促进胃黏膜修复有着重要意义.     

Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands

The specific binding of iodinated epidermal growth factor ([125I]iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites [dissociation constant (Kd) = 0.7-1.8 nM]. Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status.     

Human epidermal growth factor receptor residue covalently cross-linked to epidermal growth factor.

An epidermal growth factor (EGF) receptor monoclonal antibody (mAb), mAb LA22, was used to analyze the covalent coupling of human EGF receptors to mouse EGF by the amine-reactive cross-linking agent disuccinimidyl suberate. A soluble Mr 105,000 truncated form of the receptor secreted by A-431 epidermoid carcinoma cells and consisting of the ligand-binding extracellular domain was cross-linked to 125I-labeled EGF. Digestion of this complex with an endoproteinase that specifically cleaves at the COOH side of glutamyl residue released a single radiolabeled glycosylated fragment of Mr 18,000 that reacted with mAb LA22. As the epitope for mAb LA22 resided between Ala-351 and Asp-364 of the mature receptor, this result localized the cross-linked receptor residue(s) to the 47-amino acid interval from Phe-321 to Glu-367. The receptor residue(s) involved in the covalent coupling of rat 125I-labeled transforming growth factor alpha was similarly localized to this region of the receptor. This receptor interval, which included two glycosylated asparaginyl residues at positions 328 and 337, contained but three amino acid residues that were potentially reactive with disuccinimidyl suberate: Lys-332, Lys-333, and Lys-336. Characterization of mAb LA22-reactive 125I-EGF-labeled receptor fragments generated by an endoproteinase specific for the COOH side of lysyl residue placed the NH2 termini of the two smallest fragments between the glycosylated residues Asn-328 and Asn-337. These results indicated that disuccinimidyl suberate cross-linked the NH2 group of EGF residue Asn-1 to the human EGF receptor residue Lys-336. Our results further suggest that EGF and transforming growth factor alpha, two members of the EGF family of peptide growth factors, interact with closely apposed or identical features of the receptor.     

siRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells

Purpose This study was to investigate if downregulation of IGF1R and EGFR by RNA interference (RNAi) would sensitize human liver cancer cells (HEPG2, Huh7 ) to adriamycin. Methods HEPG2, Huh7 cell lines were transfected IGF1R siRNAs and EGFR siRNAs and IGF1R or EGFR mRNA level was determined by RT-PCR and Western-blot analysis. We investigated the effects of the adriamycin-induced apoptosis of these cells by TUNEL assay. Also we analyze caspase3, 8 and the phosphorylation levels of Akt and Erk by Western-blot. The p53 effect of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is investigated by cell growth curves. Results Transfection of an IGF1R and EGFR siRNAs resulted in substantial loss of IGF1R and EGFR mRNA of HEPG2, Huh7 cells relative to the control case. EGFR siRNA and IGF1R siRNA treatments increased the adriamycin-induced apoptosis of these cells. IGF1R siRNA and EGFR siRNA enhance a caspase-dependent cell death program. The phosphorylation levels of Akt and Erk were reduced by the combination of the two agents. The facilitation of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is p53-independent. Conclusions The results indicate that the siRNA for IGF1R has a great potential for cancer therapy when combined with either a chemotherapeutic agent or siRNAs that targets EGFR.     

Detection of epidermal growth factor-urogastrone and its receptor during fetal mouse development

