A new model for the interaction of EGF-like ligands with their receptors: the new one-two.
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Epidermal Growth Factor↗
Biomedical subjects
W J Gullick
Publications and source records attributed to W J Gullick.
Pre-implantation diagnosis of inherited predisposition to cancer.
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Female↗
c-erbB-3 and c-erbB-2 protein expression in node-negative breast carcinoma--an immunocytochemical study.
The type I growth factor receptor family has been found to play an important role in the control of normal growth and differentiation. Moreover, the epidermal growth factor receptor and the c-erbB-2 oncogene seem to be implicated in the pathogenesis and behaviour of several cancers, including breast cancer. c-erbB-3 is a new member of the type I receptor family for which there is currently little information available on its expression in neoplastic tissues, and on its possible prognostic significance. This study was undertaken to define the prognostic value of c-erbB-3 expression in a series of node-negative breast cancer (NNBC) patients when compared, by multivariate analysis, with expression of the c-erbB-2 protein and conventional clinicopathological features. cerbB-3 was recognised by the novel monoclonal antibody RTJ1, whereas c-erbB-2 was detected by the polyclonal antibody 21N, using immunocytochemical methods. We found that overexpression of c-erbB-3 occurs frequently in NNBC. Overall, 138 of 212 carcinomas (65%) had some degree of membrane RTJ1 staining, and 28 (13%) showed strong and generalised positivity ( ). Twenty-four per cent of carcinomas had membrane 21N staining, and 12% presented strong and generalised positivity ( ). c-erbB-3 protein expression was significantly associated only with that of c-erbB-2 (P = 0.05), whereas 21N positivity was significantly associated with small tumour size (P = 0.02) and ductal histotype (P = 0.04). No significant correlation between expression of either receptor proteins or relapse-free survival was observed after a median follow-up of 63 months. Applying multivariate analysis, only tumour size approached significance. Our results indicate that analysis of expression of c-erbB-3 and c-erbB-2 alone do not seem to be useful in identifying patients with NNBC at different risk of relapse or death.
Adult↗
Expression of transforming growth factor alpha, amphiregulin and cripto-1 in human breast carcinomas.
The expression of three epidermal growth factor (EGF)-related peptides, transforming growth factor alpha (TGF-alpha), amphiregulin (AR) and cripto-1 (CR-1), was examined by immunocytochemistry (ICC) in 68 primary infiltrating ductal (IDCs) and infiltrating lobular breast carcinomas (ILCs), and in 23 adjacent non-involved human mammary tissue samples. Within the 68 IDC and ILC specimens, 54 (79%) expressed immunoreactive TGF-alpha, 52 (77%) expressed AR and 56 (82%) expressed CR-1. Cytoplasmic staining was observed with all of the antibodies, and this staining could be eliminated by preabsorption of the antibodies with the appropriate peptide immunogen. Cytoplasmic staining with all of the antibodies was confined to the carcinoma cells, since no specific immunoreactivity could be detected in the surrounding stromal or endothelial cells. In addition to cytoplasmic reactivity, the AR antibody also exhibited nuclear staining in a number of the carcinoma specimens. No significant correlations were found between the percentage of carcinoma cells that were positive for TGF-alpha, AR or CR-1 and oestrogen receptor status, axillary lymph node involvement, histological grade, tumour size, proliferative index, loss of heterozygosity on chromosome 17p or overall patient survival. However, a highly significant inverse correlation was observed between the average percentage of carcinoma cells that expressed AR in individual tumours and the presence of a point-mutated p53 gene. Likewise, a significantly higher percentage of tumour cells in the ILC group expressed AR as compared with the average percentage of tumour cells that expressed AR in the IDC group. Of the 23 adjacent, non-involved breast tissue samples, CR-1 could be detected by ICC in only three (13%), while TGF-alpha was found in six (26%) and AR in ten (43%) of the non-involved breast tissues. These data demonstrate that breast carcinomas express multiple EGF-related peptides and show that the differential expression of CR-1 in malignant breast epithelial cells may serve as a potential tumour marker for breast cancer.
