Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

doi:10.1016/0022-4731(88)90378-0

Journal of Steroid Biochemistry

Volume 29, Issue 1, January 1988, Pages 69-76

https://doi.org/10.1016/0022-4731(88)90378-0 Get rights and content

Abstract

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 μM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The K_d for the high affinity binding site was 2 × 10⁻⁹ M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5α-androstane-3α,17β-diol, testosterone, estradiol and estrone. Progesterone, 5α-androstane-3β,17β-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 ± 7.6fmol of bound R1881 per 10⁶ cells (n = 34) or 2075 ± 1434fmol per mg of DNA; these values correspond to an average of 6624 ± 4577 sites per cell. Thus, using this whole cell assay system, specific androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.

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Citation Excerpt :
Because R1881 is a synthetic androgen, we next sought to determine whether the physiological androgens testosterone (T) and dihydrotestosterone (DHT) could similarly induce GnRHR-luc in vitro. DHT treatment resulted in significant induction of GnRHR-luc at 10−8 M and 10−7 M compared to untreated control (Fig. 2B, p < 0.01), but not at concentrations of 10−9 M, 10−6 M, or 10−5 M. T treatment induced GnRHR-luc activity only at higher concentrations (Figs. 2C and 10−7 M, 10−6 M, and 10−5 M, p < 0.05), likely due to its weaker affinity for AR relative to DHT and R1881 (Saartok et al., 1984; Turcotte et al., 1988). We also treated cells containing no transfected AR (empty vector control) with the highest concentration used of each hormone (10−5 M).
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Increasing information indicates that testosterone actions on cells are mediated not only through the classical intracellular androgen receptor (iAR), but also through membrane androgen receptors (mAR) on cell surfaces. Here, we investigate the expression pattern of mAR and iAR in thymic T cells, which is compared with that of splenic T cells. Thymic T cells are testosterone-sensitive in vivo, i.e. treatment of female C57BL/10 mice with testosterone for 3 weeks decreased the total number of thymic T cells by approximately 90%. The percentage of CD4⁻ CD8⁻ T cells increased, whereas that of the subsequent CD4⁺ CD8⁺ T cells was diminished. Flow cytometry and confocal laser scanning microscopy (CLSM) with different anti-iAR antibodies localized iAR predominantly in the cytoplasm, but not on the surface of thymic T cells. The iAR are functionally active since the iAR are induced by testosterone to translocate from cytoplasm to nucleus, and they bind the testosterone analogue $^{3} H$ -R1881 with high affinity (K_d approximately 2.2 nM) and saturable capacity (approximately 10,000 binding sites per cell) as determined by Scatchard analysis. By contrast, the impeded ligand testosterone–BSA–FITC (T–BSA–FITC) did not bind to the surface of thymic T cells. In accordance, testosterone was unable to induce any rapid rise in the intracellular free Ca²⁺ concentration of Fura-2 loaded thymocytes. This indicates that thymic T cells do not express any significant amounts of mAR. Conversely, splenic T cells express functionally active mAR, whereas their expressed iAR are not functional in the genomic pathway. Our results support the view of a delicately balanced developmental regulation of iAR and mAR in T cells.
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Cancer of the prostate is one of the leading causes of cancer related deaths in men. An important role in the development of prostate cancer is played by androgens and androgen ablation is therefore currently used in cancer treatment. In the past, estrogens were widely used in treatment of prostate cancer, but there are indications that estrogens could also be involved in carcinogenesis. Lately, much research has been done on the modulation of the binding of steroid hormones to their receptors by polyunsaturated fatty acids (PUFAs), which could interfere with the steroid hormone's message. Therefore, the aim of this study was to determine in whole DU-145 human prostate cells the effect of EFAs and their metabolites on the binding and affinity of the estrogen receptor (ER) and androgen receptor (AR) to estradiol (E₂) and testosterone (T), respectively. Fatty acids were dissolved in ethanol and added to the cell culture in a final ethanol concentration of 0.2% on the fourth day of incubation. The results showed that the PUFAs under investigation inhibited the AR's capacity, in contrast to the ER's capacity which was stimulated. However, the dissociation constants (K_d) of the AR and ER complexes in the presence of the PUFAs, were as follows. Except for eicosapentaenoic acid (EPA) which decreased the AR dissociation constant and EPA and α -linolenic acid (ALA) which increased the ER dissociation constant, the remaining FAs had no significant effect on the K_d values of both the AR and ER complexes. According to these priliminary results it is postulated that men should benefit with a diet rich in certain essential polyunsaturated fatty acids although its function remains to be clarified.
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The ALVA-41 cell line was derived from a bony metastasis from a human prostatic carcinoma. The line has a number of distinct, advantageous properties that should make it useful as a tool for the study of prostate cancer. It grows rapidly and is easy to work with. It has receptors for androgens and glucocorticoids but not for estrogens. Its growth is enhanced by physiological concentrations of dihydrotosterone. It does not secrete prostate specific antigen, but does not secrete prostatic acid phosphatase. Further, the secretion of prostatic acid phosphatase is enhanced by dihydrotesterone.
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Binding of androgens to adipocytes has previously been evaluated using cytosol fractions without taking into account nuclear binding, although the latter is suggested to be close to the physiological site of action. In the present study, performed in differentiated fat pad adipose precursor cells, we describe a simple, reliable and reproducible androgen binding assay in a system with intact cells. Triatiated and unlabeled methyltrienolone (R1881) were used to define specific and unspecific androgen binding. Triamcinolone acetonide was added to prevent the binding of R1881 to other types of receptors. Differentiated adipose precursor cells contain a homogeneous class of high affinity androgen binding sites, and binding is saturable and reversible. Binding apparently occurs at one site, with a K_d in the range of physiological androgen concentration (about 4 nM). Competition studies indicate that the receptor is specific for R1881, testosterone and dihydrotestosterone, which have approximately the same affinity, while progesterone, estradiol and dexametasone show much lower affinity. Androgen binding was markedly enhanced after cellular exposure to R1881 and testosterone but not dihydrotestosterone, and this increase was dependent on protein synthesis, suggesting the formation of new receptors by these androgens.
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Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [³H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotestosterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [³H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [³H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.

