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G C Chamness

Publications and source records attributed to G C Chamness.

At least 55 records · Page 3Linked to original sources
PubMed1986

17 alpha-allyl estradiol analogues as candidates for development of high-affinity fluorescein-estradiol conjugates.

In order to develop stable, high-affinity fluorescein-estradiol conjugates, the fluorescein moiety must be leashed to the estradiol molecule at a point which interferes least with estradiol's binding to the receptor. Because of the high affinity of 17 alpha-substituted estradiol (e.g. ethynyl estradiol), we investigated a series of 17 alpha-substituted estradiol compounds to determine the optimal properties of a leash at this position. Twelve estradiol derivatives bearing a three-carbon 17 alpha side chain with or without a terminal functional group and with varying degrees of unsaturation were synthesized. Initial comparison of the receptor binding affinities of some of these derivatives suggested that three factors might reduce affinity: internal hydrogen bonding of the 17 beta-hydroxyl proton with an oxygen atom of the 17 alpha side chain; hydrophilicity of the ligand; or steric interference of the side chain with receptor binding. Further comparisons were designed to evaluate the relative contribution of these factors. The results suggest that the relative affinities of these 17 alpha-substituted estradiol derivatives are influenced primarily by the steric interference of the side chains and also by their hydrophilicity. Internal hydrogen bonding involving the 17 beta-hydroxyl proton does not seem to have a profound effect.

Animals↗
PubMed1986

Androgen receptor affinity chromatography: synthesis and properties of 17 alpha-epoxypropyl-dihydrotestosterone Sepharose.

We have prepared a new affinity chromatography reagent, 17 alpha-epoxypropyl-dihydrotestosterone linked to Thiopropyl-Sepharose, with potential for use in purification of androgen receptor and other specific androgen binding proteins. The linkage is stable, and the ligand has reasonably high affinity for the receptor. Starting with 5 alpha-androstane-3 beta-ol-17-one, we synthesized in two steps 17 alpha-allyl-dihydrotestosterone, which was then oxidized to 17 alpha-epoxypropyl-DHT yielding 2 diastereomers in about a 4:1 ratio. The 17 alpha-allyl-DHT had about 50% of DHT's affinity for rat uterine androgen receptor, while the affinity of the major epoxide isomer was 9% and that of the minor isomer was 4%. Reaction of the epoxides with Thiopropyl-Sepharose-6B gave about 7 mumol of covalently bound DHT per ml of beads. These beads took up 83% of the androgen receptor from a rat uterine cytosol in a preliminary study, which more than equalled the performance of identically prepared estradiol beads successfully used for estrogen receptor purification. The use of the new DHT beads in purifications of the androgen receptor and other binding proteins is now being explored by other laboratories.

Animals↗
PubMed1986

Progesterone receptor assays in low-protein cytosols: a modified charcoal-gelatin procedure.

Quantitative measurement of steroid receptors including progesterone receptor (PgR) is usually accomplished by the dextran-coated charcoal (DCC) assay. At protein concentrations below about 1 mg/ml, however, serious underestimation of receptor content by DCC occurs, presumably because of adsorption of receptor to the charcoal and possibly to assay tubes, etc. We have therefore developed a modified charcoal-gelatin (MCG) procedure which largely avoids receptor losses even in samples with extremely low protein concentrations. In this MCG procedure, 0.1% gelatin is added to both sample and charcoal suspension, the charcoal content is increased to 1%, and dextran is no longer necessary. Comparison of the MCG procedure with the standard DCC and several other methods at decreasing protein concentrations shows that MCG retains acceptable efficiency for PgR at much lower protein than the others, even as low as 10 micrograms/ml. This MCG procedure will be useful in determining receptors for prognosis in very small human breast cancer biopsies, as shown here, but also for receptor determination in very small tissues such as specific brain regions, and for receptor assay during purification.

Breast Neoplasms↗
PubMed1986

Estrogen receptor in very small breast tumor specimens: a modified charcoal-gelatin assay.

