B. G. Mobbs

Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: can estrogen treatment increase androgen receptor content?

The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (3H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15 degrees C rather than at 0 degrees C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.

In vitro assay of androgen binding by human prostate

Abstract The in vitro binding of 5α-dihydrotestosterone by a “cytosol” fraction of human pathological tissue was investigated, and compared with binding by the rat prostate, an androgen-responsive mouse mammary tumour, and some androgen-unresponsive control tissues. Androgen-binding levels in the human control tissues were higher than those in the rat and mouse, and were not lower than androgen-binding levels in many prostatic specimens from untreated patients. Estrogen therapy and/or orchiectomy appeared to result in higher binding levels. The steroid specificity of binding in the human prostatic tissue appeared closer to that of sex hormone binding globulin than to that of the androgen receptor in the rat prostate. It was concluded that the assay used was not sufficiently specific to distinguish androgen binding by sex hormone binding globulin from that by an intracellular prostatic androgen receptor.

Use of an enzymeimmunoassay (EIA) for quantitation of cytosolic and nuclear estrogen receptor, and correlation with progesterone receptor in human breast cancer

We have adapted a commercially available enzyme immunoassay for ER (ER-EIA) for use with nuclear extracts of breast carcinoma specimens, and have examined the data obtained in relation to the results from cytosolic ER-EIA and radioligand (DCC) assays and from DCC assays for PgR. In a series of 139 carcinoma specimens, we observed a very significant correlation between the cytosolic ER concentration as measured by the EIA and DCC methods, and also between the log10 of the concentration of ER in the cytosolic and nuclear fractions assayed by the ER-EIA method. The correlation between nuclear ER and cytosolic PgR was also highly significant, but not as close as for cytosolic ER. If 130 fmol ER/mg DNA was used as a cut-off point to discriminate between specimens positive and negative for ERN, there was 87% concordance in receptor status between ERN and cytosolic ER, and 79% concordance between ERN and cytosolic PgR. Forty-one percent of specimens were positive or borderline for both ERN and cytosolic PgR, and it is suggested that this receptor combination may be a valuable predictive factor in prognosis and response to hormonal treatment.

Evaluation of the use of cyproterone acetate competition to distinguish between high-affinity binding of [3H]-dihydrotestosterone to human prostate cytosol receptors and to sex hormone-binding globulin

From preliminary experiments, it was concluded that cyproterone acetate (CA) was the most satisfactory of several anti-androgens investigated in distinguishing between high-affinity binding of [3H]-5α-dihydrotestosterone to rat ventral prostate cytosol and to human serum, using a simple incubation technique followed by removal of free steroid by Dextran-coated charcoal. Rat prostate cytosol (androgen receptor) binding of [3H]-DHT was almost eliminated by concentrations of CA which affected human serum binding to a relatively slight, though unfortunately variable, extent. n nThe total, high-affinity, and CA-inhibitable binding of [3H]-DHT by prostatic cytosol of a number of patients with benign prostatic hypertrophy and prostatic carcinoma was investigated. In the untreated patients and in some patients treated by orchidectomy and/or estrogens, the total and CA-inhibitable high affinity binding of [3H]-DHT were correlated with the endocrine status of the patient as determined by the serum testosterone level and the high affinity [3H]-DHT binding capacity of the serum (equivalent to sex-hormone binding globulin (SHBG)). n nIt was concluded that the use of cyproterone acetate to distinguish between [3H]-DHT binding to the androgen receptor and serum components in human prostate cytosol permits a semi-quantitative evaluation of the amounts of these two components.

Characterization of estrogen-induced progestin binding in cytosol of the R3327 prostatic carcinoma of the rat

High affinity binding of the synthetic steroids methyltrienolone (R1881) and promegestone (R5020) to cytosol protein from the Dunning (R3327) experimental prostatic carcinoma of the rat was investigated. Animals bearing tumours of approx 1.5 cm mean diameter were either left untreated, or were administered diethylstilbestrol diphosphate (DESP) in the drinking water in doses close to those used clinically for the treatment of human prostatic carcinoma. Tumours were excised after 10-40 days, and binding of [3H]R1881 and [3H]R5020 to tumour cytosol was characterized using Scatchard analysis, sucrose density gradient centrifugation, and steroid competition, under conditions optimal for the conservation and assay of progesterone receptor. Both ligands were bound in much higher concentrations by cytosol from DESP-treated tumours than from untreated tumours. Binding was of high affinity (Kd congruent to 1 nM), was specific for progestins, and sedimented in peaks at approximately 8S and approximately 4S in sucrose density gradients. We conclude the DESP treatment of rats bearing the R3327 prostatic carcinoma induces synthesis of progesterone receptor in this tumour.