Amphiregulin↗
A monoclonal antibody to the human c-erbB3 protein stimulates the anchorage-independent growth of breast cancer cell lines.
The c-erbB3 protein is a member of the type I growth factor receptor family. It has a widespread pattern of expression in normal tissues and is overexpressed in about 20% of breast cancers. We have raised a specific monoclonal antibody, called SGP1, against the extracellular domain of c-erbB3 which recognises the native form of the protein. The monoclonal antibody was found to modestly but significantly stimulate the anchorage-independent cloning efficiency of the breast tumour cell lines BT483 and T47D, both of which express the c-erbB3 protein. No effect was observed on 293 cells lacking expression, nor did a control isotype-matched antibody promote the growth of any of the cells tested. These results suggest that the c-erbB3 protein may normally act as a growth factor receptor.
Animals↗
eph, the largest known family of putative growth factor receptors.
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Amino Acid Sequence↗
Frequent immunohistochemical detection of EGF supergene family members in ovarian carcinogenesis.
Primary and metastatic ovarian cystadenocarcinomas, carcinomas of low malignant potential (borderline tumors), benign ovarian cystadenomas, and normal ovaries were compared for immunoperoxidase detection of the ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), cripto, and the receptors, epidermal growth factor receptor (EGF-R), and c-erbB-2. This matrix analysis of these EGF family members indicated no specific pattern of ligand or receptor expression with a specific ovarian histologic category except in the case of AR and TGF-alpha. AR was detected almost exclusively in borderline tumors, suggesting that these tumors may not arise as a pathological continuum between benign cystadenomas and invasive cystadenocarcinomas. Second, the presence of TGF-alpha immunoreactivity in the absence of coexpression of cripto or EGF appeared to be associated only with adenocarcinomas of high grade and stage.
Amphiregulin↗
Production of antisera to synthetic peptides.
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Animals↗
Characterization of high and low molecular weight forms of amphiregulin that differ in glycosylation and peptide core length. Evidence that the NH2-terminal region is not critical for bioactivity.
Human amphiregulin (AR) is a polypeptide growth regulator which acts by binding to and activating the epidermal growth factor (EGF) receptor tyrosine kinase. AR consists of an EGF-like domain and an NH2-terminal extension which contains potential glycosylation sites and nuclear localization signals. Two high molecular weight species which had molecular masses of approximately 16.5 kDa (HMW-AR1 and HMW-AR2) and a approximately 9.5-kDa low molecular weight form (LMW-AR) were isolated from the conditioned medium of phorbol 12-myristate 13-acetate-treated MCF-7 human breast carcinoma cells by sequential heparin affinity, immunoaffinity, and reverse phase-high performance liquid chromatography. HMW-AR1 and HMW-AR2 were found to possess complex or hybrid type N-linked oligosaccharide structures that contained sialic acid. Additionally, HMW-AR1 and HMW-AR2 contained the disaccharide, Gal beta(1-->3)GalNAc, linked to Ser/Thr residues. No carbohydrate moieties were detected in LMW-AR. Mapping of the peptide cores of these molecules using antipeptide antibodies revealed that HMW-AR1 and HMW-AR2 were intact molecules, whereas LMW-AR contained the EGF-like domain, but possessed a truncated NH2-terminal extension. LMW-AR, HMW-AR1, and HMW-AR2 were all found to be potent stimulators of DNA synthesis in MCF-10A human mammary epithelial cells. These results suggest that the NH2-terminal region of the AR molecule is not critical to the ability of AR to activate the EGF receptor tyrosine kinase.
Amino Acid Sequence↗
Expression of the c-erbB-3 gene product in gastric cancer.
