Epidermal Growth Factor Receptor Immunoreactivity Research Articles

Transforming growth factor-α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain

Transforming growth factor α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity is observed in the majority of neurons, and in maturing astrocytes, in the developing and adult brain of humans and different species of animals. TGF-α and EGF-R co-localize in most neurons and maturing astrocytes, suggesting that most TGF-α producing cells are EGF-R-expressing cells. TGF-α and EGF-R immunoreactivity decrease in damaged areas following different insults. However, EGF-R appears in reactive glia, mostly reactive astrocytes, within and surrounding the damaged areas. TGF-α and EGF-R immunoreactivity is found in neurons of patients affected by Alzheimer's disease and other forms of dementia, and in neurons of patients suffering from epilepsy owing to different causes, thus pointing to the conclusion that TGF-α does not play a significant role in these pathologies. However, EGF-R immunoreactivity occurs in reactive astrocytes and microglia in subacute but not chronic lesions in human cases. Since TGF-α is a membrane-anchored growth factor, which may be cleaved leading to the formation of soluble forms, and both the membrane-anchored and soluble forms have the capacity to activate the EGF-R, it is feasible that TGF-α in the nervous system may act upon EGF-R-containing neurons through different mechanisms. In addition to distant effects resulting from the release of soluble TGF-α, local effects may be produced by establishing direct cell-to-cell contacts (juxtacrine stimulation), or in cells expressing both TGF-α and EGF-R (autocrine stimulation).

TGF-alpha immunoreactivity in laryngeal carcinoma: lack of prognostic value and correlation to EGF-receptor expression.

Transforming growth factor alpha (TGF-alpha) is a polypeptide that is structurally similar to epidermal growth factor (EGF) that binds to the epidermal growth factor receptor (EGFR) and has been implicated in the development of several types of human tumours. The expression of TGF-alpha is examined in laryngeal squamous cell carcinoma (SCC) (n = 24) and non-neoplastic polyps (n = 7) using streptavidin-biotin immunohistochemistry and a monoclonal antibody to the TGF-alpha protein. These cases had been previously characterized for EGFR immunoreactivity. The carcinomas were classified as well differentiated (n = 2), moderately differentiated (n = 16) and poorly differentiated (n = 6). Tissues from metastatic tumour deposits in lymph nodes (n = 5) were also studied. TGF-alpha overexpression was defined as intense immunoreactivity in more than two-thirds of tumour cells immunostained for TGF-alpha and was present in the majority of the SCC cases (n = 15; 63%) and metastatic tumour deposits (n = 4; 80%). In contrast, although some of the vocal cord polyps showed weak (n = 2) to moderate (n = 5) immunostaining, none had evidence of strong TGF-alpha immunoreactivity. The differences in TGF-alpha immunoreactivity were significant between primary laryngeal SCC and vocal cord polyps (P = 0.013; chi 2 test with continuity correction), and between metastatic laryngeal SCC and vocal cord polyps (P = 0.023; chi 2 test with continuity correction). There was no significant difference in TGF-alpha expression between the different grades of carcinomas (P = 0.92, chi 2 test) or between non-metastatic and metastatic carcinomas (P = 0.82; chi 2 test with continuity correction). No significant correlation was found between TGF-alpha expression and patient survival or tumour recurrence (r = 0.077, r2 = 0.006, P = 0.75; simple regression analysis), or between TGF-alpha expression and EGFR immunoreactivity (r = 0.325, r2 = 0.106, P = 0.0851). In conclusion, increased TGF-alpha immunoreactivity is present in most cases of laryngeal SCC with no specific relationship to tumour grade, suggesting that it may be important in the development of laryngeal carcinomas but not in its progression. No significant correlation was found between TGF-alpha and EGFR expression in laryngeal tumours and TGF-alpha immunoreactivity is of no prognostic value.