We described recently a modified charcoal-gelatin (MCG) assay for measuring progesterone receptor activity in low-protein cytosols. We showed that pre-mixing of gelatin with sample cytosols (final gelatin concentration 0.1%) and removal of unbound steroid by a 1% charcoal suspension with 0.1% gelatin but without dextran preserves the progesterone receptor activity in dilute cytosol. For estrogen receptor (ER), as for progesterone receptor, the efficiency of the standard dextran-coated charcoal (DCC) assay drops rapidly as samples are diluted much below 1 mg protein per ml. We have therefore applied the MCG procedure to the assay of ER in breast tumor cytosols. We find that MCG is far more efficient for ER at low protein concentrations than either the DCC method or three other methods recommended previously for dilute samples, retaining at least 60% efficiency even at 0.01 mg protein per ml. The measured Kd of the receptor for estradiol is the same by MCG as by DCC. A series of human breast tumor biopsies assayed by MCG at 0.1 mg protein per ml gave about the same ER values (fmol/mg) as at 1 mg/ml, while the DCC efficiency for ER at the lower concentration averaged only 32%. In combination with 125I-labeled estradiol, this MCG method should allow accurate ER assays of extremely small breast cancer specimens.

Biopsy↗
PubMed1984

Multiple progesterone receptor assays in human breast cancer.

A review of assay results from more than 5500 patients revealed 283 patients in whom multiple breast cancer specimens were analyzed for progesterone receptor (PGR). All assays were performed in a single laboratory between 1975 and 1982 using the sucrose gradient technique. We considered only the 8S fraction of PGR. Simultaneous assays in 109 patients yielded 14% discordance [one assay with greater than 10 fmol/mg cytosol protein (PGR+) and one assay with less than 5 fmol/mg protein (PGR-)]. Among 161 sequential assays, there was an overall discordance of 19%: 8% (nine of 106) when the initial assay was PGR-, but 44% (24 of 55) when the initial assay was PGR+. Among PGR+ patients initially assayed at the time of diagnosis, there was a tendency to greater receptor loss in patients with positive axillary lymph nodes (44 versus 11%). The length of time between biopsies did not increase the discordance, but endocrine therapy within this interval did increase it (56% of initially PGR+ patients who received interim endocrine therapy were PGR- at second biopsy). to evaluate the significance of interval loss of PGR, we compared survival from first biopsy in initially PGR+ patients who subsequently lost their receptor versus those whose receptor persisted. The latter group experienced a significantly longer survival (p less than 0.02). In summary, we observed an ominous loss of PGR in sequential biopsies, particularly with intervening endocrine therapy, and those patients whose tumor cells lost PGR experienced poorer survival than did patients retaining PGR. Therefore, patients with PGR+ primary tumors require repeat biopsy for PGR upon disease recurrence for optimal treatment planning.

Biopsy↗
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

Dopaminergic stimulation of pituitary but not hypothalamic estrogen receptors in ovariectomized rats.

This study was designed to examine the role of catecholamines and serotonin in the regulation of estrogen receptors (ER) in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) of adult ovariectomized rats. Plasma PRL and LH were also determined. Injection of alpha-methyl tyrosine (alpha-MT) resulted in the significant reduction of norepinephrine and dopamine (DA) content in the MBH, and of plasma LH levels as well as in the significant elevation of plasma PRL levels. This treatment also resulted in the significant reduction of ER concentration in the AP. The elevation in plasma PRL by alpha-MT was reversed by the simultaneous injection of bromocriptine (BC), a DA agonist, which also partially reversed the alpha-MT reduction of ER concentrations in the AP. BC had no effect on plasma LH levels. The ER concentration in the MBH was not significantly changed by any of these treatments. The reduction of serotonin content in the MBH by the injection of p-chlorophenylalanine had no effect on the ER concentration in either the MBH or AP, nor did it have any effect on plasma PRL levels. However, p-chlorophenylalanine treatment did decrease plasma LH levels. Neonatal treatment of female rats with monosodium glutamate which has been reported to destroy part of the MBH, resulted in a significant reduction in body and pituitary weight and in a significant elevation of plasma PRL levels in adults (2 months old). This treatment also resulted in a significant reduction in the AP and MBH ER concentration. Injection of BC to adults reversed the effects of neonatal monosodium glutamate treatment on plasma PRL levels and on the pituitary ER concentration, but had no effect on the ER concentration in the MBH. BC had no effect on the AP or MBH ER concentration in control rats, although it did as expected reduce the plasma PRL levels in these animals. Plasma LH levels were not significantly changed by any of these treatments. Injection of the DA antagonist, haloperidol, to adult rats resulted in a significant elevation of plasma PRL and in a significant reduction of ER concentration in the AP. Haloperidol treatment did not affect the binding affinity of these receptors. Overall, these data suggest that DA is involved with the regulation of ER in the AP.

Animals↗
PubMed1983

Genetic differences in androgen receptors and in autoregulation of testicular human chorionic gonadotropin binding sites in the mouse.

