Dextran-coated Charcoal Method Research Articles

Cytomorphological grading and hormone receptor status in breast carcinoma. Visual studies of smear preparations

From 923 breast cancers sent in for hormone receptor determination, 745 diagnostically utilizable Pappenheim-stained biopsy smears at 500-fold magnification were classified into eight atypia groups (1-4 without nucleoli, 5-8 with nucleoli) visually in terms of the severity of the cellular malignancy criteria. The hormone receptor status was determined with the dextran-coated charcoal method. Values from 10 fmol/mg estrogen receptor (ER) and (or) progesterone receptor (PR) content were designated as receptor-positive. There was a decrease in the proportion of receptor-positive results with increasing degree of atypia from 97% in group 2 (cases with only small nuclei without nucleoli) to 37% in group 7 (cases with multiple prominent nucleoli). When the average atypia values of subgroups were compared, the highest values were found for the 222 receptor-negative carcinomas. The marginally receptor-positive values were on average between the receptor-negative and the receptor-positive cases with high values. Both the low-ER and the low-PR have more pronounced criteria of malignancy compared to the respective high-receptor reference group.

Glucocorticoid receptor-mediated teratogenesis and cell proliferation in the limbs and face of the chick embryo.

The susceptibility of the face region of the chick embryo to the teratogenic action of intraamniotically injected hydrocortisone contrasts with the resistance of the limbs to its action while at the same time their dysmorphogenesis may be induced by other agents. Since glucocorticoid receptors were shown to mediate face teratogenesis, their development was investigated in freshly dissected limb buds of 3-, 3.5-, and 4-day-old chick embryos in comparison with the face region. The specific binding of 3H-dexamethasone to molybdate-stabilized glucocorticoid receptors was estimated by the dextran-coated charcoal method and complemented by cytologic analysis of mitotic activity in control and hydrocortisone-treated embryos. The glucocorticoid receptors were found in both organ anlagen already on day 3 when their concentration in femtomoles per microgram DNA was significantly higher in the face region. Accordingly, on day 3 intraamniotic hydrocortisone inhibited the mitotic activity in the face without affecting the developing limbs. On days 3.5 and 4 the concentration of glucocorticoid receptors was similar in both organ anlagen. Administration of hydrocortisone on day 4 induced mitotic depression in the face as well as in the limbs. However, the degree of inhibition appeared to be dependent upon the actual mitotic rate. In the face region where the mitotic activity culminated at that time, the inhibition was much deeper and longer-lasting than in the developing limbs characterized by continuous decrease of proliferation rate in controls. These findings are consistent with a view that glucocorticoid receptors are a prerequisite, but not the only factor in receptor-mediated teratogenesis.

COMPARATIVE STUDY OF ESTROGEN RECEPTOR ASSAY IN HUMAN BREAST CANCER USING 16α-125I-ESTRADIOL AND 3H-ESTRADIOL

Estrogen receptors (ER) were determined in 50 human mammary tumor tissues by the dextran-coated charcoal method using 16α-125I-estradiol-17β (125I-E2 Kit) and the sucrose density gradient method using 3H-estradiol-17β (3H-E2). In this study, if a tumor tissue contained more than 5 fmol of ER per mg cytosol protein, it was judged to be ER-positive and if 0 to 5 fmol was present, as ER-negative. The coincident rate of positivity and negativity of ER between the two methods was 90 % (45/50) and the correlation coefficient for the binding sites of ER was r=0.863 in 19 ER-positive cases.These results showed that in ER assay, the 125I-E2 Kit was as useful as 3H-E2, and that the former was rapid, simple and practical.

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450(11) beta, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450(11) beta and may be converted to the high-spin form by the addition of deoxycorticosterone. The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxylation of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.