Increased Epidermal Growth Factor Receptor in fsn/fsn Mice

Epidermal growth factor receptors (EGF-Rs) are elevated in active human psoriatic lesions, but decrease in resolving lesions. Similar biologic responses in EGF-R levels have been demonstrated within human psoriatic skin grafted onto mice. We tested the hypothesis that flaky-skin mice (fsn/fsn), one proposed genetic animal model of psoriasis, would display EGF-R levels comparable to human psoriatic epidermis and show similar biologic responses. EGF-R levels were characterized in unperturbed sites in fsn/fsn skin and +/? skin by enzyme-linked immunosorbent assay, 125I-EGF binding, and immunostaining. Altered EGF-R levels were noted after mild trauma (tape stripping) or under resolving conditions (30 doses of 50 mJ/CM2 ultraviolet B, 2.5 mg/kg oral cyclosporin A, or daily 30 microg/ml topical EGF). Increased EGF-R immunostaining was observed in involved flaky epidermal sites before treatment. To determine whether a hyperproliferative (Koebner) reaction could be induced, we tape stripped fsn/fsn tail and non-flaky dorsal sites. EGF-R levels in dorsal epidermis increased by days 3-4 after injury by enzyme-linked immunoabsorbent assay methods. When fsn/fsn mice received one of three different treatments for 6 weeks, the skin returned to a normal phenotype both grossly and microscopically. Immunoreactive EGF-R in treated, but not untreated, sites decreased to either normal or nondetectable levels. These data indicate that fsn/fsn mice exhibit an EGF-R profile identical to that of lesional and nonlesional human psoriatic epidermis. Modulations of the flaky phenotype in response to injury and three different treatments suggest that fsn/fsn is a useful in vivo model for examining new treatment modalities for psoriasiform skin diseases.

Single-strand conformation polymorphism analysis of the epidermal growth factor receptor at codon 497.

A variant human epidermal growth factor (EGF) receptor (HER) with a transition (G to A) at codon 497, resulting in a substitution of a lysine for an arginine, was recently demonstrated in several human pancreatic cancer cell lines. In the present study, we compared the frequency of expression of wild-type (HER497R) and variant (HER497K) EGF receptors in normal and malignant pancreatic tissue samples using single-strand conformation polymorphism analysis. Both receptors were expressed in normal (42%) and malignant (46%) tissues. Three samples (two normal and one cancer) expressed only HER497K. The cancer sample that was homozygous for HER497K exhibited strong EGF receptor immunoreactivity. The high frequency of HER497K expression suggests that it is due to a relatively common polymorphism in the HER coding region. Its presence in some of the cancer samples suggests that, like the wild-type receptor, HER497K also has a role in pancreatic cancer.

EGF-receptors in human normal and pathological thyroid tissue.

Expression of the epidermal growth factor receptor (EGFR) was studied in cryosections from human thyroid tissues. Normal tissue (4 cases), nodular goitre (12), toxic goitre (9), adenoma (9), follicular carcinoma (1), papillary carcinoma (7) and poorly differentiated carcinoma (1) were used for immunohistochemistry. Northern blot analysis was performed in two nodular goitres, three adenomas, two papillary carcinomas, one follicular carcinoma and the adjacent normal tissue in five cases as well as in two cell lines from anaplastic carcinomas. Epidermal growth factor receptor immunoreactivity was detected in all tissues examined. The amount of EGFR mRNA did not differ between normal and abnormal tissues. However, the EGFR staining was weaker in normal thyroid tissue compared to the adjacent neoplastic areas suggesting an upregulation at the posttranslational level in the latter. A strong staining was also seen in hyperfunctioning thyroid glands. The EGFR location was mainly basal or basolateral in all thyroid tissues with normal histology and in toxic diffuse goitre. Pericellular and sometimes cytoplasmatic staining was seen in neoplastic tissues. In nodular goitre the staining was both basal, lateral and apical and varied in intensity. Our data suggest that a non-polarized location of EGFR probably indicates a loss of the normal epithelial cell polarity and could be interpreted as an early sign of dedifferentiation. Furthermore, a role for the EGFR is proposed, not only in the development of thyroid neoplasias but also in goitre formation.

