Galactosamine induced hepatitis induces a reduction in hepatocyte epidermal growth factor receptors.

The rapid regenerative response of the rat liver to partial hepatectomy is associated with a decline in liver epidermal growth factor receptor numbers which implies that ligand epidermal growth factor receptor interactions maybe important in initiating and/or modulating this process. The proliferative process in toxic hepatitis (where in contrast with partial hepatectomy the majority of hepatocytes have been exposed to damaging influences) has been less widely investigated. We studied the DNA synthetic response of rat livers to toxic injury induced by a 350 or 800 mg/kg ip injection of galactosamine and that caused by 70% hepatectomy, comparing the changes in epidermal growth factor receptor status. Both resulted in down regulation of epidermal growth factor receptors, suggesting similar ligand epidermal growth factor receptor binding occurs during the proliferative response after galactosamine administration and after partial hepatectomy. In vitro studies on isolated hepatocytes showed that epidermal growth factor receptor down regulation was not a direct effect of galactosamine on hepatocyte membranes.     

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues.

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.     

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.     

Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.

We analyzed the uptake and intracellular distribution of 125I-labeled epidermal growth factor, nerve growth factor, and platelet-derived growth factor in different cell lines that express or do not express the respective surface receptors for these factors. After 1 hr of incubation, all three growth factors were detected in the cytoplasmic fraction and in the nucleus, tightly bound to chromatin. The amount of chromatin-bound growth factors continued to increase during the incubation, and analysis at 48 hr revealed each chromatin-bound labeled growth factor in a nondegraded form. After limited digestion of chromatin with DNase II (10-20% digested sequences), specific release of all three growth factors was detected only after 1 hr of incubation but not after 24 and 48 hr, suggesting that the DNA regions involved in growth factor binding became nuclease-resistant. Binding of labeled epidermal growth factor and nerve growth factor to isolated chromatin was inhibited by monoclonal antibodies specific for the respective growth factor receptor. The data suggest that chromatin binding may represent an important step in the pathway of growth factor action.     

Synergistic effects of bombesin and epidermal growth factor on cancers.

Bombesin and gastrin-releasing peptide act as autocrine mitogens in various cancers. Bombesin antagonist RC-3095 inhibited growth in some cancers and slowed the progression of premalignant lesions, possibly by down-regulating epidermal growth factor (EGF) receptors. Since the EGF receptor mitogen response involves tyrosine kinase stimulation, we tested the hypotheses that bombesin stimulates, and RC-3095 inhibits, phosphorylation; EGF and bombesin promote the phosphorylation of the same substrates; and EGF and bombesin act synergistically on phosphorylation. Therefore, in vitro assays for phosphorylation were performed in the presence or absence of EGF, bombesin, RC-3095, and combinations in samples derived from tumor, tissue surrounding tumor, cell lines, and normal and transforming tissue derived from the 9,10-dimethyl-1,2-benzanthracene-induced squamous cell lesions of the hamster cheek pouch. Bombesin increased, and RC-3095 decreased, phosphorylation in these samples. In the human hepatoma sample and surrounding tissue, these ligands altered the phosphorylation of the same substrates affected by EGF. EGF and bombesin stimulated phosphorylation synergistically in the hamster samples and the hepatoma. Bombesin-induced phosphorylation was greater in tissue surrounding the hepatoma, whereas RC-3095 was more effective in inhibiting phosphorylation in the hepatoma itself. This cancer, therefore, could be endogenously stimulated by gastrin-releasing peptide. These observations support the hypothesis that bombesin stimulates growth of tissues and tumors by amplifying the phosphorylation response to EGF. The growth inhibitory response to RC-3095, or other bombesin analogues, of individual tumors may be prognosed by in vitro phosphorylation assays using the samples from the patient's tumor.     

Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.     

Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters.

Platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) decrease high affinity binding of 125I-labeled epidermal growth factor (EGF) and potentiate mitogenesis in BALB/c 3T3 cells, and both have been shown to induce the phosphorylation of the EGF receptor at threonine residues. These similarities suggest that the actions of PDGF on EGF binding may be mediated by protein kinase C, the cellular effector of PMA. We show that in density-arrested BALB/c 3T3 cells PDGF and PMA induce a rapid, transient, cycloheximide-independent loss of EGF binding activity. As has been previously shown for PDGF, the ability of PMA to reduce EGF binding was enhanced by cholera toxin, a potent activator of adenylate cyclase. In contrast to PMA, however, PDGF induced a further reduction in EGF binding that was strictly dependent upon continued protein synthesis. Furthermore, PDGF effectively reduced EGF binding in cells refractory to PMA. Cells desensitized to PMA, presumably due to the loss of protein kinase C activity, also remained mitogenically responsive to PDGF. These data suggest that the mechanism by which PDGF modulates EGF binding differs from that of PMA and thus, at least in part, is independent of protein kinase C.     

Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody.

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In the presence of 100 nM anti-EGF-receptor monoclonal IgG (designated 528), which inhibits A431 cell proliferation and blocks greater than 95% of EGF binding, EGF becomes mitogenic for A431 cells at concentrations up to 3 nM. These results suggest that a minor population of high-affinity EGF receptors may be involved in stimulation of A431 cell proliferation. Saturation binding assays with 125I-labeled EGF indicate that approximately equal to 0.1-0.2% of receptors for EGF are high-affinity receptors that bind EGF with an estimated Kd of 7 X 10(-11) M. This affinity is nearly 2 orders of magnitude higher than that of the remaining EGF receptors. Although A431 cell proliferation is maximally inhibited by nonsaturating amounts of EGF (3 nM), maximal inhibition by 528 IgG (approximately equal to 70% of maximal inhibition by EGF) requires saturating concentrations of antibody (approximately equal to 15 nM). Unlike EGF, rapid down-regulation is not observed with 528 IgG. These results indicate different mechanisms of growth inhibition of A431 cells by EGF and 528 IgG.     

Modulation of epidermal growth factor receptors by human alpha interferon.

Treatment of Madin-Darby bovine kidney (MDBK) cells with human interferon (IFN)-alpha 2 at 37 degrees C results in a dose-dependent inhibition of cell growth and a reduction of the subsequent binding of 125I-labeled epidermal growth factor (EGF) at 4 degrees C. Human IFN-beta and -gamma, which exhibit little antiviral and antiproliferative activities on MDBK cells, have little effect on cell growth or the binding of 125I-labeled EGF to these cells. The binding of EGF is decreased after exposure to IFN-alpha for greater than 8 hr. Scatchard analyses of the EGF binding data indicate that a 20-hr exposure period results in a decrease in the apparent number of cell-surface EGF receptors and a reduction in the affinity of EGF for its receptor. The rate of internalization of EGF by MDBK cells does not appear to be affected by IFN treatment.     

Presence of epidermal growth factor receptors on human thyroid membranes

The nature of epidermal growth factor (EGF) receptors in normal and pathological thyroid membranes was studied on a crude membrane fraction (10,000 X g pellet) of tissue homogenate. Optimal binding of 125I-EGF to human thyroid membranes was obtained at 25 degrees C with 1-h incubation at pH 7.4. The study revealed the presence of both high and low affinity receptors in normal and pathological thyroid membranes. The association constants of high affinity receptor (3.2 X 10(-9) mol/l) and of low affinity receptor (1.8 X 10(-8) mol/l) observed in normal thyroid membranes were similar to those of thyroid membranes from neoplastic as well as thyrotoxic thyroid tissues. [3H]thymidine incorporation into DNA of cultured human thyroid cells was stimulated by EGF in a dose-dependent manner. A half-maximal stimulation was found around 1 X 10(-10) mol/l. These results suggest that EGF may have a possible role in the regulation of thyroid growth and function in physiological and pathological situations.     

Lapatinib,a dual inhibitor of epidermal growth factor receptor and human epidermal growth factor receptor 2, potentiates the antitumor effects of cisplatin on esophageal carcinoma

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) overexpression occurs in over 30% of esophageal carcinomas. Combination therapies of EGFR‐ and HER2‐targeting agents with cytotoxic agents are considered a potential therapeutic strategy for esophageal cancer. The antitumor effects of lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2, cisplatin alone, and the combination of the two drugs on esophageal cancer cells were evaluated. The growth inhibition activity of lapatinib, cisplatin, and lapatinib plus cisplatin was measured by 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐di‐phenytetrazoliumromide (MTT) assays, and the combination index values were calculated. Additionally, cell cycle distribution and cell apoptosis treated with lapatinib or cisplatin alone and the combination of the two drugs were detected by flow cytometry analysis. The activation of EGFR and HER2 signaling pathways was monitored by Western blot analysis. These experimental data showed that the combination of lapatinib and cisplatin synergistically inhibited cell proliferation and exhibited an enhanced pro‐apoptotic effect on esophageal cancer cells. The underlying mechanisms of potentiated effects of combined treatment were associated with reduced phosphorylation of EGFR and HER2, and the downstream signaling molecules AKT and extracellular regulated protein kinases (ERK). Our findings indicated that the combination of lapatinib and cisplatin is one of the promising treatment strategies for esophageal carcinomas with EGFR and HER2 overexpression.     

Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.     

Lysomotropic amines cause intracellular accumulation of receptors for epidermal growth factor.

By direct biochemical methods, we demonstrate that the process of internalization of receptors for epidermal growth factor (EGF) occurs even without EGF stimulation and is not prevented by the lysomotopic agents methylamine or chloroquine. These agents inhibit the degradation of 125I-labeled EGF, thus preventing the rapid dissociation of EGF from cells. Furthermore, 125I-labeled EGF incubated with cells in the presence of methylamine becomes increasingly insensitive to trypsin with time, suggesting that the EGF receptor internalization is not prevented by alkylamines, but that there is an intracellular accumulation of ligand--receptor complex due to the loss of normal modes of ligand-induced receptor processing. Lysis of cells treated with methylamine results in recovery of 125I-labeled EGF binding. Fractionation of these lysates on sucrose density gradients demonstrates that EGF receptors are localized within membrane fractions having higher densities than fractions from lysates of untreated cells.     

Opposite effects of transforming growth factor alpha and epidermal growth factor on mouse placental lactogen I secretion.

This study was undertaken to determine whether transforming growth factor alpha (TGF-alpha) regulates the production of mouse placental lactogen I (mPL-I) and mPL-II in a manner that is similar to that of epidermal growth factor (EGF), which was previously shown to stimulate mPL-I secretion and inhibit mPL-II secretion. In contrast to the activity of EGF, human (h) and rat (r) TGF-alpha (each at 100 ng/ml) inhibited secretion of mPL-I by placental cells isolated from mice on day 7 of pregnancy. Maximum inhibition of mPL-I secretion occurred on the third day of a 5-day culture period and ranged between 37% and 56% in multiple trials. Incubation of cells with hTGF-alpha and EGF was not followed by a change in the mPL-I concentration of the medium, suggesting the peptides antagonized each other's effects. hTGF-alpha and rTGF-alpha inhibited secretion of mPL-II; maximum inhibition ranged between 62% and 84% in multiple trials. The lowest concentrations of hTGF-alpha that affected mPL-I and mPL-II secretion were 10 ng/ml and 1 ng/ml, respectively. EGF and hTGF-alpha bound to the same receptors on placental cells, as assessed by cross-linking, and both peptides stimulated receptor phosphorylation, as assessed by Western blot analysis. There are three types of mPL-containing cells in placental cultures: cells that contain only mPL-I, cells that contain only mPL-II, and cells that contain both mPLs. The percentage of each type of mPL-containing cell in the culture was determined by immunostaining. hTGF-alpha affected the differentiation of the subpopulations of PL-containing cells in a manner that differed from that of EGF. The data suggest that TGF-alpha and EGF do not regulate the production of mPL-I and mPL-II in a similar manner.     

Adrenergic effects on secretion of epidermal growth factor from Brunner's glands.

The influence of the sympathetic nervous system and adrenergic agonists on flow rate and secretion of epidermal growth factor (EGF) from Brunner's glands has been investigated in the rat. Chemical sympathectomy by administration of 6-hydroxydopamine increased volume secretion and output of EGF from Brunner's glands but depleted the glands of EGF. Infusion of noradrenaline, an alpha-adrenergic agonist, inhibited basal and vasoactive intestinal polypeptide (VIP) stimulated flow rate and output of EGF from Brunner's glands and increased the amount of EGF in the tissue. Vasoactive intestinal polypeptide also increased the amount of EGF in Brunner's gland tissue and this was unchanged after simultaneous infusion of VIP and noradrenaline as well as VIP and isoproterenol, a beta-adrenergic agonist. Isoproterenol had no effect on basal and VIP stimulated secretion of EGF from Brunner's glands. The presence of PAS-positive mucus in Brunner's glands was unchanged during infusion of noradrenaline whereas VIP induced a depletion of Brunner's gland mucus which in turn was prevented by simultaneous infusion of noradrenaline. This study indicates that the sympathetic nervous system influence the volume secretion, output of EGF and mucus content in Brunner's glands probably by activation of alpha-adrenergic pathways.     

Radioautographic localization of epidermal growth factor receptors in human fetal gut.