Using both a radioimmunoassay and a radioreceptor assay, we have estimated the content of mouse epidermal growth factor-urogastrone (EGF-URO) in fetal mice at 11(1/2) to 17(1/2) days of gestation. Concurrently, the amount of specific EGF-URO receptor binding was determined in crude membrane fractions from the embryos. EGF-URO receptor binding is readily detected in membranes from the youngest embryos (day 11(1/2)) and rises steadily up to parturition (18 days); the rise is more marked in embryo membranes derived from a potential target tissue, such as the maxilla and secondary palate. In the embryonic extracts, EGF-URO proved to be labile, requiring the presence of soybean trypsin inhibitor and sodium azide to stabilize the recovery of added EGF-URO in test samples. Even with added stabilizing agents, immunoreactive EGF-URO was barely detectable before day 14(1/2) (less than 20 fmol per embryo), whereas a substantial increase was observed from day 15(1/2) to 17(1/2) (from 70 to 200 fmol per embryo). In contrast, the radioreceptor assay detected appreciable amounts of an EGF-URO-like substance at 11(1/2) days (50 fmol per embryo); the values estimated by radioreceptor assay (about 10-fold higher than by radioimmunoassay) also increase markedly between days 15(1/2) and 17(1/2) (on average from 500 to 3000 fmol per embryo). We conclude that during fetal mouse development there is an increase both in the receptors for EGF-URO and in a substance (presumably a fetal growth factor) that can occupy the receptor. The differences between the radioreceptor and radioimmunoassay estimates for the EGF-URO content suggest that the fetal form of mouse EGF-URO differs from the adult molecule.     

表皮生长因子受体基因多态性与表皮生长因子受体-酪氨酸激酶抑制剂抗癌药物不良反应相关性的研究

目的:探讨表皮生长因子受体(EGFR)基因多态性与表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-Tki)抗癌药物不良反应的相关性。方法:选择2013年1月至2015年5月,在我院使用EGFR-Tki(吉非替尼)治疗的121位非小细胞肺癌(NSCLC)患者.采用直接测序法检测EGFR基因内因子区CA-SSR基因多态性(SNPs),采用放大受阻突变系统(ARMS)检测EGFR基因外显子rs2293347 G/A,以及启动区-216 G/T,-191 C/A3个基因多态性。治疗过程中观察并分析患者的不良反应发生情况,评价基因多态性与不良反应之间的关系。结果:EGFR基因内含子区CA-SSR基因多态性与发生皮疹不良反应的严重程度具有显著的相关性(P=0.003),但与不良反应发生率无关。结论:EGFR基因内含子区CA-SSR基因多态性可以考虑应用于临床评估晚期非小细胞肺癌患者使用EGFR-Tki发生皮疹毒不良反应的严重程度。     

Androgens activate mitogen-activated protein kinase via epidermal growth factor receptor/insulin-like growth factor 1 receptor in the mouse PC-1 cell line

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9?U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM.     

肝内胆管癌形成过程中表皮生长因子及其受体与转化生长因子-α的表达意义

通过免疫组织化学的方法，动态观察表皮生长因子(EGF)及其受体(EGFR)和转化生长因子-α(TGF-α)在叙利亚地鼠肝内胆管癌(ICC)形成过程中的表达情况，探讨上述因子在ICC形成过程中的作用。     

Characterization of epidermal growth factor in mouse testis.

Considerable evidence exists to suggest that epidermal growth factor (EGF) influences spermatogenesis directly. The tissue source of this EGF, however, is not yet clear. In this study we examine whether the testis itself can serve as a source of EGF. Gel filtration fractions of acid extracted testes exhibited the ability to displace 125I-EGF from testis membranes. The testicular fractions containing the 125I-EGF displacement activity coeluted within the same range as those of submandibular gland (SG) fractions containing mature EGF and prepared in an identical fashion. Next, we employed specific antisera probes to investigate first, whether the testis synthesizes this EGF displacement activity and second, to determine the cell distribution of the testicular EGF. Two types of antisera probes were employed: 1) commercially available antisera to mature EGF (EGFm), i.e. the 6,000 M(r) peptide, and 2) polypeptide specific antisera to the C-terminus of the EGF precursor (EGFp), i.e. the 140,000 M(r) integral membrane molecule which exhibits seven EGF-like repeats in addition to the EGFm. Metabolic labeling of testis with 35S-methionine was performed, followed by immunoprecipitation with the anti-EGFm antisera. Parallel studies using kidney and SG were used as positive controls. Fluorograms exhibited a prominent band at M(r) 140,000 for testis and kidney, corresponding to the EGFp. There was, in addition, a M(r) 50,000 band present for the testis. In SG, a band at M(r) 6,000, corresponding to EGFm, in addition to bands at M(r) 21,000 and 46,000 were observed also. Immunoblotting of testis, kidney, and SG membrane preparations with the specific antisera to either the EGFm or EGFp also resulted in identifying the EGFp at M(r) 140,000, as well as other lower mol wt bands. Preadsorption of anti-EGFm antisera with excess EGFm eliminated all of the specific bands that were immunoblotted. Peroxidase immunocytochemistry of testis, kidney, and SG was also performed using the specific antisera to either EGFm or EGFp. EGFp and EGFm staining in SG and kidney was identical to previously published results in which the distribution of EGFm in these tissues was established. In testis, EGFm immunostaining showed positive results in Sertoli cells, pachytene spermatocytes and round spermatids. In contrast, EGFp immunostaining was limited to pachytene spermatocytes and round spermatids. These results suggest that the testis must now be included in the list of tissues capable of synthesizing EGFp. Specifically, EGFp synthesis appears limited to the post meiotic germ cells.     

