Dextran-coated Charcoal Assay Research Articles

Steroid receptors in human endometrium. comparison of data obtained by three different methods

1. 1. To find a method for steroid receptor measurement in small endometrial tissue samples (< 100 mg), an isoelectric focusing assay has been compared with a dcxtran-coated charcoal assay for oestradiol receptor. 2. 2. The results correlated well ( r = 0.85) and this indicates that isoelectric focusing is a good technique for oestradiol receptor determination. 3. 3. The isoelectric focusing of progesterone receptor has been compared with a dextran-coated charcoal assay and sucrose density gradient centrifugation. 4. 4. Isoelectric focusing gave recoveries of 0–26% compared to receptor values obtained with the two other methods, which correlated well ( r = 0.97). 5. 5. The low recovery implies that the isoelectric focusing assay is not suitable for progesterone receptor determination.

Some practical aspects of the separation of estrogen and progesterone receptors by size exclusion high performance liquid chromatography which are relevant to quantitation

Estrogen and progesterone receptors, prepared from rodent and human tissues, have been separated on a size exclusion column (TSK-G3000SW) using a high performance liquid Chromatograph. Reproducible, minor differences in elution behaviour between various preparations of human estrogen and progesterone receptors were often observed. The trailing edges of receptor profiles obtained by size exclusion HPLC characteristically broadened the elution of individual peaks so that overlap between peaks often occurred. This broadening reduces the feasibility of automated receptor analysis based upon the pre-programmed collection of select peaks. Nonetheless, automated receptor analysis, based upon the collection of all eluant fractions remains practical. Quantitative estimates of estrogen and progesterone receptor concentration were made using the size exclusion HPLC method and compared to estimates obtained with the hydroxylapatite adsorption assay, the dextran-coated charcoal assay and sucrose gradient sedimentation separation. Size exclusion HPLC estimates of rodent and human receptor activity were in agreement with estimates obtained by the other assays. Moreover, receptor estimates obtained by size exclusion HPLC were generally greater than estimates obtained by methodologies where significant ligand dissociation can occur during the application of the assay (i.e. hydroxylapatite adsorption and sucrose gradient sedimentation). Thus, in addition to improved qualitative receptor analysis, the size exclusion HPLC methodology provides receptor quantitiation which is compatible with other techniques.

Estrogen receptors in human breast cancer: Comparative features of the hydroxylapatite- and dextran-coated charcoal assay

Samples of human breast cancer tissue were analyzed for estrogen receptor content (ER) with a hydroxylapatite method (HAP) as well as a dextran-coated charcoal method (DCC). It was found that both methods revealed artificially low ER concentrations in cytosols with low protein concentration. It was also found that the DCC assay underestimated the ER activity in the tumors compared to the HAP assay. No differences in terms of low affinity binding nor K d-values of high affinity estrogen binding were observed. A significant higher background radioactivity was obtained in the HAP assay, and in some tumors one assay would classify them as ER-negative and another as ER-positive.

Immunohistochemical Localization of Estrogen Receptors in Human Mammary Carcinoma Using Antibodies to the Receptor Protein

Abstract An immunofluorescent method has been developed for localizing estrogen receptors (ER) in frozen sections of human breast cancer biopsies which utilizes the biochemically well-characterized antibodies to the cytoplasmic ER of human breast cancer produced in rabbits. Frozen sections of sixteen breast cancer biopsies, in which the receptor content has been quantified using dextran-coated charcoal assay (DCC technique), were used to study the localization of ER. Two aspects of ER detection, namely “estrogen-binding” and ability to react with the homologous antibodies, have been analyzed and the results compared with those of DCC technique. Fluorescent labelled estrogen method (F.E 2 ) of Pertschuk was utilized to detect the distribution of estrogen binding proteins and the indirect immunofluorescent (IM-AR) method using anti-receptor antibodies to assess the antigenic sites of the receptor protein. In addition to the comparison of the results of three techniques, DCC, IM-AR and F.E 2 , for ER detection, a procedure for processing the frozen sections, which enables the study of estrogen binding characteristic as well as the antigenic reactivity of the receptor molecules, is described.

Cytoplasmic and nuclear progesterone receptors in mouse mammary tumours during pregnancy determined by an improved hydroxylapatite assay.

