Intracellular inhibition of chromatin binding and transformation of androgen receptor by 3'-deoxyadenosine

Incubation of minced rat ventral prostate with 3'-deoxyadenosine (3'-dA) prior to labeling with the androgen, tritiated 7 alpha, 17 alpha-dimethyl-19-nortestosterone, reduced the level of androgen receptor bound to chromatin and increased the level of cytosolic androgen receptor and the fraction of cytosolic androgen receptor that did not bind to DNA. This effect was specific for 3'-dA and not mimicked by adenosine, 2'-deoxy-adenosine, cytidine, guanosine, or uridine. Adenosine was a competitive inhibitor of the 3'-dA effect. Labeled cytosolic androgen receptor from 3'-dA-treated prostate had properties that were similar to those exhibited by untransformed androgen receptor from prostate cytosol prepared in the presence of Na2MoO4, an inhibitor of receptor transformation in cell-free systems. Both androgen receptors had sedimentation coefficients of 8-9 S in low-salt gradients, did not bind to DNA tightly, and had a high affinity for DEAE-cellulose. The 3'-dA effect on these properties was not observed if androgen receptor from 3'-dA-treated prostate was isolated on high-salt gradients. These findings show that androgen receptor transformation does take place in intact prostate cells and suggest that 3'-dA inhibits chromatin binding of androgen receptor by interfering with androgen receptor transformation. The transformation process appears to involve removal of components from androgen receptor. Since 3'-dA is a potent inhibitor of the synthesis, polyadenylation, and nucleocytoplasmic transport of RNA, the 3'-dA effect may indicate a role for RNA in the mechanism of receptor transformation in intact target cells.     

Effect of castration and administration of testosterone on cytosol and nuclear androgen receptor in mouse submandibular gland

The effect of castration or administration of testosterone propionate on the subcellular distribution of androgen receptor in mouse submandibular gland was investigated. Within 10 h after castration of male mice, most of the androgen receptor in nuclei was significantly reduced, the androgen receptor in cytosol increased and the increased cytosol receptor retained for at least 40 h. A single injection of testosterone propionate to female mice resulted in the translocation of cytosol androgen receptor to the nuclei by 30 min. The nuclear receptor level remained for at least 24 h and the cytosol receptor was replenished by 24-72 h. These results reveal that the endocrine manipulations such as castration and testosterone injection cause the change in the subcellular distribution of androgen receptor from mouse submandibular gland in both sexes.     

Characteristics of cytosol androgen receptor in rat thymus

Male and female rat thymic cytosol contained specific androgen receptor. The apparent dissociation constants (Kd) were 2.4 nM in males and 2.5 nM in females, and the number of binding sites (NBS) were 23.7 fmol/mg protein in males and 34.2 fmol/mg protein in females. Transformation of receptor to the DNA binding state was achieved by heat or KCl treatment of [3H]R1881-receptor complex, and the characteristics of transformed and nontransformed receptors were investigated. The nontransformed androgen-receptor complex eluted at 0.20-0.25 M KCl from DEAE-Sephacel and sedimented at 9.1 S and its molecular weight was 255,000 on agarose gel chromatography, while the transformed receptor complex eluted at 0.03-0.15 M KCl with a broad peak and sedimented at 4.5 S and its molecular weight was 80,000-85,000. The minicolumn binding assay revealed that approximately 57% of the total receptor complexes bound to DNA-cellulose following heat treatment (20 degrees C, 1 h). Castration exerted no effect on the physicochemical properties of cytosol androgen receptor, but it increased the number of binding site to the female level.     

Onset of androgen action in MCF-7 human breast cancer cells is not accompanied by receptor depletion

Quantitative and qualitative changes in estrogen receptor follow addition of estradiol to estrogen responsive MCF-7 human breast cancer cells. We asked whether similar changes would accompany treatment of these cells with physiologically relevant concentrations of androgens. Androgen receptor sites were quantified by competitive protein binding assays on whole cells or extracts at various times following hormone addition. Both direct and exchange assays were employed. The androgen receptor in all of these experiments remained in a form which is completely exchangeable and approx 85% salt extractable. Quantity of receptor was unchanged (30,000 sites/cell, Kd 0.1 nM). Responsiveness to hormone treatment was demonstrated by antagonizing the estrogen dependent augmentation of cytoplasmic progesterone receptor in the MCF-7 cells with androgens. Thus, the androgen receptor was shown to be biologically active, but no time dependent quantitative or qualitative changes were observed during the first 6 h following androgen treatment.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Androgens on the estrogen receptor II — correlation between nuclear translocation and uterine protein synthesis