The autoregulation of testicular human chorionic gonadotropin (hCG) binding sites was studied in two strains of mice known to differ in their endocrine and reproductive characteristics (C57BL/10J and DBA/2J), and in their F1 progeny (B10D2F1). Basal hCG binding levels were higher in C57BL/10J than in DBA/2J mice, while B10D2F1 mice had intermediate levels. Twenty-four h after injection, hCG produced dose-related changes in hCG binding in C57BL/10J and B10D2F1 mice not observed in DBA/2J mice. However, 72 h after treatment with hCG there was a decrease in hCG binding in all the strains studied. These results suggest the participation of genetic factors in determining basal levels, dose-related changes and temporal response of testicular hCG binding sites to hCG administration. Androgen receptor levels were measured in the same strains of mice. DBA/2J mice had higher receptor levels in the kidney and coagulating gland, and lower levels in the hypothalamus and seminal vesicle when compared to C57BL/10J mice. B10D2F1 mice had androgen receptor levels similar to those measured in C57BL/10J mice in all tissues studied, with the exception of the coagulating gland, where levels were similar to those observed in DBA/2J mice. These observations may indicate the existence of several loci coding for androgen receptors, with only one being expressed per tissue.

Animals↗
PubMed1983

Multiple estrogen receptor assays in human breast cancer.

A review of assay results from more than 6000 patients revealed 232 patients in whom multiple breast cancer specimens were analyzed for estrogen receptor (ER). All assays were performed in a single laboratory. Specimens were considered estrogen receptor positive (ER+) if the ER level was greater than 10 fmol/mg protein and estrogen receptor negative (ER-) if the ER level was less than 3. ER values between 3 and 10 fmol/mg protein were considered borderline. Simultaneous assays were performed in 58 patients with 3% major discordance (i.e., one assay ER- and one assay ER+). Major discordance for sequential biopsies was 19% (16 of 82) when the initial assay was ER+ and 13% (eight of 63) when the initial assay was ER-. (Apparent change from ER- to ER+ status was observed in five of nine patients with primary tumors less than 2 cm in diameter, suggesting that an inadequate amount of tissue may have been submitted for initial ER analysis.) There was no significant relationship between the time interval between sequential biopsies and the rate of discordance. Marked decreases in ER levels and 78% discordance were seen if rebiopsy was performed within 2 months of tamoxifen treatment. When these tamoxifen cases were excluded from the analysis, neither intervening endocrine therapy nor chemotherapy significantly altered discordance rates.

Adult↗
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

Danazol binding and translocation of steroid receptors.

Danazol, an isoxazol derivative of ethinyl testosterone which suppresses gonadotropin levels and acts as a weak androgen, is shown by competition studies to bind rat androgen receptor (Ki 10(-8M) and progestin receptor (Ki 10(-7)M) but not estrogen receptor. Effective antigonadotropin doses to the rat in vivo translocate only androgen receptor to target cell nuclei; nuclear receptor levels remain elevated more than 6 hours. The same translocation occurs when rat uteri are incubated with danazol in vitro, showing that the action of danazol is direct and probably does not require metabolic conversion of the drug.

Animals↗
PubMed1980

Are histochemical methods for estrogen receptor valid?

Because of the great usefulness of estrogen receptor determinations in selecting therapy for breast cancer patients a number of histochemical and immunohistochemical methods for visualizing bound estrogen in cells and tissue sections have been proposed. We discuss all of these histochemical methods in the light of the known properties of the estrogen receptor and other estrogen binders, and we consider some criteria that must be met if such methods are to be considered valid for receptor. In spite of the great potential value of histochemical methods, we are forced to conclude that, in their present form, none of them are likely as yet to be detecting estrogen receptor.

Breast Neoplasms↗
PubMed1979

Androgen receptor in the rat brain--assays and properties.

The existence and relevance of an androgen receptor in the developing brain has been a matter of controversy. We here describe both sucrose density gradient and hydroxylapatite assays which clearly define a distinct androgen receptor in the 24-day-old rat hypothalamus, amygdala and preoptic region, but not in the cortex. This receptor has considerable affinity for estradiol-17beta, thus perhaps accounting for some uncertainty about its nature, but none for diethylstilbestrol or other estrogens, antiestrogens or glucocorticoids. Its Kd for both dihydrotestosterone and testosterone is about 1 X 10(-9) M and for estradiol about 2 X 10(-8) M. Its properties are generally consistent with those of androgen receptor reported for other tissues.

Amygdala↗