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Biomedical subjects

D P Edwards

Publications and source records attributed to D P Edwards.

At least 127 records · Page 7Linked to original sources
PubMed1983

Monoclonal antibodies against three major glycoproteins of varicella-zoster virus.

Varicella-zoster virus (VZV) codes for three prominent glycoproteins--gp62, gp98, and gp118--in infected cell cultures. To characterize individually these known immunogens, we first inoculated BALB/c mice with crude VZV extracts, produced hybridoma cultures by Köhler-Milstein cell-fusion technology, and screened culture supernatants by indirect immunofluorescence for reactivity directed against unfixed VZV-infected cells (FAMA assay). Supernatants from five independently derived and subcloned hybridomas with a high VZV-FAMA titer but no reactivity against either uninfected or herpes simplex virus-infected cells were further analyzed by immunoprecipitation of [3H]fucose-labeled and detergent-solubilized VZV antigen preparations. Fractionation of the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that four monoclonal antibodies reacted with both gp62 and gp98, and one precipitated only gp118. The profiles were unchanged whether performed under reducing or nonreducing conditions. When assayed for neutralizing activity, the secretory product of the single anti-gp118 hybridoma, but not the supernatants from the four anti-gp62/gp98 clones, inhibited VZV plaque formation by greater than 80%. Thus, at least one of the glycosylated antigens detected by the FAMA assay is a determinant which elicits neutralizing activity.

Animals↗
PubMed1983

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity.

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.

Animals↗
PubMed1983

Purification of an estrogen-regulated breast cancer protein by monoclonal antibody affinity chromatography.

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. This rapid, two-step procedure produces purified protein in high yield and demonstrates the power of monoclonal antibodies in protein purification.

Amino Acids↗
PubMed1983

Detection of a Mr 24,000 estrogen-regulated protein in human breast cancer by monoclonal antibodies.

Monoclonal antibodies have been prepared that can specifically detect an estrogen-regulated protein with a molecular weight of 24,000 in the MCF-7 human breast tumor cell line and in human breast tumor biopsies. These antibodies are of the immunoglobulin G1 subclass, exhibit a high affinity for the Mr 24,000 protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to confirm estrogen regulation of the Mr 24,000 protein in MCF-7 cells and to detect Mr 24,000 protein in certain human breast tumors.

Animals↗
PubMed1982

Immunohistochemical detection of an estrogen-regulated protein by monoclonal antibodies.

It has been shown previously that estradiol regulates the synthesis of a Mr 24,000 cytosol protein in MCF-7 human breast cancer cells. This protein is an attractive probe to study the mechanisms of estrogen regulation in human breast cancer. We have produced monoclonal antibodies against the Mr 24,000 protein which show immunocytochemical localization in the cytoplasm of MCF-7 cells grown as solid tumors in nude mice. They do not react with normal mouse tissue or with MDA-MB-231 cells, an estrogen receptor-negative cell line. Moreover, the monoclonal antibodies react against certain human breast tumor biopsy samples. These data suggest that the monoclonal antibodies obtained will be useful to detect the estrogen-regulated protein by immunohistochemistry.

Antibodies, Monoclonal↗
PubMed1981

Estrogen regulation of growth and specific protein synthesis in human breast cancer cells in tissue culture.

Taking advantage of the fact that estrogens can stimulate proliferation of antiestrogen-inhibited MCF-7 cells has enabled us to study molecular events involved in steroid-mediated growth of tumor cells, using a breast cancer cell line which otherwise is affected very little in its growth by estrogens. Under these growth conditions, estradiol stimulates DNA polymerase activity in a manner analogous with reported estrogen effects on enzyme activity in normal target tissues. We also observed dissociation of estrogen effects on cell growth and PgR stimulation, indicating that PgR induction and estrogen-mediated cell division may involve separate control mechanisms. The relative rate of synthesis of the 24,000 molecular weight protein is also increased under these conditions of estrogen-stimulated cell growth. Since increased synthesis of the 24,000 molecular weigh protein was determined not to be a reflection of different stages of cell growth, we suggest that this protein is regulated specifically by estrogens and that it may be a marker of estrogen stimulated growth of human breast tumor cells.

Breast Neoplasms↗
PubMed1981

Failure of estradiol immunofluorescence in MCF-7 breast cancer cells to detect estrogen receptors.