Overexpression of transforming growth factor-alpha and epidermal growth factor receptor, but not epidermal growth factor, in exocrine pancreatic tumours in hamsters.

Using immunohistochemistry, Northern blotting and a semi-quantitative PCR technique, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) expression were studied in the pancreas of N-nitrosobis(2-oxopropyl)-amine (BOP)-treated hamsters. After initiation pancreatic carcinogenesis was modulated by a high fat diet or by injections with the cholecystokinin analogue caerulein. Autopsies were performed 6 and 12 months after the last injection with BOP. Immunohistochemistry revealed a weak expression of TGF-alpha in nomal acinar cells and a stronger expression in ductular and centro-acinar cells. Over-expression of TGF-alpha was observed in advanced putative preneoplastic lesions (classified as borderline lesions) and in ductular adenocarcinomas. EGFR immunoreactivity was present only in ductular adenocarcinomas. EGF peptide expression was observed both in acinar and ductular normal and tumorous cells and the level of expression did not change significantly during carcinogenesis. Moreover, the post-initiation treatments did not cause differences in EGF, TGF-alpha or EGFR peptide or mRNA levels, except for a significantly lower expression of TGF-alpha mRNA in hamsters fed a high fat diet when compared with those fed a low fat diet. TGF-alpha mRNA levels increased, whereas EGF mRNA levels decreased significantly in total pancreatic homogenates of BOP-treated hamsters in comparison with untreated controls. Also, in ductular adenocarcinomas TGF-alpha and EGFR (but not EGF) mRNA levels were significantly higher than in normal pancreatic homogenates. In pancreatic homogenates obtained 6 months after the last BOP injection, these differences were less pronounced in comparison with those obtained after 12 months. The present results indicate that TGF-alpha (but not EGF) might act in a paracrine or autocrine manner in pancreatic tumours in BOP-treated hamsters via simultaneously expressed EGFR. However, TGF-alpha, EGF and EGFR do not seem to be involved in the modulating effects of a high fat diet or caerulein treatment on pancreatic carcinogenesis in BOP-treated hamsters.

Transforming growth factor-alpha, epidermal growth factor, and epidermal growth factor receptor in human testis obtained from biopsy and castration: immunohistochemical study.

We analyzed expression of transforming growth factor (TGF)-alpha, epidermal growth factor (EGF) and their receptor, EGF receptor (EGFR), by immunohistochemistry in the human testis to determine the possible roles of these growth factors in human testicular function. Specimens were obtained from 17 patients including 9 patients with infertility, 4 patients with prostatic carcinoma and 4 patients with contralateral testicular tumor. EGF immunoreactivity was positive in the hyperplasic Leydig cells of one patient but negative in the other cases. On the other hand, strong TGF-alpha immunoreactivity was observed in Leydig cells, with weak staining in Sertoli cells and germ cells in cases with normal spermatogenesis. EGFR immunoreactivity was observed in the Leydig and peritubular cells, appearing as membrane staining. Marked immunoreactivity for TGF-alpha was observed in the Sertoli cells in testes with decreased spermatogenesis, especially in the Sertoli-cell-only syndrome. This finding may indicate a compensatory increase of TGF-alpha expression in the Sertoli cells accompanying a decrease in spermatogenesis. No significant correlation was found between the degrees of spermatogenesis and immunolocalization of the EGF receptor. These findings suggest that TGF-alpha is a locally produced growth factor that is involved in spermatogenesis in the human testis via an autocrine and/or paracrine mechanism.

