Number Of Androgen Binding Sites Research Articles

Down-regulation of androgen receptor by progestins and interference with estrogenic or androgenic stimulation of mammary carcinoma cell growth

The regulatory influence of medroxyprogesterone acetate (MPA) on estrogen and androgen receptors of the human breast cancer cell lines MCF-7 and EFM-19 was explored in conjunction with the growth-promoting properties of these steroids. In the absence of steroidal stimulation, up to 1 microM MPA had no effect on the proliferation of the MCF-7 cell strain used and of EFM-19 cells. Under stimulation with 10 nM 17 beta-estradiol or 1 microM dihydrotestosterone, dose-dependent inhibition of the cell proliferation rates by 0.1-1 microM MPA was observed. Binding of MPA to the androgen receptor (Kd = 2.1 nM) but not to the estrogen receptor was demonstrable. During incubation of MCF-7 or EFM-19 cells with 1 microM MPA for 7 days, the estrogen and androgen receptor contents were down-regulated by approximately 50% and 60%, respectively. Likewise, the number of androgen-binding sites was reduced to 35% of the untreated controls after incubation of MCF-7 cells with 1 microM synthetic progestin R5020 for 7 days. The results indicate down-regulation of estrogen and androgen receptors by progestins in the absence of stimulatory effects on the proliferation of mammary carcinoma cells.

Age-related changes in the concentration of cytosolic androgen receptors in the epididymis, vas deferens and seminal vesicle of maturing male mice.

In this study, changes in the number of androgen binding sites that occur in cytosols of epididymis, vas deferens and seminal vesicle of mice from 10 to 90 days of age are described. Specific saturable binding of [3H]R-1881 by cytosols of the three organs at all time points studied and age-related differences in the number of binding sites measured were observed. Cytosolic androgen receptor levels in all three organs studied were found to decrease with increasing age, regardless of whether the binding was expressed relative to weight of tissue, cytosolic protein or cellular DNA. The most pronounced change in androgen receptor levels (from 442 to 50 fmol/mg protein) was observed in the epididymis between 10 and 30 days of age. In these three organs there was no significant correlation between androgen (testosterone + dihydrotestosterone) levels and the concentration of androgen binding sites.

Mediation by the Androgen Receptor of the Stimulatory and Antiandrogenic Actions of 17β-Estradiol on the Growth of Androgen-Sensitive Shionogi Mammary Carcinoma Cells in Culture

Increasing concentrations of 17 beta-estradiol (E2) led to a maximal 7-fold stimulation of growth of the highly androgen-sensitive clone (SEM-1) of the mammary carcinoma Shionogi cell line. Half-maximal stimulation by the estrogen was observed at 100 nM E2. Diethylstilbestrol (DES), on the other hand, a synthetic estrogen with no affinity for the androgen receptor, had no significant stimulatory effect on cell growth but caused growth inhibition at concentrations above 1 microM. Mediation of the action of E2 by the androgen receptor is indicated by the absence of interference of E2 action by the antiestrogen LY156758 while the antiandrogen hydroxyflutamide (3 microM) caused a 50% inhibition of E2 action. While increasing concentrations of E2 led to a progressive increase in cell growth, a progressive shift in the ED50 value of action of dihydrotestosterone (DHT) was observed at intermediate (10-100 nM) concentrations of E2 while 10 microM E2 completely inhibited DHT action. At those high E2 concentrations, however, E2 itself led to a stimulation of cell growth equivalent to approximately 50% of the maximal value achieved by DHT. E2 competed with the specific uptake of [3H]testosterone in intact cells at an inhibition constant (Ki) value of 15 nM, thus indicating direct interaction of E2 with the androgen receptor. Preincubation with E2 had no influence on the apparent affinity of testosterone for the androgen receptor nor on the number of androgen binding sites. The present data demonstrate that both the stimulatory and antiandrogenic action of E2 on the growth of the androgen-sensitive mammary carcinoma cell line SEM-1 are mediated through direct interaction of the estrogen with the androgen receptor. Such data may offer an explanation for the subjective improvements reported in prostate cancer patients receiving a high dose of E2 when relapsing after castration.