An indirect immunofluorescence assay was used to detect estradiol in MCF-7 breast cancer cells to determine if the estradiol-specific fluorescence observed represented estrogen receptor-bound estradiol. Appropriate controls were used to demonstrate the immunological specificity of our assay procedures. Initial studies of estradiol binding in MCF-7 cells were performed at 20 degrees for 1 hr with different concentrations of estradiol. Cytoplasmic and nuclear staining were observed following treatment with 10 nM estradiol, but not with lower concentrations which were nevertheless still sufficient to saturate estrogen receptor. The staining intensity increased with higher estradiol concentration, which is consistent with estradiol binding to lower-affinity binding sites. In order to further determine if estradiol binding by estrogen receptor was being detected, we pretreated MCF-7 cells with 5 nM diethylstilbestrol at 37 degrees for 1 hr to translocate all estrogen receptor to the nucleus and then administered estradiol at varying concentrations for 4 hr at 4 degrees. The estradiol was still primarily detected in the cytoplasm, although virtually all of the estrogen receptor was found to be present in the nucleus by standard [3H]estradiol binding assays. Additional immunochemical studies using sucrose gradient analysis to detect antibody-estradiol-receptor complexes clearly established that these complexes could not be detected. The present results suggest that, although immunocytochemical assays can specifically detect estradiol in MCF-7 cells, the estradiol is bound to lower-affinity binding sites rather than to estrogen receptor. Saturation analyses of intact viable MCF-7 cells performed at 37 degrees for 30 min using [3H]estradiol at concentrations ranging from 0.1 to 93 nM revealed an additional lower-affinity estradiol-binding site besides the receptor, perhaps analogous to the Type II sites reported in the rat uterus and human breast cancers.

Binding Sites↗
PubMed1980

17 alpha-Estradiol is a biologically active estrogen in human breast cancer cells in tissue culture.

The biological effects of 17 alpha-estradiol (17 alpha-E) and its interaction with estrogen receptors were studied in the MCF-7 human breast cancer cell line. Competition for [3H]17 beta-estradiol ([3H]17 beta-E) binding shows that 17 alpha-E binds to receptor with high affinity and has a dissociation constant (Kd) estimated to be 0.7 nM. Upon binding with 17 alpha-E, the cytosol receptor is translocated to the nucleus and is then rapidly depleted or processed in the same manner as the 17 beta-E-receptor complex. The nuclear 17 alpha-E-receptor complex was determined to be biologically active by its ability to stimulate an increase in the progesterone receptor content and to reverse antiestrogen inhibition of cellular proliferation and DNA polymerase activity. The estrogenic potency of 17 alpha-E, estimated from the dose-response curves as the median effective dose in stimulating progesterone receptor content and in reversing antiestrogen inhibition, is about one tenth the potency of 17 beta-E. Competition curves show that 17 alpha-E binds to the cytosol estrogen receptor with about one third the affinity of 17 beta-E, so the correlation between relative binding affinity and biological potency is not perfect. The correlation, however, is reasonably good compared with that in animal studies, in which 17 alpha-E displays negligible biological activity. Gas chromatography and mass spectrometry rule out the possibility that the observed estrogenic activity of 17 alpha-E was due to contamination of the preparation with the more active 17 beta-E. The enhanced estrogenic potency of 17 alpha-E in MCF-7 cells raises the question of whether the stereospecificity and, thus, the sensitivity of the estrogen receptors in breast cancer cells may be different than those in normal target tissues. Enhanced estrogenic activity may also be due simply to the nature of the tissue culture system, which allows continuous exposure of the cells to hormone; a condition which may not be achieved in some animal studies.

Binding, Competitive↗
PubMed1980

Effects of estrogen and antiestrogen on DNA polymerase in human breast cancer.

We have investigated the effects of estrogens and antiestrogens on cellular DNA-dependent DNA polymerase activity in human breast cancer, using as a model the MCF-7 human breast cancer cell line which contains estrogen receptor. 17 beta-Estradiol had little if any effect on cytosol DNA polymerase activity or growth (total DNA per flask) of MCF-7 cells. Incubation of the cells for 4 to 6 days with the antiestrogen nafoxidine, however, resulted in a dose-dependent reduction in cytosol DNA polymerase activity to one-half that observed in untreated cells. Enzyme activity in antiestrogen-treated cells was restored to levels contained in untreated cells by removing antiestrogen from the growth medium and incubating the cells for an additional 4 days with 17 beta-estradiol. The restoration required estrogenic steroids specifically, and the time course, magnitude, and dose dependence of the response were similar to estrogen-stimulated increases in DNA polymerase activity described in other estrogen target tissues. Estrogen-mediated reversal of antiestrogen suppression of DNA polymerase activity was paralleled by increases in total DNA synthesis.

Breast Neoplasms↗