Epidermal Growth Factor Receptor Immunoreactivity in Gallbladder and Extrahepatic Biliary Tract Tumours

Elevated levels of epidermal growth factor receptor (EGFR), a transmembrane protein tyrosine kinase receptor, are found in carcinomas from various sites of the body. We investigated the expression of EGFR in carcinoma of the gallbladder (n = 13), common bile duct (n = 7) and ampulla of Vater (n = 9). Non-malignant conditions investigated include chronic cholecystitis (n = 11), gallbladder dysplasia (n = 3) and adenoma (n = 1), and ampullary carcinoma in situ (CIS) (n = 2). Routine immunohistochemistry was employed using a monoclonal antibody against the EGFR protein. Immunostaining was assessed according to both intensity and extent of staining of cells. There was strong immunoreactivity for all gallbladder carcinoma and adenoma, the majority of common bile duct (n = 6; 86%) and ampullary (n = 6; 67%) carcinoma. In contrast, all cases of gallbladder dysplasia, the majority of cases of chronic cholecystitis (n = 7; 64%) and ampullary CIS (n = 2; 67%) had only weak to moderate immunoreactivity. The pattern of immunoreactivity was one of diffuse cytoplasmic and membranous staining. In conclusion, EGFR expression is increased in both gallbladder, common bile duct and ampullary carcinomas but not in non-malignant conditions of the gallbladder and biliary tract. These findings suggest that EGFR overexpression occurs late in the sequential development of gallbladder and biliary tract cancers.

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations.

We have assessed the multiple biological variables on breast carcinoma FNA specimens using a Cytoblock technique. The growth fraction (MIB1), oestrogen receptor (ER), progesterone receptor (PR), p53 mutant protein, c-erbB-2, epidermal growth factor receptor (EGFR), NCRC11/epithelial membrane antigen (EMA) and DNA plopidy were examined. Objective quantification using image analysis (CAS 200) was applied as appropriate. Fifty cases were examined in this preliminary study. Excellent correlation between the Cytoblock preparations and parallel tissue sections was seen. Of the cancers, 81% were aneuploid with only 19% diploid in character, but 67% of the carcinomas were of histological grade 3. The mean nuclear area staining with MIB1 was 31.3% and with ER was 26.7%. Twenty-four percent (24.1%) of the nuclear area showed immunoreactivity with PR. Significant EGFR and EMA, respectively. A significant association between histological grade of the resected tumours and both MIB1 (P = 0.04) and EGFR (P = 0.02) expression in the Cytoblock samples was seen. p53 (P = 0.03) and EGFR (P = 0.01) immunoreactivity showed an association with tumour size. EGFR (P = 0.04) immunostaining also showed a relationship with the lymph node status of the patient. The technique is, we believe, a useful one for the assessment of multiple variables on breast cytology specimens; these preliminary data suggest that some of these may be useful in predicting prognosis in breast cancer patients.

Expression of amphiregulin and transforming growth factor alpha mRNA in gestational tissue at term

Among epidermal growth factor (EGF) family growth factors, EGF, transforming growth factor-α (TGF-α) and amphiregulin (AR) are considered to exert their effects through binding to epidermal growth factor receptor (EGFR). Cripto (CR) is homologous to EGF in its structure, but it is postulated that it might not bind with EGFR. We examined localization of TGF-α, AR, CR and EGFR in human chorionic villi of various stages. Chorionic villi were collected from thirty-four cases. Immunohistochemistry was performed on 4% paraformaldehyde fixed and paraffin embedded specimens using an avidin biotin complex-perioxidase technique. TGF-α immunoreactivity was intense in the cytoplasm of the cytotrophoblast (CT), especially in first trimester. AR immunoreactivity was observed predominantly in the nuclei of the CT and some syncytiotrophoblast (ST) only in first trimester. EGFR immunoreactivity was intense in the ST, and weak in the CT and its frequency appeared to decrease as gestational period progressed. CR immunoreactivity was mainly observed in the ST, but marked heterogeneity was observed in its expression and its frequency appeared to increase as pregnancy progressed. Based on these findings, TGF-α is considered to act mainly in a paracrine manner in first trimester chorionic villi. AR is produced to modify the biological function of the trophoblast cells during the first trimester. In contrast, CR may modify ST function, especially during the third trimester.

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

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

Emergence of salivary gland cell lineage diversity suggests a role for androgen-independent epidermal growth factor receptor signaling.

