Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding.

The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin.     

Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding.

Epidermal growth factor (EGF) receptor monomers and noncovalently associated dimers were isolated by sucrose density gradient centrifugation, and their respective binding and autophosphorylation activities were determined. We find that monomers are low-affinity receptors and dimers are high-affinity receptors. In the absence of EGF, dimers exhibit a 4-fold higher autophosphorylation activity than do monomers. Addition of EGF increases autophosphorylation on monomers an average of 4.8-fold but has a minimal effect on autophosphorylation of dimers. Furthermore, EGF binding shifts the receptor monomer-dimer equilibrium to the dimer form. We conclude that EGF stimulates in vitro receptor autophosphorylation by inducing kinase-inactive receptor monomers to associate and form receptor dimers, in which conformation the autophosphorylation activity is enhanced.     

Mechanism of biological synergy between cellular Src and epidermal growth factor receptor

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T½ murine fibroblasts that contain kinase-deficient (K−) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.     

Induction of differentiated trophoblast function by epidermal growth factor: relation of immunohistochemically detected cellular epidermal growth factor receptor levels

The effects of epidermal growth factor (EGF) on the production and secretion of hCG and human placental lactogen (hPL) by cultured placental tissues were investigated in relation to immunohistochemical measurements of cellular EGF receptor levels in the placenta. Explants of trophoblastic tissues obtained from normal early and term placentas were cultured in the presence or absence of EGF (100 ng/mL) with or without processing inhibitors (bacitracin, 1 mg/mL; colchicine, 100 microM; chloroquine, 100 microM) for 5 days, with EGF present for the first 2 days. Addition of EGF to the medium increased the release of hCG, hCG alpha, and hPL by the cultured early placental tissues. This EGF-stimulated hCG, hCG alpha, and hPL release was markedly inhibited by concomitant treatment with processing inhibitors. The time course of EGF effects indicated that the EGF-stimulated increase in hCG alpha secretion required a lag period of approximately 1 day, whereas significant increases in hCG and hPL secretion became apparent only after 3 days of EGF treatment. By contrast, in term placental tissues EGF stimulated only hCG alpha and hPL release, with a lag period of approximately 3 days. A possible direct action of EGF on the cultured placental tissues was reinforced by the immunohistochemical demonstration of EGF receptors in the placenta. When determined using the avidin/biotin immunoperoxidase method with monoclonal antibody to the mouse EGF receptor, EGF receptors were found predominantly on the syncytiotrophoblasts. Immunohistochemical measurements of cellular EGF receptor levels in the syncytiotrophoblasts revealed remarkably higher levels in early placenta compared to those in midterm and term placentas. Since EGF is likely to interact with its receptor, the lesser biological effects of EGF in cultures of term placental tissues may be due to the lower cellular EGF receptor levels in term placenta. These results demonstrate that EGF, via its receptors on the syncytiotrophoblasts, stimulates the release of both hCG and hPL in normal early placenta. They also suggest that EGF may play a significant role in the induction and regulation of the differentiated function of trophoblasts.     

Studies of epidermal growth factor receptor inhibition in breast cancer

Until recently, there has been little knowledge on the growth control of oestrogen receptor (ER)-negative ductal carcinoma in situ (DCIS) and invasive breast cancer. The recent development of DCIS models, such as transgenic mice, cell-line xenograft models and, importantly, in vivo human DCIS xenograft models has facilitated the investigation and understanding of the control of growth of early pre-invasive breast lesions. Recent studies have shown that ER-negative DCIS, unlike ER-positive DCIS, is hormone independent and does not respond to anti-oestrogen treatment. Moreover, DCIS of the comedo type utilises type I tyrosine kinase growth factors, such as epidermal growth factor receptor (EGFR) and c-erbB-2, in receptor signalling for growth. New data underscore the importance of EGFR as the major modulating growth factor receptor in the control of proliferation in the breast. Pre-clinical studies performed on human DCIS xenografts in nude mice suggest a potential role for EGFR tyrosine kinase inhibitors (EGFR-TKIs). More specifically, ZD1839, a novel orally active and selective EGFR-TKI, has been shown to produce a response in DCIS through a decrease in epithelial proliferation. These findings have enhanced our knowledge of signal transduction pathways in cancer and indicate that tyrosine kinase blockade of EGFR has potential for the treatment and chemoprevention of DCIS. It is hoped that further advances in this area and evaluation of EGFR-TKIs in Phase II/III clinical trials will allow their therapeutic potential as anticancer agents to be appreciated.     