Effects of alcohol feeding on androgen receptors in the rat pituitary gland

Specific binding of testosterone-1β, 2β- 3H by cytosol from anterior pituitary gland of alcohol-fed, isocaloric control, and castrated control and alcohol-fed rats with or without testosterone treatment has been investigated by charcoal assay. The number of androgen binding sites was significantly reduced in alcohol-fed rats (8±1.0 fmoles/mg cytosol protein), when compared to the isocaloric control value (13.2±2.1 fmoles/mg protein), with no significant change in Kd (0.7±0.14 nM). Castration significantly increased the number of receptor sites in control rats and when castrated control animals were treated with testosterone the binding sites were decreased to the intact control level. In contrast, castration or testosterone given to castrated alcohol-fed rats did not alter alcohol-induced reduction of the receptor sites. The binding affinity (Kd) is identical in all groups. The concentration of serum luteinizing hormone (LH) was significantly lower in alcohol-fed rats when compared to that of normal controls. An increased serum LH level with a decreased testosterone level was noted in castrated control rats. However, castration of alcohol-fed rats had little or no effects on the concentrations of LH and testosterone. Administration of testosterone suppressed castration-induced high LH in control rats but alcohol-induced reduction of LH level was not altered by this treatment. These findings indicate that alcohol exerts a suppressive effect on the content of androgen receptors and secretory functions of gonadotropins in the pituitary gland.

Thyroidal modulation of androgenic expression in mice submandibular gland.

We investigated the influence of testes and thyroid gland on the maintenance of biochemical parameters and of [3H]R1881 binding sites of adult mice submandibular gland (SMG). Castration (Cx) performed at beginning of puberty prevented sex-dependent SMG development without interfering with maximal androgen binding capacity. Thyroidectomy (Tx) had strong effects on SMG, mainly by lowering the number of androgen binding sites. All alterations could be fully reverted after treatment with testosterone (5 mg/animal, single dose) or with thyroxine (T4, 250 micrograms/animal per day during 5 days). The effects of Cx on SMG could be reverted by therapy with testosterone, T4, or with both hormones (testosterone + T4) in a non-synergistic fashion. It is shown the importance of thyroidal activity on the physiological maintenance of androgen receptors in the murine SMG; the role played by thyroid gland seems to be essential for the full expression of the androgen-dependent SMG activity in adult mice.

Anabolic Steroids and Training

Based on the currently known actions of androgens as anabolic compounds, we conclude that skeletal muscle represents a relatively minor target for anabolic steroids, if the androgen receptor is considered a limiting step in steroid action. The possibility exists that steroids are able to exert other cellular effects such as through regulation of post-transcriptional events or through the glucocorticoid receptor. Muscle overload by weight training may also increase the number of androgen binding sites, thus making the trained muscles more susceptible to anabolic compounds. Women with fewer circulating androgens and more androgen receptors than men are likely to be influenced to a greater extent than men by anabolic steroid administration. However, a great deal of research is needed to explore these possibilities and to further substantiate the biochemical functioning and mechanisms of anabolic steroid action in muscle.

Ontogenesis of androgen receptors in the mouse submandibular gland: correlation with the developmental profiles of circulating thyroid and testicular hormones.

Specific binding of the synthetic androgen, [17 alpha-methyl-3H]methyltrienolone, to the cytosol fraction of the submandibular salivary gland (SMG) of male mice was studied in relation to the developmental profiles of testosterone and thyroid hormones in blood. The peak rise of serum triiodothyronine (T3) at prepubertal age was closely related to both the increase of maximal androgen-binding capacity in SMG and the conspicuous surge of proliferative activity as indicated by increased rate of glandular DNA content. Also, 2-month thyroidectomized mice had an age-related, strong reduction in the number of androgen-binding sites. On the other hand, the development of the secretory functions of the gland could be better related to the rise of circulating testosterone by days 25-30 of age. The results suggest that thyroid hormones play a very important role in the early induction and further maintenance of androgen receptors in the murine SMG.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland.

Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 degree C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5'-phosphate (5 mmol/l) from nuclear fractions with 93-95% efficiency. The exchange of the bound steroids occurred by 24-48 h at 0 degree C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0.8 and 0.9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females.

Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone ( 3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 ± 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 ± 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 ± 0.6 nM) on the other hand was not different from that (1.7 ± 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.