Relationship between estrogen receptor concentration and cytomorphometry in breast cancer

This study aimed to evaluate the relationship between estrogen receptor (ER) concentration and cytomorphometric parameters in breast carcinoma. Primary breast cancer specimens were both imprinted on cytologic slides and submitted to ER determination with a dextran-coated charcoal method. Patients were stratified by ER levels ranging from 0 to greater than or equal to 200 fmol/mg protein. Measurements were performed on cytologic imprint technique smears in five cases from each ER strata, using a computer-assisted tracing device. Nuclear, cytoplasmic, and cellular parameters were measured on 50 cells per case. The cytometric findings were correlated with ER concentrations. A statistically significant correlation between decreasing area (P = 0.011), perimeter (P = 0.015), maximum diameter (P = 0.034), minimum diameter (P = 0.008), and volume (P = 0.01) of nuclei and increasing ER levels was found. With regard to whole cells, the following parameters significantly decreased versus increasing ER levels: area (P = 0.038), perimeter (P = 0.046), minimum diameter (P = 0.011), and volume (P = 0.044). A statistically significant relationship between the decreasing cytoplasmic perimeter (P = 0.025), i.e., nuclear plus cellular perimeter, and increasing ER content was found. Cytoplasmic area and nuclear to cytoplasmic ratio (N/C) did not correlate with the amount of ER. In all classes of different ER concentration, cells and nuclei displayed a regular shape closer to a circle than to an ellipse. The results of the current investigation indicate that tumors with higher ER concentration are composed of smaller cells with smaller nuclei than are tumors with lower ER content.

Estrogen and progesterone receptors in breast carcinoma and in nonmalignant breast tissue.

Since 1983 we have studied the relationship, in the same patient, between receptor status in breast carcinoma and in nonmalignant breast tissue. Fifty patients have been evaluated to date. The total unoccupied cytosol estrogen and progesterone receptors were determined by a dextran-coated charcoal method. In nonmalignant breast tissue we found a measurable receptor concentration above the sensitivity of the method in 62% of cases for estrogen receptors and in 44% of cases for progesterone receptors. No relationships were found between the receptor level of each tumor and that of the corresponding benign tissue. The data suggest that the levels of the receptors in the tumor and in the nonmalignant tissue are totally independent.

Biochemical and histochemical analysis of steroid hormone binding sites in human primary breast cancer.

Mammary carcinoma tissue from 514 primary breast cancer patients were all biochemically and histochemically analyzed for both estrogen receptors and progesterone receptors. The dextran-coated charcoal (DCC) method measured the ER and PR as defined by Scatchard analysis, ligand competition experiments and target organ specificity. The ligands, estradiol-6-carboxymethyloxime-BSA-fluoresceine isothiocyanate and hydroxyprogesteronehemisuccinate-BSA-tetramethylrhodamine isothiocyanate, used for histochemistry, did not bind to either ER or PR and were mainly bound to the membrane fraction of isolated breast cancer cells. Fluorescence was not specifically inhibited by estrogens or progestogens. In addition, "estrogenic" always coincided with "progestogenic" fluorescence. The binding of the fluoresceine compounds to tissue slides depended on the large steroid hormone substitution on the bovine serum albumin molecule. Clinical parameters, known to be related to ER and PR did not correlate with the histochemical results. The observations indicated the impossibility of specific steroid receptor detection by the histochemical method. Therefore, up to the present, evaluation of hormone dependency and prognosis in human breast cancer cannot be based on this approach.

Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Correlation with biochemistry and clinical endocrine response.

Breast cancer specimens from 114 patients were assayed for the presence of estrogen receptors (ER) utilizing highly specific, monoclonal antiestrophilin antibodies and the peroxidase-antiperoxidase technique. Results were compared with conventional ER determinations by the dextran-coated charcoal method (DCC) and were in agreement as to positivity and negativity in 86%. Semiquantified immunocytologic assay results were in accord with the level of ER as measured by DCC in 66%. The tumors studied included 43 from patients with Stage IV disease where clinical response to hormonal manipulation was known. In the latter group, the immunohistologic method had a sensitivity similar to that of DCC but showed a superior positive predictive value and a significantly better specificity. These results indicate that this new method is a valuable laboratory tool, enabling prediction of hormone responsiveness in advanced mammary carcinoma and capable of performance at the community hospital level.