Characterization of the epidermal growth factor receptor in the gastric mucosa

The effects of epidermal growth factor (EGF), a potent mitogen involved in mucosal protection, are mediated by specific cellular receptors. Here, we present the characteristics and binding properties of EGF receptors in the gastric mucosa. The studies were conducted using cell membranes isolated by subcellular fractionation of rat stomach mucosal scrapings. Specific binding of [125I]-EGF to the membrane preparations was assessed at room temperature for various periods of time and at different pHs. The results showed that the binding was proportional to the incubation time up to 1 h and was not affected by a pH change between 4.0 and 7.0. Scatchard analysis of the binding data infer the presence of 2 binding sites, one of high affinity (Kd = 1.34 nM, Bmax = 34 fmol/mg protein) and the other of low affinity (Kd = 484 nM, Bmax = 2.29 pmol/mg protein). Cross-linking experiments using disuccinimidyl suberate to link the [125I]-EGF to gastric membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed that the major receptor for EGF was a protein of 170 kilodaltons. When the solubilized membranes were subjected to wheat germ agglutinin affinity chromatography, the purified material was found to act as substrate for EGF-stimulated phosphorylation. The major component which was labeled by the [gamma-32P]-ATP was also found to be a 170-kilodalton protein. The data are the first to provide evidence that the gastric mucosa possesses a functional EGF receptor and describe its binding characteristics.     

Targeting the epidermal growth factor receptor in metastatic colorectal cancer

Although significant advances have been made in the treatment of metastatic colorectal cancer (CRC), prognosis remains poor, with a 5-year survival of less than 10%. Monoclonal antibodies that target the epidermal growth factor receptor (EGFR) have shown clinical benefit as single agents and in combination with standard chemotherapy in the refractory setting, with tolerable toxicity. This article will discuss the role of the EGFR pathway in the pathogenesis of CRC, the data supporting the current use of cetuximab and panitumumab in the treatment of CRC, and clinical trials of EGFR tyrosine kinase inhibitors in CRC. Novel strategies of targeting the EGFR pathway to improve efficacy, as well as ongoing research in identifying molecular predictors of response to anti-EGFR agents, will also be reviewed.     

Localization of epidermal growth factor receptor in hepatocyte nuclei

Experiments undertaken to investigate the binding of epidermal growth factor by hepatocyte nuclei showed that: (a) isolated nuclei from both normal and regenerating rat liver are capable of binding 125I-epidermal growth factor, (b) the nuclear epidermal growth factor-binding protein is similar in molecular weight to the plasma membrane epidermal growth factor receptor, (c) monoclonal antibodies produced against the plasma membrane epidermal growth factor receptor recognize the nuclear epidermal growth factor receptor and (d) the nuclear receptor has an affinity for epidermal growth factor comparable to that of the plasma membrane receptor, but fewer (approximately 10%) nuclear receptors are available per protein unit compared with the plasma membrane.