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Biomedical subjects

F E May

Publications and source records attributed to F E May.

At least 37 records · Page 2Linked to original sources
PubMed1997

Close physical linkage of the genes encoding the pNR-2/pS2 protein and human spasmolytic protein (hSP).

Trefoil proteins contain a conserved domain of distinctive structure. Three human trefoil proteins have been described to date of which the human spasmolytic polypeptide (hSP) and pNR-2/pS2 proteins have a similar pattern of expression in normal tissues. The genes encoding these two proteins were isolated from a human DNA library. Preliminary experiments suggested that some recombinants contained both genes. Southern hybridisation showed that all the recombinants were derived from a single stretch of DNA spanning 45 kb and suggested that the hSP gene was located downstream of the pNR-2/pS2 gene. Further experiments demonstrated that the two genes are transcribed in the same direction and that the distance between the 3' end of the pNR-2/pS2 gene and the 5' end of the hSP gene is 12.5 kb. The close linkage of these two genes is evidence that they have evolved by gene duplication and that their similar pattern of expression in normal tissues could result from the retention of common regulatory elements.

Chromosomes, Human, Pair 21↗
PubMed1996

Involvement of phenylalanine 23 in the binding of IGF-1 to the insulin and type I IGF receptor.

The hydrophobic residue Phe-23 lies on a surface of insulin like growth factor 1 (IGF-1) which may be involved in binding to the type I IGF and insulin receptors. The possibility that Phe-23 participates directly in binding to these receptors has been investigated by comparing the properties of [F23G]IGF-1, a mutant of insulin-like growth factor 1 in which Phe-23 has been replaced by Gly, with those of IGF-1 and insulin. [F23G]IGF-1 has a 48-fold lower affinity for the type I IGF receptor, a markedly reduced affinity for the insulin receptor and a 100-fold reduced affinity for insulin-like growth factor binding proteins (IGFBPs) compared to IGF-1. [F23G]IGF-1 is a full IGF-1 agonist as measured by its ability to stimulate cell proliferation. Its potency was only 4.5-fold less than IGF-1, probably because its bioavailability was increased as a result of its reduced affinity for IGFBPs. The large reduction in the affinity of [F23G]IGF-1 for the type I IGF receptor and insulin receptor contrasts with the lack of effect of the corresponding alteration to insulin [FB24G]. Phe-B24 is not thought to be involved directly in the binding of insulin to the insulin receptor and the C-terminus of the B-chain of insulin is suggested to be displaced on binding. We suggest that for IGF-1, the C-terminus of the B chain cannot be displaced because of the presence of the C-domain and the large reduction in the binding affinities of [F23G]IGF-1 suggest that the Phe-23 side chain may be directly involved in binding of IGF-1 to the type I IGF and insulin receptors.

Binding Sites↗
PubMed1996

Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol.

Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5-fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor.

Breast Neoplasms↗
PubMed1995

Mutations at positions 11 and 60 of insulin-like growth factor 1 reveal differences between its interactions with the type I insulin-like-growth-factor receptor and the insulin receptor.

Insulin-like growth factor 1 (IGF-1) and three analogues ([V11I]IGF-1, [V11T]IGF-1, and [Y60F]IGF-1), constructed by site-directed mutagenesis, were expressed as fusion proteins and secreted into the periplasmic space of Escherichia coli. Purified IGF were obtained following IgG Sepharose affinity and cation-exchange chromatographies of the products of hydroxylamine cleavage of fusion proteins. The properties of the mutants were assessed using (a) quantification of affinities for the human insulin receptor overexpressed on NIH 3T3 cells, (b) quantification of affinities for the type I IGF receptor via competition for binding to a monolayer of MDA-MB-231 cells, (c) promotion of the in vitro mitogenesis of growth-arrested MCF-7 cells in the presence of 17-beta-oestradiol, and (d) a competition assay for binding to IGF-binding proteins secreted by MCF-7 cells. The mutants exhibited decreases in affinity for the insulin receptor, relative to IGF-1, of 2.6-, 3.8- and, 8.8-fold for [Y60F]IGF-1, [V11I]IGF-1, and [V11T]IGF-1, respectively. IGF-1, [V11I]IGF-1, and [Y60F]IGF-1 were of equal potency in the growth assay and in affinity for the type I IGF receptor. [V11T]IGF-1 exhibited a three fold loss of potency in the type I IGF receptor-binding and growth assays. The mutants did not differ significantly from IGF-1 in their affinities for the IGF-binding proteins. The full-activity of [Y60F]IGF-1 at the type I IGF receptor, in contrast to the weakened receptor affinity of IGF-1 with a Leu substitution at this position, indicates a requirement for an aromatic ring, rather than a hydroxyl group, in the interaction of IGF-1 with the type I IGF receptor. The decrease in affinity for the insulin receptor of all the mutants indicates that, as in insulin, the residues Val11 and Tyr60 are important for the interaction of IGF-1 with the insulin receptor. The unchanged or minor changes in the affinities of the mutants for the type I IGF receptor contrast with the more deleterious effects of the mutations on insulin receptor binding and with the properties of analogues of insulin mutated at equivalent sites: 3-fold and 5-10-fold reductions in biological activity for [VB12I]insulin and [YA19F]insulin, respectively. Thus, the results obtained using the mutants indicate important differences between the IGF-1/type I IGF receptor and insulin/insulin receptor interactions.