Prognostic value of estrogen receptors determined by radiochemical vs. histochemical methods in breast cancer

Estrogen receptors (ER) were evaluated in 634 breast cancer patients by the dextran-coated charcoal method (DCC). In 206, ER and progesterone receptors (PR) were also tested by cytochemistry (Lee method), and in 124 ER were tested by immunofluorescence (Pertschuk method). The median follow-up is 3.7 years. Comparisons have been made between receptor content and: anatomical and clinical features, disease-free survival (DFS), and survival. The following conclusions can be drawn: there is no correlation between ER determinations by DCC and by immunofluorescence or cytochemical methods; there is no evidence of association between ER and PR determined by Lee's method and anatomical and clinical features; a highly significant positive association was found between ER rich specimens and age, post-menopausal status, lobular and tubular histologic types; there is no association between ER values and TNM stage, WHO grading, pathologic prognostic factors of primary tumor and of lymph nodes; and the DFS was not affected by ER status, except for tumors with more than 50 fmol/mg protein.

Radioimmunoassay of bencyclane in human serum.

A radioimmunoassay of bencyclane in human serum was developed. Male rabbits were immunized with p-(3-carboxy-propoxy)bencyclane-bovine serum albumin conjugate, giving antisera with high titers. 125I-p-Hydroxybencyclane with a high specific activity was prepared as a labelled antigen by a chloramine-T method. In the radioimmunoassay procedure, a mixture of serum sample, diluted antiserum and 125I-antigen solution were incubated at 4 degrees C for 18 h, and bound-free separation was carried out by a dextran-coated charcoal method. The detection limit of bencyclane in human serum was 1.0 ng/ml, and the cross-reactivity of the antiserum with metabolites was found to be very low. Serum samples from healthy volunteers dosed orally with bencyclane fumarate were analyzed by both of the radioimmunoassay and gas chromatography-mass spectrometric methods. An excellent correlation was observed between the values obtained by both methods.

Steroid receptors in intracranial tumors.

Cytosolic estrogen (ER), progesterone (PR), androgen (AR), and glucocorticoid (GR) receptors were evaluated in 6 meningiomas and 12 gliomas (6 astrocytomas and 6 glioblastomas) using a dextran-coated charcoal method. Thirty-three percent (two) of the meningiomas and 17% (two) of the gliomas contained positive (greater than or equal to 10 fmol/mg protein) but low ER levels. On the other hand, positive PR, GR, and AR levels were detectable in 100% (six) of the meningiomas. PR was present in 50% (six), AR in 42% (five), and GR in 58% (seven) of the gliomas. PR, GR, and AR values were statistically higher in meningiomas than in neuroepithelial tumors.

Estrogen receptors in brain tumors.

We examined the cytosolic estrogen receptor (ER) level in tumor tissue from 77 patients: 36 meningiomas, 20 gliomas (12 glioblastomas, 2 cerebellar astrocytomas, 2 ependymomas, and 4 medulloblastomas), 8 neurinomas, 7 pituitary adenomas (2 prolactin-producing adenomas, 1 growth hormone-producing adenoma, and 4 nonfunctioning adenomas), and 6 metastatic brain tumors (1 from breast cancer, 4 from lung cancers, and 1 from colon cancer). Nuclear ER levels were assayed in 11 meningiomas and 2 glioblastomas. ER was determined by the dextran-coated charcoal method and calculated by Scatchard analysis. Cytosolic ER was detected in 100% of the pituitary adenomas, 50% of the meningiomas, 50% of the metastatic brain tumors, 25% of the neurinomas, and 15% of the gliomas. In gliomas, only medulloblastomas had ER activity. Nuclear ER was found in three premenopausal women with meningioma. The dissociation constant of the ER complex was, in each case, less than 10(-9) M. These observations suggest that some brain tumors may be responsive to estrogen via the cellular ER.

