Are fluorescein-conjugated androgens appropriate for a histochemical detection of prostatic androgen receptors?

Histochemical fluorescence techniques could be of great value in order to assay androgen receptors (AR), particularly in punch biopsies from human prostatic carcinoma (PCA). Therefore, prostatic tissue was examined for specific binding of fluorescein-labeled Sα- dihydrotestosterone derivatives (FDHT) to AR. Using a 17β-fluoresceinated DHT derivative (17-FDHT), variable fluorescence was found in human and rat prostates at high 17-FDHT concentrations. This fluorescence could be blocked by unlabeled DHT in 11 and 73% of human and rat prostatic tissue, respectively. Control studies of receptor and organ specificity were conducted and showed that preheated slices from rat prostates displayed no decrease of fluorescent staining. No difference in fluorescence intensity could be seen between prostates from castrated and uncastrated rats. Unstained tissue slices frequently showed a considerable intensity of autofluorescence. An appreciable amount of fluorescence in both rat liver and spleen was found. From these results and various general methodical problems inherent in fluorescent receptor assays, we conclude that the fluorescence techniques described are inappropriate for demonstration of AR.     

Mechanisms of action of androgens and antiandrogens: Effects of antiandrogens on translocation of cytoplasmic androgen receptor and nuclear abundance of dihydrotestosterone

The effects of antiandrogens on the translocation of androgen receptor and the accumulation of nuclear dihydrotestosterone in rat ventral prostate were investigated. Using an isotope-exchange assay and radioimmunoassay it was found that 1 hr after the injection of 70 nmol of dihydrotestosterone into 1-day castrated rats, 19,000 ± 2,350 (mean ± SE) receptor molecules and 99,300 ± 8,200 molecules of dihydrotestosterone were recovered per nucleus. When equivalent doses of antiandrogens were substituted for dihydrotestosterone only 0–1,500 receptor molecules were translocated. After injection of antiandrogens followed 1 hr later by injection of dihydrotestosterone, the inhibition of translocation given by the reduction in the mean numbers of molecules of receptor and dihydrotestosterone, respectively, was as follows: 17β-estradiol (0, 0); megestrol acetate (2000, 0); diethylstilbestrol (6000, 4000); cyproterone acetate (5500, 27000); flutamide (10000, 29000); R2956 (10000, 28000). With the exception of the effect of cyproterone acetate on receptor translocation, the reductions produced by the latter three antiandrogens were all statistically significant (P < 0.5). Since less than 20% of the dihydrotestosterone in the nucleus is bound to receptor in dihydrotestosterone-treated rats, and since the concentrations of receptor and dihydrotestosterone are unequally affected by some antiandrogens, we infer that the nuclear abundance of dihydrotestosterone may be regulated in part through nonreceptor-mediated processes.     

Molecular modelling of the androgen receptor axis: rational basis for androgen receptor intervention in androgen-independent prostate cancer

Androgen depletion in combination with antiandrogenic agents is initially highly effective for treating prostate cancer, and is the recommended treatment for more advanced or higher-grade tumours. However, many tumours eventually become insensitive to androgens, even though the androgen receptor (AR) continues to be expressed. Computational chemistry combined with structural analysis of nuclear receptors and determination of binding affinities of natural and designed coregulators (coactivators and corepressors) provides the theoretical framework for the rational design of novel therapeutic agents directed at the AR. Adding alternative groups to various sites throughout the receptor can alter the conformation of the molecule and its functional binding with coactivators or corepressors. Possible molecules can be identified thoroughly and systematically using intelligent high-throughput screening and FASTrack chemistry (three-dimensional crystallography). Applying these techniques should eventually result in therapeutic agents for androgen-independent prostate cancer that can block binding of AR coactivators while simultaneously increasing binding of AR corepressors.     

Synthesis and evaluation of immobilized androgens for affinity chromatography in the purification of nuclear androgen receptor

Seven biospecific adsorbents containing immobilized androgens were synthesized: dihydrotestosterone-17 beta-succinyl agarose, and both the unsubstituted and the 17 beta-acetyl derivatives of dihydrotestosterone-7 alpha-undecanoyl agarose, testosterone-7 alpha-undecanoyl agarose, and 19-nortestosterone-7 alpha-undecanoyl agarose. The retention capacities for nuclear androgen receptor were generally between 40-80% with little variation in reproducibility; the amount of binding was greatest with dihydrotestosterone-17 beta-succinyl agarose and dihydrotestosterone-17 beta-acetoxy-7 alpha-undecanoyl agarose. Rapid flow rates were obtained with all gels, and no tendency for decomposition was observed over a period of 1 year. Factors that affected retention included the concentration of immobilized androgen, length of the linker arm, occupation of receptor sites, interval of contact with the gel, and temperature of incubation. Chemical dissociation of androgens from androgen receptor complexes with 0.2 mM mersalyl increased the retention of receptor by dihydrotestosterone-17 beta-succinyl agarose. Two elutants showed promise for the dissociation of gel-bound receptor: 1) 0.2 mM mersalyl in the presence of 1.5 mg/ml of ovalbumin; 2) 10% (v/v) dimethylformamide:water containing 30 microM [1,2-3H] dihydrotestosterone and 0.5 M sodium thiocyanate.     