Diversity of cell lineages within glandular organs is generated postnatally by differentiation of committed progenitor cells. Fundamental regulatory aspects of this process are not understood. The mouse submandibular salivary gland (SSG) served as model to assess the role of epidermal growth factor (EGF) receptor signaling during emergence of cell lineage diversity. Temporal fluctuations in EGF receptor mRNA levels coincident with crucial differentiative cell lineage transitions were revealed by RNase protection analyses. Between days 2 and 5, when proacinar cells are maturing and striated duct cells emerge, EGF receptor mRNA levels were highest and all differentiating cells exhibited EGF receptor immunoreactivity. EGF receptor mRNA levels then declined sharply and immunoreactivity became confined to ductal cells. At day 11 in male mice, and days 11 and 16 in females, a second increase in EGF receptor mRNA was detected coincident with emergence of granular convoluted tubule (GCT) cells. With completion of androgen-dependent GCT cell differentiation at the onset of puberty, EGF receptor mRNA levels and intensity of immunoreactivity decreased. Androgen effects on EGF receptor mRNA or immunoreactivity could not be detected. These temporally distinct patterns of EGF receptor expression suggest that this signaling pathway is a mechanism of potential importance in emergence of cell lineage diversity in a glandular organ.

Preparation of a monoclonal antibody to a glycidic epitope of the epidermal growth factor receptor that recognizes inhibitors of astrocyte proliferation and reactive microglia.

A mouse monoclonal antibody (5B9), directed against a carbohydrate epitope of human epidermal growth factor receptor (EGFR), recognized an 81-kDalton glycoprotein in buffer-soluble and detergent-solubilized rat brain extracts (BE). The glycoprotein was more abundant in extracts prepared from injured brain than in those from normal tissue. Removal from BE of the antigens recognized by 5B9 increased their astrocyte mitogenic activity. Sections of injured rat brain and cultures derived from damaged brain, enriched in microglia, showed 5B9 immunoreactivity in ED1-positive cells. The abundance of the glycoprotein recognized by 5B9 in injured, relative to normal, tissue, suggested that molecules with EGFR immunoreactivity may be expressed in reactive microglial cells and released after injury.

Comparative studies of epidermal growth factor, epidermal growth factor receptor, K-ras, and P53 in gallbladder carcinomas with and without an anomalous junction of pancreticobiliary duct