The binding of [3H] 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to progesterone receptors in cytosol and nuclear extracts (0·6 m-KCl) of the pregnancy-dependent, TPDMT-4 mouse mammary tumour was measured at various stages of pregnancy. Compared with conventional dextran-coated charcoal (DCC) assays, a hydroxylapatite assay with DCC pretreatment and precharging of the cytosol with unlabelled R5020 (4 × 10−8 mol/l, for 3–4 h at 4 °C) showed the highest level of binding. The DCC treatment markedly increased the level of R5020 binding in both cytosol and nuclear extracts by allowing the receptor to bind to hydroxylapatite. The DCC pretreatment apparently removed a heat-stable and non-dialysable factor which prevented the receptor from binding to the hydroxylapatite. Using this assay R5020 binding reached a steady state in 24 h at 4 °C, with complete exchange of radioactive for non-radioactive ligand by 20 h. Nuclear extracts did not require precharging and complete exchange was more rapid. Scatchard analysis (without precharging) disclosed a single class of binding sites with a dissociation constant for cytosol of 3·2 ± 0·8 (s.e.m.) × 10−9 mol/l (n = 3) and 4·7 ± 0·6 × 10−9 mol/l (n = 5) for the nuclear extract. Binding was hormone-specific and progesterone translocated binding from the cytoplasm to the nucleus both in vivo and in vitro. Translocation, however, led to a substantial loss of total (nuclear + cytoplasmic receptors. During pregnancy, cytoplasmic progesterone receptor levels were unchanged and low compared to nuclear progesterone receptors which increased by sevenfold from days 1 to 11 and then decreased at day 16. Compared with recent data on cytoplasmic progesterone receptors in normal mammary gland, our results suggested that this tumour may have a reduced sensitivity to the down-regulatory activity of progesterone. This lesion may, in part, account for the failure of the tumour to differentiate during pregnancy.

The detection of estrogen receptors in gynecologic tumors using immunoperoxidase and the dextran-coated charcoal assay.

The dextran-coated charcoal receptor assay for demonstrating functional estrogen and progesterone receptors was used to evaluate the receptor content of gynecological tumors. An immunocytochemical method, the immunoperoxidase antiperoxidase method, that may detect estrogen receptors, was also employed on the same specimens utilizing sections from formalin-fixed paraffin embedded blocks. The results by the two methods were compared and were correlated with the state of differentiation of the tumors. According to the dextran-coated charcoal method, two cases of well differentiated endometrial adenocarcinoma were strongly positive for both estradiol and progesterone receptors, and three moderately differentiated cases contained lesser amounts of both types of receptors. Six cases of undifferentiated adenocarcinoma ranged from no detectable receptors to very high values, while of five cases of squamous cell carcinoma of the cervix, only two were positive for estradiol receptors by the dextran-coated charcoal method. Staining of tissue sections from these same cases using the immunoperoxidase method, demonstrated a positive correlation to the dextran-coated charcoal assay for estrogen receptors. The Dextran-coated charcoal method is presently being used clinically as a screening measure for statistical probability of a patient's response to hormone therapy. The degree of positive correlation shown here suggests that use of the immunoperoxidase method may have further potential for diagnostic and clinical use, and merits further investigation.

Quality control for estrogen receptor quantification by dextran-coated charcoal assay: a single laboratory's experience.

Wide fluctuations in results obtained by different laboratories for the content of estrogen receptors from a single source of tissue powder were reported by several investigators at the 1979 NCI Consensus Committee Meeting for Steroid Receptors in Breast Cancer and were further supported by data collected by our laboratory for the Eastern Cooperative Oncology Group. In a multilab study the inconsistency in the receptor content is a product of the variations associated with the assay procedures used by different laboratories and those due to unavoidable daily changes in the experimental conditions within each laboratory. In this report we present data on the variation of estrogen receptor measurements obtained by a single laboratory for any single source of tissue powder repeatedly analyzed. A retrospective analysis of the data collected on the receptor content of several different batches of calf uteri analyzed on different days (inter-assay variation) under uniform conditions revealed a coefficient of variation averaging +/- 35% (standard deviation divided by mean X 100). Experiments showed that the fluctuations in the receptor yield from aliquots of the same tissue powder are primarily an effect of the homogenization step that precedes cytosol preparation. Therefore, for establishing quality control, distribution of lyophilized cytosols should precede lyophilized powders to test the efficiency of performance of laboratories. A homogenizer designed to minimize thermal denaturation is also essential.