The present investigation was undertaken to study the localization and accessibility of epidermal growth factor binding sites in the human fetal gut (15-19 weeks of gestation) using light-microscopic and quantitative autoradiography. Exposure of colonic explants to 5 nmol/L 125I-labeled epidermal growth factor for 60 minutes at 22 degrees C revealed extensive accumulation at binding sites in undifferentiated cells of the crypts and at the base of the villus, as well as in the inner circular layer of the muscularis externa bordering the submucosa. Some labeling was also present in the mesenchymal and vascular elements of the lamina propria. Labeling was virtually absent on the brush border at all levels of the epithelium. Quantitative analysis revealed a distinct gradient in grain density along the various compartments of the crypt-villus axis. Epithelial cells in the deep portions of the crypts showed the highest grain density (9.2 grains/microns 2) with values gradually decreasing to 6.5 in upper crypt and 3.9 in lower villus cells. The upper third of the villus showed very little labeling (0.4 grains/microns 2). Cellular distribution of silver grains in lower villous cells revealed a polarization of labeling in the basolateral infranuclear region. Experiments performed at 4 degrees C and at various incubation times showed similar results. Using isolated loops of intact colon and jejunum, segments in which labeled epidermal growth factor was only accessible on the serosal side showed extensive labeling and distribution similar to that found in explanted tissue. On the other hand, labeled epidermal growth factor could not access these same receptor sites when infused into the lumen, either at 22 degrees C or 4 degrees C. These results show that in the human fetal gut (a) the greatest concentration of epidermal growth factor binding sites is found in regions of high proliferative activity and (b) binding sites are absent from the brush border, suggesting that, under normal circumstances, systemic but not luminal epidermal growth factor has free access to its specific receptor.     

Prostatic epidermal growth factor receptors and their regulation by androgens

Prostatic membranes contain high affinity [dissociation constant (KD) = 1.16 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I]iodo-EGF is specific since it is displaced by excess EGF but not by insulin, fibroblast growth factor, platelet-derived growth factor, or multiplication-stimulating activity. Affinity labeling with [125I]iodo-EGF and subsequent cross-linking with disuccinimidyl suberate demonstrated the specific binding of [125I]iodo-EGF to a macromolecule with a mol wt of 170,000. Castration of mature rats resulted in a 3- to 6-fold increase in [125I]iodo-EGF binding, while treatment of 7-day castrated rats with 5 alpha-dihydrotestosterone decreased the number of binding sites. Administration of estrogen or progesterone produced a slight decrease in EGF binding sites but not nearly to the extent observed with 5 alpha-dihydrotestosterone, suggesting that the observed effect is androgen specific. These results demonstrate that rat prostate contains specific binding sites for EGF and that their level is modulated by androgens.     

表皮生长因子受体抑制剂埃罗替尼对胰腺癌血管生成的影响

目的 观察表皮生长因子受体抑制剂埃罗替尼对胰腺癌新生血管生成的影响，探讨其对胰腺癌生长抑制的作用机制。方法 ①应用小管形成实验观察埃罗替尼（终浓度100μmol／L）对血管生成的影响，并与对照组（加入无血清培养液）进行比较。②建立胰腺癌细胞株BxPC-3裸鼠移植瘤模型，用埃罗替尼灌胃，每天100mg／kg，共4周。每周测量移植瘤体积，4周后处死裸鼠，计算抑瘤率，并与未用埃罗替尼对照组裸鼠比较。RT-PCR法检测不同浓度（5、50、100、200／μmol／L）埃罗替尼处理后BxPC-3细胞血管内皮生长因子（VEGF）表达的变化。采用Ⅷ因子免疫组化染色评估瘤组织中微血管密度（MVD）。结果小管形成实验中，埃罗替尼组细胞数显著少于对照组，中空闭合管状结构缺如。埃罗替尼灌胃4周后，治疗组平均瘤重（0．397±0．550）g，显著低于对照组的（1．5704±1．060）g，抑瘤率为74．5％。浓度≥50μmol／1．的埃罗替尼各组BxPC-3细胞中VEGFmRNA相对表达量较对照组下调，移植瘤组织VEGF表达亦较对照组显著下调，瘤组织MVD（1．86±0．43）显著低于对照组（5．98土1．27，P〈0．01）。结论 埃罗替尼可抑制裸鼠移植瘤生长和胰腺癌体内、体外血管的生成，可作为胰腺癌的辅助治疗方法之一。