AIM: We reported a 6 to 10 times higher incidence of gallstone disease in curatively gastrectomized patients for cancer (Gastroenterology 104: A362, 1993) and a possible factor for the lithogenesis to be bile infection (Gastroenterology 106: A340, 1994). The aim of this study is to experimentally investigate whether bile infection and postgastrectomy gallstone disease is related with methods of gastrectomy for cancer, i.e., a conservative pylorus-preserving gastrectomy (PPG) and a radical Billroth II subtotal gastrectomy (B-II). METHOD: Five mongrel dogs underwent PPG with the truncal vagi and the hepatic branch intact and 6 dogs underwent B-I1 with wide bursectomy and bilateral truncal vagotomy. Other 6 dogs undergoing only laparotomy (sham operation) were served as the controls. The dogs were maintained with an ordinary laboratory canine diet for 1 year. Gallbladder (GB) bile was collected by sterile fine needle puncture during laparotomy at the time of surgery, 6 months and 12 months. The obtained bile was cultured for bacteriology and analyzed for chemical constituents. Bile acids were quantified by GLC. The presence or absence of gallstone (GS) was consecutively examined by ultrasound at 2 week intervals and by inspection of the aspirated bile. GS's were examined with infrared spectral analysis for classification. RESULTS: The PPG and control dogs incurred no bile infection nor gallstone disease. Three Of the 6 B-II dogs incurred bile infection (E. coli, Bacteroides and Enterococcus). GS's (black stones containing calcium bilirubinate) developed in 3 of the 6 B-II dogs (1 identified at 6 months and 2 at 12 months). Two of the 3 B-II dogs with GS were with bile infection but one without. Total bile acids and bile acid fractions did not significantly differ among the three animal groups. Free bile acids was detected in all of the B-II dogs and in 2 of the 5 PPG dogs but in none of the control dogs. Lithogenicity calculated was within the micellar zone for all dogs. CONCLUSION: Calcium bilirubinate stone developed only in the B-II gastrectomized dogs. Positive bacterial culture and detection of free bile acids were most common in the B-II dogs, which suggested that (1) bile infection is involved in postgastrectomy gallstone disease, (2) B-II subtotal gastrectomy is more liable to bile infection and gallstone disease and (3) PPG may preserve a better sphincter of Oddi motor function, thereby preventing retrograde biliary infection. COMPARATIVE STUDIES OF EPIDERMAL GROWTH FACTOR, EPIDERMAL GROWTH FACTOR RECEPTOR, K-RAS, AND P53 IN GALLBLADDER CARCINOMAS WITH AND WITHOUT AN ANOMALOUS JUNCTION OF PANCREATICOBILIARY DUCT. Keiji Hanada, Masaki Itoh, Kiyomu Fujii, Akira Tsuchida, Shouhei Ishimaru, Manabu Hirata, Toshiyasu Iwao, and Goro Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. AIMS. Previously, we reported that mutations of Kras and p53 contributed to carcinogenesis in gallbladder mucosa with an anomalous junction of the pancreaticobiliary duct (AJPBD). In this study, we examined the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR), and mutations of K-ras or p53in gallbladder carcinoma with and without AJPBD. METHODS. We used l0 gallbladder carcinomas with AJPBD (Group A), and 25 gallbladder carcinomas without AJPBD (Group B). EGF and EGFR were examined using immnohistochemistry. Genomic DNA was extracted from routinely processed tissues. Polymerase chain reaction (PCR) restriction fragment length polymorphism was performed for mutations of K-ras. Mutations of p53 were examined using PCR single strand conformation polymorphism. RESULTS. The percentage of immunoreactivity for EGF and EGFR in Group A (70%) was higher than that in Group B (40%). The incidence of mutation of Kras in Group A (30%) was higher than that in Group B (7%). As for p53, although mutations were found in only exon 7 and 8 in Group A, they were found in exon 5, 6, and 7 in Group B. CONCLUSIONS. i) The autocrine loop of EGF and EGFR may play an important role in tumor growth of gallbladder carcinomas associated with AJPBD. 2) Mutations of K-ras may strongly contribute to carcinogenesis of gallbladder carcinomas associated with AJPBD. 3) Mutation spots of p53 in gallbladder carcinomas associated with AJPBD may be different from that in gallbladder carcinomas without AJPBD.

Epidermal growth factor receptor (EGFr) expression in non-small cell lung carcinomas correlates with metastatic involvement of hilar and mediastinal lymph nodes in the squamous subtype

Epidermal growth factor receptor (EGFr) levels were evaluated in paraffin-embedded tumour specimens of non-small cell lung cancer (NSCLC) from 176 patients who underwent surgical resection. The EGFr expression was evaluated by immunocytochemical assay using a monoclonal antibody which recognises the external domain of the receptor. EGFr immunoreactivity was significantly higher in squamous than in non-squamous cell carcinomas (P = 0.0009). Hilar and/or mediastinal nodal involvement was found in 29 of 105 (27.4%) squamous cancers, and in this group of patients, the mean of EGFr positive cells was significantly higher than that of patients without nodal involvement (P = 0.01). No significant correlations were found between the expression of EGFr and other clinicopathological or biological parameters such as T-status, grading, proliferative activity. EGFR is suggested to represent a useful indicator of nodal metastasis in NSCLC.