Isoelectric focusing of oestradiol receptor protein from human mammary carcinoma—A comparison with dextran coated charcoal assay

Two simple methods for the detection of specific oestradiol—17β receptor protein in breast tumour tissue were investigated. Isoelectric focusing of receptor protein in polyacrylamide gel was examined as an alternative micromethod to dextran-coated charcoal receptor assay. In a series of 32 cytosols prepared from unselected breast tumour tissue, assay results agreed on the presence or absence of receptors in 97% of cases. Quantitative analysis of the positive results showed a correlation coefficient, r = 0.88. Isoelectric focusing gave significantly lower receptor concentrations than the dextran-coated charcoal method. Twenty-six Tru-Cut biopsy tumour specimens were analyzed for receptor by isoelectric focusing and the results were compared with those obtained by the dextran-coated charcoal method on the same tumour. In 88% of the specimens examined, both methods agreed as to the presence or absence of receptor protein. A correlation coefficient, r = 0.62. was calculated from the 15 positive results. There was no significant difference between the results from the two methods. Quantitative results were related to total protein concentration in the cytosol. Negative receptor status was defined as less than 5 fmol/mg cytosol protein for both methods.

Immunohistochemical localization of estrogen receptors in human mammary carcinoma using antibodies to the receptor protein

An immunofluorescent method has been developed for localizing estrogen receptors (ER) in frozen sections of human breast cancer biopsies which utilizes the biochemically well-characterized antibodies to the cytoplasmic ER of human breast cancer produced in rabbits. Frozen sections of sixteen breast cancer biopsies, in which the receptor content has been quantified using dextran-coated charcoal assay (DCC technique), were used to study the localization of ER. Two aspects of ER detection, namely “estrogen-binding” and ability to react with the homologous antibodies, have been analyzed and the results compared with those of DCC technique. Fluorescent labelled estrogen method (F.E 2) of Pertschuk was utilized to detect the distribution of estrogen binding proteins and the indirect immunofluorescent (IM-AR) method using anti-receptor antibodies to assess the antigenic sites of the receptor protein. In addition to the comparison of the results of three techniques, DCC, IM-AR and F.E 2, for ER detection, a procedure for processing the frozen sections, which enables the study of estrogen binding characteristic as well as the antigenic reactivity of the receptor molecules, is described.

Distribution of estrogen and progesterone receptors on primary tumor and lymph nodes in individual patients with breast cancer.

Primary breast cancer tissue and lymph nodes were obtained from 55 patients, Histologically, 34 of these patients had positive and 21 negative lymph nodes. Estrogen receptors (ER) and progesterone receptors (PR) were determined by a dextran-coated charcoal assay. The tumor tissue was ER positive in 58% of the cases and PR positive in 34%. The malignant lymph nodes were ER positive in 56% and PR positive in 24%. ER in 14% and PR in 5% of the benign lymph nodes could be detected. The primary tumor tissue and the corresponding malignant lymph nodes showed an identical ER and PR status, i.e. both tumor sites were receptor positive or both receptor negative, in 68 and 74%, respectively. However, 21% of the patients had receptor-positive tumors but receptor-negative lymph nodes. Receptor-positive lymph nodes in combination with receptor-negative tumors occurred in only 11% for ER and 6% for PR. These data show that receptor-positive malignant lymph nodes mostly display the same receptors status as the corresponding primary tumor, whereas receptor-negative lymph nodes may be combined with receptor-positive tumors.

The effects of a second specific estrogen binding site on estrogen receptor quantitation in human breast cancer cytosol

Cytosol from pooled human breast cancer was used to evaluate the accuracy of different assay methods for estrogen receptor (ER). Saturation analysis over a range of ligand concentrations sufficient to allow the resolution of two-component binding systems yielded accurate estimates. Saturation analysis over the limited range of ligand concentrations generally used yielded inaccurate estimates because of the presence of a second estrogen binding site in breast cancer cytosol. Since the breast tumor specimens often do not yield enough tissue for a wide-range saturation analysis, single saturating-dose assay methods were also evaluated. It was demonstrated that the use of ligand concentrations above 2–4 nM could cause overestimation of the ER concentration even when the dextran-coated charcoal assay was used. Exposure of the cytosol to nonequilibrium conditions during the assay apparently protected against overestimation. The analytical method of choice remains the sucrose density gradient sedimentation method since it combines appropriate ligand concentration during the labeling period and controlled non-equilibrium conditions during the separation step. If a sound, quantitative basis for the interpretation of ER results is to be developed, reproducible and accurate methods for ER quantitation must be used.