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextrancoated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5α-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [ 3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated K d for methyltrienolone (R1881) of 5 nM at 0°C. Scatchard analysis of [ 3H]Dx binding by RAG cytosol showed a K d of 6 nM for Dx and 44 nM for corticosterone at 0°C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [ 3H]testosterone and [ 3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [ 3H]testosterone. There was little conversion of DHT to androstanediol.

Quantitative aspects of the E2 receptor assay for human breast tumour cytosol using dextran-coated charcoal.

Misclassification of the oestrogen status of a human breast tumour cytosol, arising from different sources and magnitudes of error in the dextran-coated charcoal (DCC) method, have been investigated using both practical and computer simulated data analysed by Scatchard and Mass Action models. The minimum detectable receptor site concentration, relative or absolute numerical bias and imprecision which are complex and integral functions of misclassification, have been calculated from practical data and for a range of experimental conditions likely to be encountered in practice. The Mass Action model was found to be superior and the computer program, designed to investigate the effects of methodological errors on quantitative aspects of the assay, may be a useful aid for analytical design and internal quality control of the receptor assay.

A sensitive and specific radioimmunoassay for 1-β- d-arabino-furanosylcytosine

In order to determine the blood level of 1-β- d-arabinofuranosylcytosine (Ara-C), an antileukemic agent, a sensitive and specific radioimmunoassay (RIA) system using anti-Ara-C serum, [5- 3H] Ara-C and a dextran-coated charcoal method has been developed. The anti-Ara-C serum obtained from a guinea pig was hardly cross-reactive with 1-β- d-arabinofuranosyluracil (Ara-U), tetrahydrouridine (THU) and other Ara-C analogues. The RIA system for Ara-C could detect concentrations as low as 60 pg/ml in plasma. Average of the intra- and inter-assay variancies at 5, 10, 20 ng/ml were 4.3% and 5.6%, respectively. Ara-C in blood samples obtained from human patients who orally received N 4-palmitoyl-1-β- d-arabinofuranosyl-cytosine (PL-AC) was determined by the present RIA system.

Receptor heterogeneity of human breast cancer as measured by multiple intratumoral assays of estrogen and progesterone receptor

Multiple intratumoral tissue samples from the primary mass of 30 consecutive invasive breast cancer patients were assayed for estrogen receptor (ER) and progesterone receptor (PR) by the dextran-coated charcoal method following frozen section histopathological examination. Steroid receptor status of each sample was classified as positive (R+) or negative (R−), based only upon quantitative guide lines form the ER/PR results. Four out of 32 (12.5%) of the invasive cancers had an intratumoral sample classified as R+ and one sample as R−. R+ invasive ductal carcinomas has a highly significant degree of tubule formation ( P = 0.005) when compared with R− invasive ductal cancers. While the quantitative ER content ( r = 0.18) and the degree of quantitative variation in ER content ( P = 0.04) did not correlate with the tumor cellularity of the individual samples, tumor cellularity ( P = 0.005) and ER content ( P = 0.005) were lower in the samples from the tumor border than from the central tumor samples. Variations in ER and PR content may be found on a regional basis within a breast tumor mass resulting from heterogeneity of tumor subpopulations and/or differences in tumor cellularity.