Vascular Endothelial Growth Factor and Glioma Angiogenesis: Coordinate Induction of VEGF Receptors, Distribution of VEGF Protein and Possible In Vivo Regulatory Mechanisms

We have previously suggested that tumor angiogenesis in human gliomas is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and flt-1 (VEGF-receptor 1). VEGF, an endothelial-cell-specific mitogen, is abundantly expressed in glioma cells which reside along necrotic areas, whereas flt-1, a tyrosine-kinase receptor for VEGF, is expressed in tumor endothelial cells, but not in endothelial cells in normal adult brain. Recently, a second tyrosine-kinase receptor which binds VEGF with high affinity, designated KDR or flk-1, has been described. We performed in situ hybridization for VEGF mRNA, flt-1 mRNA and KDR mRNA on serial sections of normal brain, low-grade and high-grade glioma specimens. We show that KDR mRNA is co-expressed with flt-1 in vascular cells in glioblastoma but not in low-grade glioma. Since flt-1 and KDR are not expressed in endothelial cells in the normal adult brain, the coordinate up-regulation of 2 receptors for VEGF appears to be a critical event which controls tumor angiogenesis. Immunocytochemistry with a monoclonal anti-VEGF antibody revealed significant amounts of VEGF protein in the same glioma cells that expressed VEGF mRNA. The largest amount of VEGF immunoreactivity, however, was detected on the vasculature of glioblastomas, the site where VEGF exerts its biological functions. These findings suggest that VEGF is produced and secreted by glioma cells and acts on tumor endothelial cells which express VEGF receptors. To further characterize VEGF-producer cells in vivo, we investigated cellular proliferation, immunoreactivity to the p53 tumor-suppressor gene product and epidermal-growth-factor-receptor (EGFR) expression on serial sections by immunocytochemistry. VEGF-producer cells did not show increased cellular proliferation, p53 immunoreactivity or EGFR immunoreactivity as compared with glioma cells which did not express VEGF. Our studies therefore do not demonstrate evidence for a growth advantage of VEGF-producer cells in vivo or VEGF induction by p53 mutation or EGFR over-expression.

Immunocytochemical study of epidermal growth factor receptor, transforming growth factor alpha, and “squamous differentiation” in human endometrial carcinoma

The expression of epidermal growth factor receptor (EGFr) and transforming growth factor alpha (TGF-α) was compared with the presence of “squamous differentiation” (SD) visualized in various histotypes of endometrial carcinoma by using a panel of monoclonal antibodies. The results of the current study demonstrate that EGFr and TGF-α are present in routinely processed endometrial carcinoma. The highest positive EGFr and TGF-α expression was seen in the group of adenocarcinomas with SD. The more intense EGFr and TGF-α immunoreactivity was observed in “squamous” foci both in adenoacanthomas (AA) and in adenosquamous carcinomas (AS). These EGFr- and TGF-α-positive squamous areas prevalently displayed a “stratification-related” cytokeratin (CK) immunoprofile characterized by the expression of CKs 1, 4, 5, 10, 13, 14, and 16. No correlation was found between EGFr- and TGF-α-positive status and depth of myometrial invasion or surgical stage. These results clearly demonstrate that EGFr and TGF-α expression is related remarkably to endometrial carcinoma with “squamous” areas both morphologically and immunophenotypically. This specific association leads us to suggest that EGFr and TGF-α expression in endometrial carcinoma may be prevalently involved in the equilibrium of cell differentiation of the “squamous” foci commonly observed in this group of neoplasias.

Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms.