Saccharin and cyclamate inhibit binding of epidermal growth factor.

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibition of 17 beta-estradiol-induced increases in rat uterine epidermal growth factor receptor binding activity and gene expression

Treatment of immature female Sprague-Dawley rats with 17 beta-estradiol (5 micrograms/animal) resulted in an increase in uterine epidermal growth factor (EGF) receptor binding activity. Moreover, in a separate study it was also shown that 17 beta-estradiol increased steady-state levels of rat uterine EGF receptor mRNA as determined by Northern analysis. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a dose-response decrease in constitutive rat uterine EGF receptor binding activity and this was paralleled by a decrease in steady-state levels of uterine EGF receptor mRNA. Cotreatment of the animals with both TCDD (16 nmol/kg) and 17 beta-estradiol (5 micrograms/rat) gave results which showed that TCDD significantly inhibited the estrogen-induced increases in rat uterine EGF receptor binding activity and EGF receptor mRNA levels. These results further extend the range of antiestrogenic properties of TCDD and suggest that the inhibition of growth factor expression may play a role in the growth-inhibiting properties of TCDD in estrogen-responsive tissues or cells.     

Insulinlike growth factor binding proteins and tumor hypoglycemia.

Hypoglycemia associated with nonislet cell tumors is a relatively rare metabolic disorder, which develops in the presence of low or unmeasurable serum insulin levels. Understanding of the pathogenetic mechanisms has been obscured by the lack of appropriate analytic methods. It now appears that this disorder can be classified as a paraneoplastic syndrome caused by the oversecretion of unprocessed (big) insulinlike growth factor (IGF) II propeptide by the tumor. In contrast to other paraneoplastic syndromes, however, this oversecretion does not lead to grossly elevated IGF II RIA values in the sera of the patients, but is masked by (a) a negative feedback that suppresses the production of mature (7.5-kD) IGF II, so that the total IGF II level remains more or less unchanged and (b) an altered distribution of the hormone between two specific IGF binding protein complexes in the circulation so that its bioavailability is essentially enhanced.     

Increased binding of epidermal growth factor at preimplantation sites in mouse uteri

The presence of receptors for epidermal growth factor (EGF) in nonpregnant uteri and the elevation of EGF levels in blood during early pregnancy suggest that EGF and its receptor may play important roles in the early stages of pregnancy. We determined the distribution of EGF receptors in uteri of nonpregnant and pregnant mice during the late preimplantation period (days 4.5-5.0 of pregnancy) using radioautograph and quantitative binding techniques. Radioautography of [125I]EGF binding to cornua from nonpregnant mice showed low levels of specific binding evenly distributed throughout the cornua. In contrast, radioautographs of cornua from pregnant mice showed bands of elevated binding encircling the lumen at sites of preimplantation. Results from radioautography were supported by quantitative analysis of [125I]EGF binding to uterine homogenates from nonpregnant and pregnant mice. Binding of [125I]EGF to uterine membranes was highly specific and time dependent. The average level of specific EGF binding calculated from Scatchard plots of nonpregnant uteri (27 +/- 13 fmol/mg protein) was significantly (P less than 0.05) lower than that in pregnant superovulated uteri (106 +/- 67 fmol/mg protein). Furthermore, specific binding of EGF was significantly (P less than 0.05) higher in preimplantation sites than in the intervening nonimplantation regions from the same uteri (42 +/- 6 vs. 29 +/- 4 fmol/mg protein, respectively). Differences in EGF binding appear to be due to changes in the number of EGF receptors, since half-displacement values (1 nM) were similar in all samples. These results demonstrate that alterations of EGF receptor levels occur at sites where implantation will occur in mouse uteri and support the concept that the transforming growth factor-alpha/EGF receptor and its ligands are involved in implantation of concepti.     

表皮生长因子受体相关蛋白在肿瘤靶向治疗中的应用前景

靶向治疗是一种比较新的治疗方法,特别是干预表皮生长因子受体(epidermal growth factor receptor,EGFR)家族成员,可以有效抑制肿瘤的生长,但目前应用的靶向治疗药物,只能作用于EGFR或HER-2,不能同时作用于二者,并且肿瘤多表达不同水平的EGFR及不同的EGFR家族成员,而表皮生长因子受体相关蛋白是EGFR及其家族成员的共同的抑制剂,有望成为有效的靶向治疗药物.     