The pattern of c-erbB-3 gene product was studied in 91 advanced gastric carcinomas, adjacent hyperplastic mucosa, intestinal metaplasia and dysplasia and in normal controls, using immunohistochemistry in archival material. All tumours showed positive c-erbB-3 staining in both cytoplasm and membrane. No significant differences of expression were observed between intestinal and diffuse-type carcinomas or any other clinical parameters. Of interest is the expression in the adjacent mucosa, which is extensive, cytoplasmic, and of lower intensity than in the tumours. Further studies are currently being carried out to clarify the role of this protein in tumour behaviour and gastric carcinogenesis.
Adult↗
Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media.
The present studies were aimed at determining if the use of a cell culture medium that supports proliferation of human mammary epithelial cells of the luminal lineage would allow routine isolation of breast cancer cells from primary and metastatic tumor specimens. Results obtained with mammary epithelial cells derived from reduction mammoplasty specimens and primary breast carcinomas indicated that growth of cells on type I collagen-coated dishes in Ham's F-12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, cholera toxin, and 5% fetal bovine serum resulted in the growth and serial passage of cells that stained positively for the luminal cell marker cytokeratin 19. By contrast, growth of mammary epithelial cells in a growth factor-supplemented serum-free medium resulted in the emergence of mammary epithelial cell colonies that were uniformly negative for keratin 19. Filter isolation methods were used to isolate individual keratin-19-positive colonies from primary cultures derived from breast cancer specimens. All of the luminal mammary epithelial cells isolated from breast cancer tissues expressed characteristics of normal cells. Keratin-19-positive colonies isolated from several different tumors all grew rapidly for 30 to 60 days in culture and then senesced. Cells were isolated from one tumor that was known to have undergone a loss of heterozygosity at a specific locus in the p53 gene. All colonies isolated from this specimen contained both p53 alleles, which was consistent with their origin from normal luminal cells. Cells were also isolated from one tumor in which the c-erbB2 protein was drastically overexpressed in the neoplastic cells. Once again, keratin-19-positive colonies isolated from this tumor did not overexpress the c-erbB-2 protein. Experiments were then performed with cells derived from pleural effusions and metastatic lymph nodes. Results obtained with these specimens indicated that the growth conditions that support the growth of normal luminal mammary epithelial cells do not support the growth of neoplastic cells. However, the omission of cholera toxin, epidermal growth factor, and type I collagen substratum resulted in the isolation of two long-term cell lines. Both cell lines have population doubling times of approximately 100 h, are hyperdiploid, and stain positively for cytokeratin 19. Thus, culture conditions that support the growth of normal luminal mammary epithelial cells do not, in general, support the growth of breast cancer cells.
Base Sequence↗
Intrahepatic distribution of transforming growth factor-alpha (TGF alpha) during liver regeneration following carbon tetrachloride-induced necrosis.
The distribution of transforming growth factor-alpha (TGF alpha) in rat liver during regeneration was studied immunohistochemically using two antibodies, one a polyclonal (26T) raised against a synthetic peptide corresponding to the 17 C-terminal amino acids of the mature rat protein, and the other a monoclonal (Ab-2) raised against recombinant human protein. In normal liver, immunoreactive TGF alpha was detected in perivenular hepatocytes using both antibodies. No sinusoidal cells were found to contain the peptide. In response to carbon tetrachloride (CCI4)-induced necrosis, an initial increase in the intensity of immunoreactivity was noted at 24 h following exposure to the toxin. This coincided with the period immediately preceding the peak of hepatocyte proliferation; Ab-2 immunoreactive cells outnumbered 26T-positive cells. Thereafter there was a reduction in the number of TGF alpha-positive cells, but by day 4 the level of immunoreactivity had returned to that of normal liver. Using bromodeoxyuridine labelling, spatial and temporal relationships between TGF alpha expression and cell proliferation were identified, supporting the concept that this peptide plays an important role in the in vivo regenerative response to hepatic injury via an autocrine mechanism.