We have previously suggested that tumor angiogenesis in human gliomas is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and flt-1 (VEGF-receptor 1). VEGF, an endothelial-cell-specific mitogen, is abundantly expressed in glioma cells which reside along necrotic areas, whereas flt-1, a tyrosine-kinase receptor for VEGF, is expressed in tumor endothelial cells, but not in endothelial cells in normal adult brain. Recently, a second tyrosine-kinase receptor which binds VEGF with high affinity, designated KDR or flk-1, has been described. We performed in situ hybridization for VEGF mRNA, flt-1 mRNA and KDR mRNA on serial sections of normal brain, low-grade and high-grade glioma specimens. We show that KDR mRNA is co-expressed with flt-1 in vascular cells in glioblastoma but not in low-grade glioma. Since flt-1 and KDR are not expressed in endothelial cells in the normal adult brain, the coordinate up-regulation of 2 receptors for VEGF appears to be a critical event which controls tumor angiogenesis. Immunocytochemistry with a monoclonal anti-VEGF antibody revealed significant amounts of VEGF protein in the same glioma cells that expressed VEGF mRNA. The largest amount of VEGF immunoreactivity, however, was detected on the vasculature of glioblastomas, the site where VEGF exerts its biological functions. These findings suggest that VEGF is produced and secreted by glioma cells and acts on tumor endothelial cells which express VEGF receptors. To further characterize VEGF-producer cells in vivo, we investigated cellular proliferation, immunoreactivity to the p53 tumor-suppressor gene product and epidermal-growth-factor-receptor (EGFR) expression on serial sections by immunocytochemistry. VEGF-producer cells did not show increased cellular proliferation, p53 immunoreactivity or EGFR immunoreactivity as compared with glioma cells which did not express VEGF. Our studies therefore do not demonstrate evidence for a growth advantage of VEGF-producer cells in vivo or VEGF induction by p53 mutation or EGFR over-expression.

Expression of transforming growth factor-alpha and epidermal growth factor receptor is increased following bleomycin-induced lung injury in rats.

To investigate the potential role of transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R) in the fibroproliferative response to acute lung injury, we determined lung steady-state TGF-alpha and EGF-R mRNA levels, TGF-alpha protein levels, and the distribution of TGF-alpha and EGF-R immunoreactive protein of bleomycin-injured and control rat lungs. At 2 and 4 days after a single intratracheal injection of bleomycin, TGF-alpha mRNA levels increased to 159% and 184% of control values, respectively. EGF-R mRNA levels increased to 163%, 314%, and 170% of control values at 1, 7, and 14 days after bleomycin instillation. TGF-alpha protein levels in whole lung extracts increased to 230% of control values at 4 days after bleomycin administration. TGF-alpha and EGF-R immunoreactivity was detected in macrophages, alveolar septal cells, and airway epithelium of control and bleomycin-injured animals with an apparent increase in the intensity and number of specifically immunostained cells following lung injury. TGF-alpha and EGF-R immunoreactive proteins were detected in foci of cellular proliferation and in areas of intraalveolar fibrosis. We conclude that TGF-alpha and the EGF-R are present in normal and bleomycin-injured rat lung and that the expression of this growth factor and its receptor are up-regulated following lung injury. These results suggest that increased expression of TGF-alpha and the EGF-R may be an important mechanism that modulates the fibroproliferative response to acute lung injury.

Expression of epidermal growth factor receptor (EGFr) immunoreactivity in human cutaneous nerves and sensory corpuscles.

The epidermal growth factor receptor (EGFr) binds both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha), which are currently considered among putative growth factors playing a role in the nervous system. EGFr and their ligands have been localized in the mammalian peripheral nervous system. The present study was undertaken to investigate whether nerves and sensory corpuscles supplying human glabrous skin express EGFr. Formaldehyde fixed, paraffin embedded samples of finger-tip digital skin obtained from adult healthy subjects were processed for indirect PAP immunohistochemistry using a monoclonal antibody against an epitope of the intracellular domain of EGFr. To ascertain the localization of EGFr immunoreactivity, neurofilament proteins (NFP), S100 protein (S100P), and epithelial membrane antigen (EMA) were studied in parallel to label axons, Schwann cells, and perineurial cells, respectively, as well as their corpuscular derivatives. A variable intensity of EGFr immunostaining was regularly observed in the perineurium and Schwann cells, and occasionally in the axons of nerve bundles. EGFr immunoreactivity was also present in the axon and lamellar cells of Meissner corpuscles, and within the axon, inner-core, outer-core, and capsule of Pacinian corpuscles. Present results demonstrate that human cutaneous nerves and sensory corpuscles express EGFr suggesting a role for peptides able to bind EGFr, i.e., EGF and TGF alpha, in the human peripheral nervous sensory system.