3T3 Cells↗
PubMed1995

Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58.

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

Amino Acid Sequence↗
PubMed1995

Estrogen regulated messenger RNAs in human breast cancer cells.

A large number of estrogen-regulated mRNAs have been identified in human breast cancer cells. Some of these encode proteins whose regulation by estrogen had been established previously. Others have been identified by differential screening of cDNA libraries established from estrogen-stimulated breast cancer cells. The regulation of these RNAs by estrogens in different cell lines is reviewed. The possible role of their gene products where known is discussed as is the possibility that the proteins mediate the proliferative effects of estrogens on breast cancer cells. The possibility that measurement of one or more of these estrogen-regulated gene products in breast tumours might allow prediction of the likely benefit of patients from endocrine therapies is evaluated. Finally the impact that they have had on current understanding of how estrogens and antiestrogens regulate transcription of endogenous genes in human breast cancer cells is considered.

Biomarkers, Tumor↗
PubMed1995

Determination of oestrogen responsiveness of breast cancer by competitive reverse transcription-polymerase chain reaction.

Competitive polymerase chain reaction assays have been developed for the quantitation of oestrogen receptor mRNA and two oestrogen-regulated mRNAs (progesterone receptor and pNR-2/pS2) in breast cancer cells. These assays are more sensitive than traditional hybridisation techniques, do not require the use of radioisotopes, measure absolute amounts of messenger RNAs and can be used to measure the expression of mRNAs in small numbers of tumour cells obtained by fine-needle aspiration (FNA). These assays should prove useful for predicting the hormone responsiveness of breast cancer from tumour cells obtained by FNA at diagnosis and could be particularly useful in the management of elderly/frail patients who receive primary tamoxifen, or in other patients for whom tumour tissue for standard biochemical measurements is not available.

Base Sequence↗
PubMed1994

Mitogenic activity of procathepsin D purified from conditioned medium of breast-cancer cells by affinity chromatography on pepstatinyl agarose.

A simple procedure for the affinity purification of procathepsin D from tissue culture medium conditioned by breast-cancer cells is described. This procedure yielded 2 micrograms of procathepsin D/100 ml medium. The procathepsin D was approximately 95% pure as judged by silver staining of polyacrylamide gels, the major contaminant being mature cathepsin D. The ability of procathepsin D to stimulate the proliferation of oestrogen-responsive MCF-7 breast cancer cells was determined. The purified procathepsin D had no mitogenic effect alone or in combination with oestradiol or other growth factors. These data suggest that procathepsin D does not act as an oestrogen-regulated autocrine growth factor for malignant breast epithelial calls.

Breast Neoplasms↗
PubMed1994

Role of insulin-like growth factors in steroid modulated proliferation.

The mechanism by which steroids influence cell proliferation is poorly understood although an understanding of this process might facilitate the development of strategies to modulate the tissue-specific activity of steroid hormones. In this article, the evidence that steroid hormones interact with the insulin-like growth factor (IGF) signal transduction pathway is reviewed for three different tissues. In osteoblasts, oestradiol stimulates the production of IGF-I which appears to act as an autocrine growth factor. In uterine tissue, oestradiol increases the synthesis of IGF-I in the stroma which then modulates the proliferation of epithelial cells although there is also evidence that oestradiol can modulate the sensitivity of uterine epithelial cells to IGFs. In breast cancer, oestrogens may increase IGF-II synthesis in epithelial cells, increase the sensitivity of breast cancer cells to IGFs (possibly by modulating type I IGF receptor levels) as well as resulting components of the IGF signal transduction pathway resulting in induction of immediate early genes. There therefore appears to be a variety of ways in which oestradiol interact with the IGF signal transduction pathway and these may be applicable to other malignant and normal tissues and other groups of steroid hormones.

Animals↗
PubMed1994

Turnover at the top: CNOs and hospital characteristics.

Considering the importance of chief nursing officer (CNO) turnover, little is known. Using data published annually by the American Hospital Association and other public sources, a national sample of 102 hospitals was studied for a five-year period to determine CNO turnover rate and hospital characteristics affecting turnover.

Analysis of Variance↗
PubMed1993

The human cathepsin D-encoding gene is transcribed from an estrogen-regulated and a constitutive start point.