In the immature rat uterus, occupation of the androgen and estrogen receptor sites after injection of 5 α dihydrotestosterone (DHT = 17β-hydroxy-5α-androstan-3-one) were compared to the biological responses induced by the androgen on protein synthesis. Injection of 100 μg DHT induced a maximal occupation of androgen receptor sites (RA) but was totally ineffective in translocating the estrogen receptor sites and in increasing general protein synthesis. Conversely, high doses of androgen (? 3 mg) translocated the estrogen receptor (RE) and stimulated general protein synthesis. In addition, these high doses induced a specific uterine protein undistinguishable from that induced by estradiol treatment (IP). These results strongly suggest that the uterotrophic response of the uterus to androgen is correlated with the nuclear translocation of the estrogen receptor and not with that of the androgen receptor which is present in much smaller amounts.     

Time-courses of the appearance/disappearance of nuclear androgen + receptor complexes in the brain and adenohypophysis following testosterone administration/withdrawal to castrated male rats: relationships with gonadotropin secretion

We characterized the temporal dynamics of brain and pituitary cell nuclear androgen receptor binding and serum androgen and gonadotropin levels associated with the implantation and removal of testosterone (T)-filled Silastic capsules into performed s.c. flank pouches of castrated, awake male rats. These capsules produced serum T levels in the physiologic range. The number of cell nuclear androgen + receptor complexes, as measured in an exchange assay using [3H]R1881, increased 15-fold at 0.5 h after capsule insertion in the HPAS (combined hypothalamus, preoptic area, amygdala and septum) and anterior pituitary gland, but then showed a second progressive rise within the next 8 h. This pattern suggests that T exerts an initial action in the tissues to alter the affinity and/or number of available androgen receptors. There was a lag time of 2-4 h to the first indication of negative feedback suppression of LH secretion. Serum LH levels declined only slightly at 4 h after capsule insertion but continued to fall thereafter, reaching undetectable values by 24 h. In contrast, serum FSH levels declined only slightly after 24 h of T exposure. After removal of the T capsules, serum T levels declined to castrate values within 2 h at which time the level of androgen + receptor complexes had fallen to 60% in the brain and pituitary. Serum LH and FSH concentrations were unchanged at 2 h after capsule removal, but rose significantly within the next 2 h. The data indicate that the occupation of androgen receptors rapidly changes in response to variations in circulating T in a fashion that implicates their involvement in the expression of this steroid's negative feedback actions on gonadotropin secretion.     

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.     

Androgen metabolism in hirsute patients treated with cyproterone acetate

Cyproterone acetate (CPA) in association with percutaneously administered estradiol has been used for the treatment of 150 hirsute patients for periods ranging from 6 months to 3 years. A spectacular clinical improvement ensued. Plasma testosterone (T) and androstenedione (A) fell from 69.0 +/- 24 to 33.0 +/- 8 and 210 +/- 103 to 119 +/- 25 ng/dl (mean +/- SD) respectively after 3 months of treatment and remained low thereafter. In contrast, T glucuronide (TG) and 3 alpha-androstanediol (Adiol) remained high during the whole course of treatment: 37 +/- 9 and 115 +/- 43 micrograms/24 h respectively. In vitro T 5 alpha-reductase activity (5 alpha-R) in pubic skin decreased from 147 +/- 34 to 79 +/- 17 fmol/mg skin after 1 year of treatment. To elucidate the discrepancy between plasma and urinary androgens levels, T production rate (PR) and metabolic clearance rate (MCR) were measured with the constant infusion technique in 7 patients before and after 6 months of treatment. PR decreased from 988 +/- 205 to 380 +/- 140 micrograms/24 h (mean +/- SD). In contrast MCRT increased from 1275 +/- 200 to 1632 +/- 360 1/24 h; this increase in MCRT explains the striking plasma T concentration fall and the high TG and Adiol excretion relative to the decrease in PR. Antipyrine clearance rate (n = 8) increased from 36.3 +/- 5.2 to 51.5 +/- 7.4 ml/min whereas 6 beta hydroxycortisol remained unchanged. In conclusion, CPA acts through several mechanisms: (1) it lowers the androgen input to the target cells by (a) depressing T production through its antigonadotropic effect and (b) accelerating T metabolic inactivation due to a partial enzymatic inducer effect on the liver; (2) at the target cell level it competes with any remaining T for the receptor binding sites; (3) the decrease in the androgen-dependent skin 5 alpha-R is a consequence of both actions of androgen suppression and androgen receptor blockade; it reinforces the antiandrogenic effect of CPA.