Animals↗
Expression of the c-erbB-3 protein in gastrointestinal tract tumours determined by monoclonal antibody RTJ1.
We have examined the expression of the c-erbB-3 protein in a wide range of tumours of the gastrointestinal (GI) tract using immunocytochemical staining. Two antibodies were employed, a polyclonal antibody, 49.3, and a new monoclonal antibody, RTJ1, both raised to a synthetic peptide from the cytoplasmic domain of the human c-erbB-3 protein. The antibodies recognize c-erbB-3 by immunoprecipitation and Western blotting of lysate prepared from a cell line engineered to overexpress the protein. Both antibodies also detect expression of the protein in formalin-fixed, paraffin-embedded human tissues. The RTJ1 monoclonal antibody gave superior staining, showing lower background and more pronounced cell surface immunoreactivity. The c-erbB-3 protein was found in normal epithelial cells throughout the GI tract, in squamous epithelium of the oropharanyx and oesophagus, in the parietal cells of the stomach, and in the surface enterocytes of the small and large bowel. Seventy-six tumours arising at these sites were examined for c-erbB-3 protein expression. Widely varying levels of expression were seen, from absent to intense staining with cell membrane accentuation.
Adenocarcinoma↗
Relationship between EGF-R, c-erbB-2 protein expression and Ki67 immunostaining in breast cancer and hormone sensitivity.
The expression of the epidermal growth factor receptor (EGF-R), c-erbB-2 protein product and Ki67 have been evaluated in 105 breast cancers of known responsiveness to endocrine therapy using immunohistochemistry. EGF-R staining was observed in 62 of the tumours and was significantly associated with elevated rates of cell proliferation (%Ki67 positive cells) and loss of hormone sensitivity. In contrast, c-erbB-2 expression was not correlated with cell proliferation rates and was less strongly related to hormone insensitivity. Subdivision of the EGF-R data according to c-erbB-2 measurements revealed an association between c-erbB-2 immunostaining and worsened patient outlook and hormone insensitivity in moderately EGF-R-positive tumours. c-erbB-2 immunostaining in highly EGF-R-positive tumours did not further contribute to the already poor prognosis of these patients. These data confirm the prognostic importance of EGF-R measurements in breast cancer and may infer a functional interaction between this protein and the c-erbB-2 protein product in the aberrant growth of a subset of breast tumours.
Breast Neoplasms↗
Expression of transforming growth factor alpha in human hepatocellular carcinoma.
Transforming growth factor alpha (TGF-alpha) is a mitogenic polypeptide which acts on the epidermal growth factor receptor (EGFR). The aim of this study was to examine the expression of TGF-alpha in human hepatocellular carcinoma (HCC) and surrounding cirrhotic tissue, and to compare it with normal liver. Immunoreactive TGF-alpha was detected using two antibodies raised against its C terminus, a polyclonal antibody 26T and a monoclonal antibody Ab-2. In normal liver immunoreactive TGF-alpha was localised strongly to bile duct epithelium and weakly in occasional parenchymal cells but was notably absent from perisinusoidal and Kupffer cells. Eight out of twenty-eight (28%) cases of HCC expressed TGF-alpha as demonstrated by cytoplasmic staining with both antibodies and in four cases additional membrane immunoreactivity was demonstrated using 26T. However, where cirrhotic tissue surrounding TGF-alpha positive tumours was available for analysis immunoreactive TGF-alpha was detected in only 1/7 cases. TGF-alpha synthesis by malignant hepatocytes was supported by the detection of specific RNA by Northern blotting from two cases with TGF-alpha immunoreactivity. These results implicate bile duct epithelium as an important source of TGF-alpha in human liver. Furthermore, in HCC the expression of TGF-alpha in some cases, together with paucity of TGF-alpha immunoreactivity in surrounding cirrhotic tissue, suggests that TGF-alpha may play a role in continued cell proliferation in human hepatocarcinogenesis.