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.     

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.     

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.     

Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha

Persistent hepatitis C virus (HCV) infection is associated with the development of human hepatocellular carcinoma (HCC), although the mechanism of HCV-related hepatocarcinogenesis remains unclear. Recently, however, the close relationships between the development of HCC and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) cascade have been described. In the present study, we investigated the effects of HCV core protein on this MAPK/ERK cascade. HCV core protein significantly activated the MAPK/ERK cascade, including Elk1. We also examined whether HCV core protein acted synergistically along with hepatocyte mitogen-mediated MAPK/ERK activation. Interestingly, Elk-1 activities were further enhanced by the tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate (TPA), but not by hepatocyte mitogens (epidermal growth factor [EGF] and transforming growth factor alpha [TGF-alpha]) in NIH3T3 cells and HepG2 cells expressing HCV core protein. Moreover, the MAPK/ERK activation by HCV core protein was blocked in the presence of the specific MEK1 inhibitor, PD98059. These results indicate that ERK activation by HCV core protein may be independent of hepatocyte mitogen-mediated signaling but synergistic with TPA, and HCV core protein may function at MEK1 or farther upstream of that component.     

Heparin-binding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function

Peptide growth factors have been implicated in progression of prostate cancer (PCa) to the androgen-independent state; however, much of the evidence linking diffusible mitogens and survival factors to this process remains circumstantial. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate stroma-derived factor, promotes survival, proliferation, and neuroendocrine differentiation of androgen-dependent LNCaP PCa cells in vitro. To test whether sustained exposure to HB-EGF can confer an androgen-independent phenotype, we generated stable populations of LNCaP cells that express constitutively a secreted form of HB-EGF (LNCaP/sHB). LNCaP/sHB cells proliferated more rapidly under androgen-depleted conditions in vitro and formed larger tumors with higher frequency in intact and castrated severe combined immunodeficient mice, in comparison to control cells. LNCaP/sHB tumors also expressed higher levels of the neuroendocrine marker, neuron-specific enolase, compared with control tumors. In castrates, increased neuron-specific enolase expression in LNCaP/sHB tumors was associated with reduced androgen receptor (AR) levels. In vitro, AR protein levels were reduced in LNCaP/sHB cells, and in transient transfection assays using an androgen-responsive promoter (mouse mammary tumor virus-long terminal repeat), LNCaP/sHB cells showed reduced sensitivity to dihydrotestosterone compared with controls. This is the first demonstration that continuous exposure of AR-positive PCa cells to a single growth factor can promote an androgen-independent phenotype in vivo. These findings also emphasize the potential role of pathways other than the AR axis in acquisition of androgen independence.     

Release and binding of epidermal growth factor in the pancreas of rats.

Previous studies showed that EGF is produced by salivary and duodenal glands and released in saliva and duodenal secretion. Using specific radioimmunoassay of EGF, this study showed that the salivary glands and duodenal mucosa contain high levels of EGF, reaching, respectively, about 38 and 4 micrograms/g of tissue weight. EGF immunoreactivity was also found in high amounts in the pancreatic tissue (20 micrograms/g) and the pancreatic juice (32 ng/mL), where the content of EGF was found to increase in response to feeding, cholecystokinin, or bombesin and to decrease after the administration of atropine and somatostatin. Studies on the binding of EGF revealed that pancreatic acinar membranes possess the specific and saturable EGF receptors with a high affinity sites with Kd of about 4.3 nM and binding capacity of about 62 fmol/mg of protein, and with low affinity sites with Kd of 21 nM and binding capacity of about 180 fmol/mg of protein. The observed high content of immunoreactive EGF in the pancreatic tissue and the presence of high and low affinity binding sites for EGF in the pancreatic acinar membranes, as well as the high EGF output in the pancreatic juice and its alterations in response to hormonal and postprandial stimulation, suggest an important role of EGF in pancreatic physiology.     

A variant epidermal growth factor receptor exhibits altered type alpha transforming growth factor binding and transmembrane signaling.

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. We compared the binding and biological actions of EGF and TGF-alpha in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibited two orders of binding sites with radioiodinated EGF (125I-EGF). Similar results were obtained with 125I-TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with 125I-TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhanced tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibited an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha (5 nM) inhibited growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates that binding characteristics of TGF-alpha.