Steroid receptors in cns: Estradiol(ER) and Progesterone(PR) receptors in human spinal cord tumors

To investigate the role of steroid hormones in the occurrence and growth of human spinal cord tumors and to study the biochemical basis of the sex related difference in the incidence of the various spinal oncotypes, ER and PR have been measured in 24 human spinal cord tumors (12 neurinomas, 6 meningiomas, 5 ependymomas and 1 astrocytoma) by means of sodium thiocyanate exchange assay at low temperature and with dextran-coated charcoal method and R5020 for PR. Cytoplasmic and nuclear receptors have been evaluated. Neurinomas are the oncotypes which contained both ER (usually in the nuclear fraction) and PR: estradiol binding has been found only in one meningioma; ependymomas contained cytoplasmic ER and the astrocytoma had nuclear ER and cytoplasmic PR. The dissociation constant is in favour of a high affinity binding ( K ind = 0.15 × 10 −9 M) for ER c; K d = 3.85 × 10 −9 M for ERn; K d = 8.07 × 10 −9 M for PRc). The overall incidence of steroid receptors in the spinal oncotypes studied is higher in male than in female patients (63.64 vs 46.15% for ER and 100 vs 71.43% for PR). Further studies on a greater number of patients will help to define the correlation between different spinal oncotypes and steroid receptors as well as the possibility of an endocrine therapy as adjuvant treatment for selected patients.

Estrogen and progesterone receptors and progesterone metabolism in canine mammary tumours.

Twenty-seven canine mammary tumours of varying histological types were examined for the presence of estrogen and progesterone receptors and aspects of [ C] -progesterone metabolism. Using dextran-coated charcoal method with the binding parameters of the reaction determined by Scatchard plots, a half of the 27 mammary tumours were shown to contain both receptors. The majority (69%) were benign tumours. This figure fell to 25% for estrogen receptor negative and/or progesterone receptor-negative tumours. After incubation of [ C]-progesterone with cell-free homogenates of mammary tumours and normal glands, 5α-pregnanedione, 17α-hydroxy-5α-pregnanedione, 5α-pregnanediols, and 5α-andro-stanedione were identified as the main metabolites. Although the enzyme activities relevant to the metabolism varied amoung the tissues examined, the most active enzyme demonstrated was 5α-reductase in both tissues. This enzyme activities were significantly greater in the estrogen receptor positive tumours than in the normal glands. These data indicate that some of canine mammary tumours may well be hormone-dependent and these possess active steroid metabolising enzymes.

Human breast carcinoma (MCF-7) serially transplanted into nude mice.

The tumor cells (0.5 ml, 1 X 10(7) of MCF-7 line were inoculated into the subcutaneous tissue or intraperitoneum of female BALB/c nude mice. Primarily transplanted mice were treated with 17 beta-estradiol dipropionate (E2) in a dose of 5 mg/kg and 17 alpha-hydroxy progesterone caproate (Pg) in a dose of 250 mg/kg once a week. After the transferable strain was established, tumors were transplanted into female and male mice treated with E2, Pg, and E2 + Pg. The tumors treated with E2 or E2 + Pg grew exponentially while tumors in the other group regressed. Pg was assumed to play some role in the growth of MCF-7, in the presence of estrogen. Although cytosol estrogen receptors (ERc), nuclear estrogen receptors (ERn), and progesterone receptors (PgR) were detected by dextran coated-charcoal method and exchange assay in the growing tumors, ERn and PgR of regressing tumors was usually negative. This MCF-7 strain in nude mice may be a promising animal model for studying chemo-hormone therapy for human breast carcinomas.

Specific estrogen binding sites in human lymphoid cells and thymic cells

The binding of estrogen in preparations of human peripheral blood mononuclear cells, as well as by splenic and thymic cells is demonstrated by three different approaches (Dextran-coated charcoal method, whole cell assay, and gel filtration on a sepharose 4B column). Scatchard's analysis of [ 3H]-moxestrol (R2858) and [ 3H]-estradiol binding proves the existence of a single class of receptor sites having a dissociation constant of 0.18−2.4 × 10 −9 M. Physicochemical properties of the binder, including binding capacity and steroid specificity, are quite similar to those reported for the thymus of small mammalian species or human thymoma.