Blotting, Northern↗
Prognostic value of epidermal growth factor receptor expression in cervical carcinoma.
AIMS: To investigate the pattern of epidermal growth factor receptor expression and its prognostic value in the three main types of cervical carcinoma. METHODS: 62 cases of stage IB/IIA cervical carcinoma, all with a minimum of five years of follow up, were studied. Representative sections were stained for mucin to permit accurate tumour typing and a standard avidin-biotin immunoperoxidase technique using the polyclonal antibody 12E was used to demonstrate the presence of epidermal growth factor receptor. RESULTS: A proportion of all three tumour types expressed epidermal growth factor receptor, it being most common in squamous cell carcinomas (50%). Overall, there was a correlation between epidermal growth factor expression and mortality. This was particularly obvious in the absence of lymph node metastases. When the individual tumour types were considered this association with prognosis was not demonstrable for squamous cell carcinomas or adenocarcinomas but was a very prominent feature of adenosquamous carcinomas. CONCLUSIONS: Immunohistochemical demonstration of epidermal growth factor receptor expression may be useful in identifying those patients with a poor prognosis, particularly those with adenosquamous carcinomas which have not metastasised to the regional lymph nodes.
Adenocarcinoma↗
Specific short transmembrane sequences can inhibit transformation by the mutant neu growth factor receptor in vitro and in vivo.
The neu oncogene is activated by a point mutation within its transmembrane domain that results in the substitution of glutamic acid for valine at position 664, and is associated with constitutive activation of the tyrosine kinase. It has been proposed that the mutation allows for stabilization of homodimers of the receptor that are necessary for transduction of the mitogenic signal. To investigate the role of the alpha-helical transmembrane sequence in the function of neu, we constructed an expression vector to produce a variety of short transmembrane neu proteins, lacking ligand binding or intracellular kinase domains. Such sequences should interact with full-length receptors and prevent receptor dimerization and thus act as specific inhibitors of function. These small proteins all included a pentapeptide from position 661-665, which has been proposed to be necessary for packing. We show that the short transmembrane molecules are expressed at the cell surface and can retard the growth of neu-transformed cells in monolayers, as colonies in soft agar and as tumours in animals. As predicted by molecular modelling, the magnitude of inhibition depended on the nature of the packing surface, suggesting that the neu transmembrane domain is directly involved in neu protein dimerization.
3T3 Cells↗
Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumors.
Thirty-six primary human colorectal tumors, 43 noninvolved colon samples that were adjacent to either carcinomas of adenomas, 22 adenomas, and nine normal colon specimens were immunohistochemically examined for the presence and localization of two epidermal growth factor-related peptides, amphiregulin (AR) and cripto. Within the primary tumors, 18 (50%) showed moderate levels of AR expression. Approximately 60% of the tubular and tubulovillous adenomas were positive for AR expression, whereas only 15% of the adjacent, noninvolved colon mucosa expressed AR. A greater proportion of well-differentiated tumors (71%) were positive for AR expression than were poorly differentiated tumors (18%). All of the nine normal colon specimens were positive. Consequently, AR expression appeared to be associated with both normal and malignant epithelial cells that were more differentiated. The distribution of cripto expression was different. Seventy-nine % of the colon tumors expressed cripto with a frequency of expression that was approximately equivalent between well-differentiated and poorly differentiated tumors. Approximately 86% of the tubulovillous adenomas, but only 43% of the tubular adenomas, were positive for cripto expression. In contrast, whereas AR was expressed in normal colon specimens, none of these tissues expressed cripto, and only 12% of the noninvolved normal colon samples adjacent to tumors or adenomas were positive for cripto. Cripto expression therefore appeared related to neoplasia. These data suggest that AR and cripto may be functioning as potential autocrine and/or paracrine growth factors in the colon and that the differential expression of cripto may serve as a potential tumor marker for colonic carcinogenesis.
Adenoma↗