Hypothalamic-hypophyseal-gonadal axis in the streptozotocin-induced diabetic male rat

In the present work it was observed that the diabetic state alters the hypophyseal response to castration, without the expected increase in the LH serum levels, as found in the control rats. On the other hand, the stimulation of the hypophysis with LHRH resulted in a lower response in the case of the diabetic animal. The results presented herein are in good agreement with the finding of a reduction in the number of androgen binding sites and also with a diminished activity of the 5α-reductase in the hypophysis from diabetic animals. The present results indicate an alteration in the hypophyseal gonadotropin production as well as in the overall process of hypophyseal response in experimental diabetes.

Androgens control androgen-binding sites in rat epididymis.

Previous results suggested that the number of androgen-binding sites in rat epididymis was controlled by androgens. Using an exchange technique for receptor determination, we now report that cytoplasmic receptor number decreased to about half the control value after 4 days of castration and reached its lowest level (30% of the control value) after 12 days of castration. The Kd for [3H]methyltrienolone for cytoplasmic receptor varied from 2.8 X 10(-9) M in controls to 0.6 X (10-9) M (P less than 0.001) after 6 or more days of castration. Nuclear binding sites were reduced to 17% of the control value from the second day of castration on, but no change in the affinity for methyltrienolone was noted. The administration of testosterone propionate (400 micrograms/day) to rats castrated for 20 days significantly increased the number of nuclear and cytoplasmic binding sites from the second and third days of treatment, respectively. Labeled thymidine incorporation into DNA rose significantly after a 4-day latency period. The proportion of total binding sites capable of translocation into the nucleus was elevated during the early phase (2-4 days) of androgen treatment. These results also suggest the heterogneity of the cytoplasmic binding site population. Control epididymides were composed of 61.5% receptor-containing epithelial cells, and this proportion was significantly reduced to 53.2% after 20 days of castration. Androgen administration elevated this percentage after an 8-day latency period. Proteolytic activity in cytosol was increased over control values after castration (for 20 days) and until the fourth day after the onset of androgen treatment. However, this activity does not seem to be an important factor in the decrease in binding sites caused by orchidectomy.

High affinity binding of labeled androgens in the androgen-target tissues of the male rhesus monkey

High affinity testosterone (T)-specific and dihydrotestosterone (DHT)-specific binding sites exist in a 1:2 ratio in the cytosol fractions of the caput epididymidis, prostate, seminal vesicles and the ductus deferens of the rhesus monkey. The number of androgen-binding sites in the caput epididymis is 3 times greater than that of the other 3 tissues.

Androgen receptor in breast tissues—I. A new approach for the estimation of androgen receptors in the normal and cancerous mammary gland of women

Optimum temperature, time and dose of androgenic steroids (5 α-dihydrotestosterone (5 α-DHT) and testosterone) required for quantitative estimation of nuclear and cytosol androgen receptor (AR), before and after affecting nuclear translocation of AR, was investigated in normal and cancerous breast tissues of women. Translocation of AR was temperature dependent, being maximum at 37°C. Optimal conditions required for estimation of AR in nuclei and cytosol are described. Three methods, single saturation dose analysis, sucrose gradient analysis and multidose saturation analysis, were used to estimate AR. The results of the three methods had a high coefficient of correlation (⪢0.95). Immunoabsorption of plasma proteins significantly reduced population of specific androgen binding sites in cytosol without effecting number of AR translocated to nucleus. 5 α-DHT estimated 1.51 times the number of androgen binding sites compared with testosterone. Majority ( $ ̃ 80%) of specific androgen binding sites in situ were distributed as free sites in cytosol and a significant percentage was occupied by endogenous steroid either in cytosol or nuclear fraction. An important finding was thai only a part and a highly variable percentage (19–100%) of specific androgen binding sites in the cytosol were translocated to the nucleus even after immunoabsorption of plasma proteins from the cytosol. Based on these findings, a new approach, involving the estimation of translocatable androgen receptors/100 μg nuclear DNA. by the easy and simple procedure of single dose saturation analysis, with either 5 α-DHT or testosterone, is suggested. This method will help in determining the total population of functionally active receptor sites in a cell. which can be more accurately used as a diagnostic test in the endocrine treatment of the patient.