The 5' flanking sequences, exon 1 and part of intron 1 of the human cathepsin D (CTD)-encoding gene (CTD) have been cloned and sequenced. RNase protection experiments identified two major transcription start points (tsp) located 14 and 63 nucleotides upstream of the start codon. The proximal -14, but not the distal -63 tsp has upstream near-concensus TATAAA and CCAAT sequences. Estrogens increase transcription from the -14 tsp, but not the -63 tsp and CTD is therefore unique among estrogen-regulated genes in having estrogen-regulated and constitutive transcription. Sequencing approximately 800 bp upstream and 600 bp downstream of the tsp failed to identify a consensus 13-bp palindromic estrogen-response element (ERE); however, four half-palindrome GGTCA motifs were located within 340 bp upstream of the -14 bp tsp. Thus, estrogen regulation of CTD may not be mediated by a consensus ERE.

Base Sequence↗
PubMed1993

int-2 amplification in breast cancer: association with decreased survival and relationship to amplification of c-erbB-2 and c-myc.

Amplification of the int-2 oncogene was measured in a series of breast tumours and related to amplification of the c-myc and c-erbB-2 oncogenes, histopathological features and relapse-free and overall survival. int-2 was amplified in 11%, c-myc in 20% and c-erbB-2 in 27% of the tumours assessed. int-2 amplification was associated with large tumour size (p < 0.05) and reduced relapse-free (p < 0.05) and overall (p < 0.0005) survival. c-myc amplification was associated with poor tumour differentiation (p < 0.05) but had no association with prognosis. c-erbB-2 amplification was associated with low levels of expression of oestrogen receptor mRNA (p < 0.05), poor tumour differentiation (p < 0.05) and shortened relapse-free (p < 0.0001) and overall survival (p < 0.0001). This is the first report of an association between amplification of the int-2 oncogene in breast tumours and a significantly increased risk of death from breast cancer, and suggests that int-2 may be useful for identifying breast-cancer patients having a poor prognosis.

Breast Neoplasms↗
PubMed1993

Isolation and characterization of an oestrogen-responsive breast-cancer cell line, EFF-3.

A novel oestrogen-responsive breast-tumour cell line, EFF-3, has been established from a pleural exudate of a patient with metastatic breast cancer. The cells show morphological and immunohistochemical features consistent with their origin from a metastatic breast carcinoma. The cells aggregate and form sheets in culture, and electron microscopy confirms the presence of cell-surface microvilli and intercellular tight junctions. The epithelial origin of EFF-3 cells was confirmed by their expression of low-molecular-weight cytokeratins and carcinoembryonic antigen. The karyotype of the cells is markedly abnormal and there are large numbers of structurally abnormal chromosomes. EFF-3 cells express oestrogen receptor, oestrogen-receptor mRNA, their growth is oestrogen-responsive, and specific genes are regulated by oestrogens. The pNR-2/pS2 and pNR-25 oestrogen-regulated mRNAs are induced 15- and 13-fold respectively by oestrogen, whereas the oestrogen-receptor and cathepsin D mRNAs are not regulated. This pattern of regulation differs from that reported previously for other cell lines. The EFF-3 cell line should be useful for studying the mechanisms involved in oestrogen-stimulated proliferation and the factors determining the regulation of specific genes by oestrogens.

Breast Neoplasms↗
PubMed1993

Type I IGF receptor and acquired tamoxifen resistance in oestrogen-responsive human breast cancer cells.

Tamoxifen inhibited the oestrogen-stimulated proliferation of MCF-7 cells but had little effect on the oestrogen-stimulated proliferation of two tamoxifen-resistant variants (RL-3 and AL-1). The lack of oestrogen antagonist activity in the resistant cells was largely a result of an increased oestrogen agonist activity of tamoxifen on cell proliferation. Proliferation of the tamoxifen-resistant cells was also stimulated by 4-hydroxytamoxifen but not by ICI 164,384, a structurally distinct pure anti-oestrogen. Tamoxifen does not have increased oestrogen agonist activity for the induction of a series of oestrogen-regulated RNAs, and this suggests that the increased agonist activity may be restricted to key components involved in the proliferative response. Tamoxifen-stimulated cell proliferation was dependent on insulin-like growth factor I (IGF-1) in the resistant cells, suggesting that tamoxifen stimulates cell proliferation by sensitising cells to the proliferative effects of IGF-1. This may involve induction of the type-I IGF receptor.

Breast Neoplasms↗
PubMed1992

c-erbB-2 oncogene expression in hepatocellular carcinoma and cholangiocarcinoma.

The c-erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the epidermal growth factor receptor. This protein can be localized immunohistochemically in formalin-fixed paraffin-embedded material using a monoclonal antibody NCL-CB11; positive membrane staining correlates with gene amplification and protein overexpression in breast cancer. Using this technique we have shown that only 2/26 (8%) of hepatocellular carcinomas, 0/10 (0%) of cholangiocarcinomas and 0/2 (0%) hepatoblastomas overexpressed c-erbB-2 as evidenced by membrane staining. Moreover c-erbB-2 mRNA was not detected in seven hepatocellular carcinomas examined by Northern blot analysis. c-erbB-2 overexpression is, therefore, unlikely to be contributing to the malignant phenotype in hepatocellular carcinoma and cholangiocarcinoma.

Adenoma, Bile Duct↗