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^☆: This paper was presented, in part, to the 28th Annual Meeting of the Canadian Federation of Biological Societies, Toronto, Ontario, 1985, Abstract no. 289.

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Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells☆

Abstract

J. Urol.

Biochem. biophys. Res. Commun

J. Urol.

Molec. Cell. Endocr

Molec. Cell. Endocr.

Molec. Cell. Endocr.

Analyt. Biochem.

J. steroid Biochem.

J. steroid Biochem.

J. steroid Biochem.

J. steroid Biochem.

J. biol. Chem.

J. steroid Biochem.

J. steroid Biochem.

J. steroid Biochem.

Steroids

Benign hypertrophy and carcinoma of the prostate: occurrence and experimental production in animals

Surgery

The physiology of the prostate gland

Physiol. Rev.

Comparison of spontaneous and experimentally induced canine prostatic hyperplasia

J. clin Invest.

Morphologic and endocrine aspects of prostatic function

The Prostate

Characterization of the specific androgen receptors in the human prostate gland

J. Endocr.

Androgen receptors and androgen-dependent initiation of protein synthesis in the prostate

Vitam. Horm.

Dihydrotestosterone in prostatic hypertrophy I. The formation and content of dihydrotestosterone in the hypertrophic prostate of man

J. clin. Invest.

DHT in prostatic hypertrophy II. The formation and content of DHT in the hypertrophic canine prostate and the effect of DHT on prostate growth in the dog

J. clin Invest.

Tissue content of dihydrotestosterone in human prostatic hyperplasia is not Supranormal

J. clin Invest.

Dihydrotestosterone concentration of beagle prostatic tissue: Effect of age and hyperplasia

Endocrinology

Androgen receptor content of the normal and hyperplastic canine prostate

J. clin Invest.