Partial androgen insensitivity syndrome with thermolability in the androgen receptor

We report case of partial androgen insensitivity syndrome in a 12-year-old boy referred to our clinic complaining of bilateral gynecomastia and left undescended testicle. Laparoscopy for undescended testicle and bilateral mastectomy were performed, and the left testicle was absent. When skin fibroblasts of the scrotum obtained during surgery were cultured to analyse the androgen receptors, a slight thermolability was observed. Genomic examination of the androgen receptor gene could not detect any mutations.     

One and the same androgen for all?towards designer androgens

The introduction of designer oestrogens as a treatment modality in hormone replacement in women has invited toconsider the concept of compounds with selective androgenic effects for male hormone replacement therapy. The fullspectrum of the actions of testosterone may not be necessary of even undesired for certain indications for testosteronetreatment. To define for what indications certain androgenic properties are desired and undesired more insight in basicandrogen (patho)physiology is required. There is convincing evidence that aromatization of androgenic compounds tooestrogens might be an advantage for maintenance of bone mass and it might also mitigate negative effects of androgenson biochemical parameters of cardiovascular risks; the potentially negative effects of oestrogens on prostate pathology inageing men needs further elucidation. While the role of dihydro-testosterone (DHT) for the male sexual differentiationand for pubertal sexual maturation is evident, its role in mature and ageing males s     

The synthetic androgen mibolerone induces transient suppression of the transformed phenotype in an androgen responsive human prostatic carcinoma cell line: Das synthetische Androgen Miboleron induziert eine transiente Suppression des transformierten Phänotyps in einer Androgen-abhängigen menschlichen Prostata-Karzinom-Zellinie

Summary.  The synthetic androgen mibolerone elicits a set of distinct changes in the behaviour of an androgen responsive human prostatic carcinoma cell line (LNCaP). Inhibition of cell proliferation, induction of morphological change and of a prostate specific mRNA, and inhibition of colony formation in soft agar are induced by very low concentrations of mibolerone. The natural androgen dihydrotestosterone is much less effective. The changes in growth characteristics and morphology are reverted by excess antiandrogen, i.e. cyproterone acetate or hydroxyflutamide. Cell lines lacking androgen receptors (PC-3, DU 145 and MRC-5) are completely unresponsive to mibolerone. Taken together, our results indicate androgen receptor mediated suppression of the transformed phenotype in LNCaP cells.
Zusammenfassung.  Das synthetische Androgen Miboleron löst eine Reihe bestimmter Veränderungen im Verhalten einer Androgen-responsiven menschlichen Prostata-Karzinom-Zellinie (LNCaP) aus. Hemmung der Proliferation, Induktion morphologischer Veränderungen und einer Prostata-spezifischen mRNA, sowie die Hemmung der Kolonie-Bildung im Weichagar werden von niedrigen Konzentrationen von Miboleron hervorgerufen. Das natürliche Androgen Dihydrotestosteron ist viel weniger effektiv. Die Veränderungen im Wachstumsverhalten und der Morphologie werden durch zusätzliches Antiandrogen, z.B. Cyproteronazetat oder Hydroxyflutamid, rückgängig gemacht. Zellinien ohne Androgenrezeptor (PC-3, DU 145 und MRC-5) sprechen auf Miboleron überhaupt nicht an. Zusammenfassend zeigen unsere Versuche eine Androgenrezeptor-mediierte Suppression des transformierten Phänotyps bei LNCaP-Zellen.     

Modulation of androgen receptor protein by culture conditions of human skin fibroblasts

Cultures of skin fibroblasts show variation of androgen binding with culture conditions; binding variations are usually avoided by using confluent cultures. In this work, we analysed the effect of cell density and mitogenic agents on the level of androgen receptor (AR) of cultured human skin fibroblasts. Results demonstrated that in cultures of human skin fibroblasts, cellular binding of dihydrotestosterone was higher in cells grown at low than at high cell density. The reduction in binding resulted from a decrease in the number of high affinity receptors and not from a change in receptor affinity. Immunocytochemistry for AR showed greater staining intensity in cells grown at low than at high cell density. Additionally, immunoblot analysis demonstrated more AR protein in low cell density cultures. On the other hand, it was observed that cells grown at low cell density showed diminished androgen binding capacity after 24 h of treatment with insulin-like growth factor (IGF-l), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or granulocyte-colony stimulating factor (G-CSF); this effect of growth factors was not observed in cells grown at high cell density. In conclusion, we found that cell density of cultures and mitogenic agents can regulate AR binding activity in human fibroblasts. While we do not yet know how changes in cell density affect the amount of AR, we conclude that the mechanism could be mediated by activation of the tyrosine kinase pathway, as the effect was reproduced by mitogens.     

前列腺癌组织雄激素受体表达的意义

目的 探讨Pca发生、发展过程中AR的变化及其临床意义。方法 采用免疫组化和图象分析技术检测PCa(N=40)和NP(N=40)中AR相对含量。结果 PCa中AR相对含量显著高于NP组(0.24±0.03 VS 0.13±0.01 P<0.05);随着PCa病理分级增高,AR相对含量有明显降低趋势;AR阳性表达者的生存期明显较阴性者为长(9.3 VS 21.4月,P<0.05)。结论 在前列腺癌发生发展过程中,AR及其基因存在着复杂的变化,检测AR相对含量对判断患者预后有一定意义。