Quantitation of the cytosolic glucocorticoid receptor in human normal and neoplastic leukocytes using isoelectric focusing in polyacrylamide gel

The glucocorticoid receptor has been studied in cytosol from normal human lymphocytes, human leukemic cells and rat liver using isoelectric focusing in polyacrylamide gel. The proteolytic receptor fragments obtained after incubation of the [ 3H]-dexamethasone-receptor complex with 0.5 μg trypsin/A 280–310nm focused at pI 5.9–6.1 (major peak) and at 6.1–6.4 (minor peak), respectively. Isoelectric focusing following trypsin digestion gave a better recovery of total steroid-receptor complexes than focusing of the non-trypsinized steroid-receptor complex. Furthermore, the digested receptor fragments formed sharper peaks on the gel than the native receptor. The glucocorticoid receptor in normal human lymphocytes had an apparent dissociation constant of 18.8 ± 3.8 nM and the lymphocytes had a receptor content of 305.0 ± 73.9 fmol/mg protein or 6070 ± 2610 receptor sites/cell. A comparison between conventional dextran-coated charcoal assay and isoelectric focusing in quantitating the glucocorticoid receptor in rat liver cytosol showed that isoelectric focusing gave significantly higher values. The isoelectric focusing patterns of the cytosolic glucocorticoid receptor in leukemic cells from one patient with acute myelomonocytic leukemia and one patient with chronic lymphatic leukemia were very similar to the patterns seen in cytosol from normal human lymphocytes and rat liver. It is concluded that isoelectric focusing in polyacrylamide gel following trypsin digestion is a convenient method to quantitate the glucocorticoid receptor in normal human lymphocytes and human leukemic cells.

Oestrogen receptor assay—Limitation of the method

The variability of estimates of K d and binding capacity ( fmol oestrogen bound/ mg cytosol protein) of oestrogen receptors (RC) which may arise from different cytosol protein concentrations ( 0.25–5 mg/ml), different charcoal concentrations ( 0.25–5 mg/ml) and cytosols from different, histologically comparable portions of the same tumour has been studied using a dextran-coated charcoal (DCC) assay. K d value correlated positively to protein concentration and negatively to charcoal concentration, whereas the binding capacity remained constant over a wide range of protein concentrations. In receptor-poor tumours, low cytosol protein contents precluded a distinction between specific and non-specific oestrogen binding. Both in receptor-rich and receptor-poor tumours, increasing charcoal concentrations added to the incubation mixture decreased the cytosol protein concentration and thus removed RC complexes as well as excess of oestrogen. Both in receptor-rich and receptor-poor tumours, a wide variation in recptor content was observed within histologically comparable portions of the same tumour (from 0 to 300 fmol/mg protein).

Simultaneous measurement of estrogen and progesterone receptors in tumor cytosols with use of 125I-labeled estradiol and of 3H-R5020.

We describe a dual-isotope assay for measuring the concentration of estrogen receptors and progesterone receptors in tumor cytosols. The concentrations of these receptors are derived from separate Scatchard-plot analyses of a single dextran-coated charcoal assay that incorporates both radioiodinated estradiol and tritiated R5020 as the labeled ligands. The two isotopes, radioiodine and tritium, are easily measured with a liquid-scintillation counter. The concentrations of estrogen and progesterone receptors derived from the dual-label assay are identical to values derived from the respective single-label assays. Moreover, values obtained from an assay with dextran-coated charcoal that incorporates radioiodinated estradiol are identical to those obtained from an assay in which tritiated estradiol is used as the labeled ligand. The dual-isotope assay requires both less time and less tissue.

Glucose-6-phosphate dehydrogenase activity in human breast cancer. Lack of association with oestrogen receptor content.

The correlation between glucose-6-phosphate dehydrogenase activity and different tumour characteristics was investigated in human breast cancer tissue. The enzyme activity was measured by a histochemical method and the oestrogen receptor content by a dextran-coated charcoal assay. The proliferative activity of the tumours correlated positively with the glucose-6-phosphate dehydrogenase activity. No correlation was found between enzyme activity and tumour type and menopausal status, age, TNM-class nor oestrogen receptor content. Based on this investigation it is concluded that the activity of glucose-6-phosphate dehydrogenase reflects the proliferative status of the tumour but not the hormone dependency of the tumour.

Estrogen receptor: a prognostic factor in breast cancer.

Two-hundred-seventeen women with primary breast carcinoma had an estrogen receptor determination tested by both the dextran-coated charcoal assay and sucrose density gradient. The results were correlated with the disease-free interval, survival, response to hormone therapy or chemotherapy, and site of recurrent disease. The disease-free interval (DFI) was significantly longer in premenopausal patients with estrogen receptor positive (ER+) determination compared with premenopausal patients with estrogen receptor negative (ER-) determinations, irrespective of nodal involvement (P less than 0.05). There was no difference between the postmenopausal patients. The survival of the ER+ patients was statistically longer than that of the ER- patients (P less than 0.05). Statistical significance remained when the patients were grouped according to menopausal status or nodal involvement (P less than 0.002 or less). Sixty-two patients were treated with hormonal therapy, either ablative or additive. Forty-eight percent of patients with ER+ responded compared with 6% of patients with ER- (P less than 0.0005). Seventy-nine patients received chemotherapy; 52% of the ER+ and 57% of the ER- patients responded (P less than 0.5). ER+ tumors had a predilection to metastasize in skin and bone, while ER- tumors metastasized more commonly to the viscera and brain.

Estrogenic effects of nafoxidine on ovarian-dependent and independent mammary tumor lines in the mouse.

Nafoxidine (1-[2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]ethyl]-pyrrolidine hydrochloride); a triphenylethylene derivative] and estradiol cause translocation of the estrogen receptor to the nucleus, elevate the levels of cytoplasmic progesterone receptors, and do not effect levels of nuclear progesterone receptor in hormone-dependent and -independent MXT-3590 mammary tumor lines. Cytoplasmic and nuclear receptor levels were measured by the dextran-coated charcoal assay and a modified protamine sulfate nuclear exchange assay, respectively. Both estradiol (2.5 microgram) and nafoxidine (40 microgram) stimulate partial growth in the independent line. These data are the first evidence of the estrogenic effects of a so-called antiestrogen (nafoxidine) on the quantity of progesterone receptors in a hormone-independent experimental mammary tumor. The growth effects of antiestrogens demonstrated here in ovariectomized mice should alert clinicians to be watchful of these effects when using such drugs in the treatment of human breast cancer. These results also confirm recent suggestions that growth and progesterone receptor synthesis are controlled separately by estrogenic compounds. (Endocrinology 108: 668, 1981)

Identification, partial characterization and age-related changes of a cytoplasmic androgen receptor in the rat penis

A cytoplasmic androgen receptor has been identified in the penis of the immature rat utilizing a dextran-coated charcoal assay and sucrose density gradient centrifugation. The receptor sediments at 8S, is specific for androgens and binds with high affinity to 5α-dihydrotestosterone, K d = 5.52 × 10 −1U M. In addition, this penile receptor is found in very high concentration in the immature period and decreases with aging to reach almost undetectable levels at adulthood. This diminution in the concentration of the penile receptor from birth to adulthood appears to be consistent with the clinical and experimental observation that the maximum androgen-induced response in the penis occurs during the immature period while the penis is relatively unresponsive to androgenic stimulation at adulthood. The identification of an androgen receptor in the penis should provide an important probe into the elucidation of the endocrine events that govern the ultimate size of the adult penis.

Stabilization of human breast cancer progesterone (5020) receptor by sodium molybdate

Inclusion of the salt, sodium molybdate, at 20 mmol/l concentration during the determination of progesterone receptor in human breast cancer cytosols was associated in a number of samples with marked stabilization of binding by the radioactive ligand, 5020, to 8S and 4S receptors. Both the percent of positive patients and the absolute amount of PR were increased, with the appearance of 8S peaks where none had been evident in controls without the salt, and increased amounts of 8S and 4S binding in other samples. In addition, results from some dextran-coated charcoal assays were altered, and positive samples incubated in the presence of the salt frequently yielded higher values of PR. Finally, estrogen receptor values determined by SDG assays have been increased in the presence of the salt. Routine addition of sodium molybdate to samples analyzed for PR by centrifugation overnight can be expected to yield an increased percent of receptor-positive patients with higher average concentration of receptor, compared with results of previous methodology. Its presence during the dextran-coated charcoal assay can also alter the measured binding. Although the stability of ER during overnight centrifugation is greater, in some patients additional receptor can be detected when buffers include this salt.

A comparative study on estradiol receptor assays in human breast cancer tissue

Standard dextran-coated charcoal assays for measuring the estrogen receptor content in human breast cancer tissue are compared. A single-point assay, using the estrogen antagonist nafoxidine as competitive inhibitor, showed good qualitative agreement with a three-point Scatchard plot assay, using radio-inert estrogen to assess the non-specific binding. Based on statistically evaluated dividing points, 116 (66.3%) of the 175 analysed primary tumor specimens were positive in the single-point assay compared to 121 (69.1%) in the Scatchard plot assay. Use of the additional dissociation constants in the evaluation of the Scatchard plot assay data gives a complete qualitative agreement between the two assays. We conclude that, in spite of the lack of specific controls inherent in the Scatchard plot assay, the single-point assay using diethylstilbestrol instead of nafoxidine is equally useful, especially if biopsy specimens are too small to provide the larger number of aliquots necessary for multiple steroid receptor analyses. Factors affecting the quantitative differences between the two assays are discussed.