[Role of androgen receptors in hormone-refractory prostate cancer: molecular basics and experimental therapy approaches]. / Die Rolle des Androgenrezeptors im hormonrefraktären Prostatakarzinom : Molekulare Grundlagen und experimentelle Therapieansätze.

The development of hormone-refractory prostate cancer cells is one of the major causes for the progression and high mortality rates in advanced prostate cancer (PCA). While the loss of the androgen receptor (AR) is the predominant mechanism for development of a hormone-insensitive disease in vitro, the first in vivo studies showed that the AR is still expressed or is even overexpressed in hormone-refractory PCA. In view of the increasing cases of PCA in the industrialized Western countries, a series of cell and molecular biological studies has led to the identification of various new factors and mechanisms that play a role during the development of hormone-refractory tumors. These findings should lead to the development of new therapeutic strategies.

Effect of testosterone on plaque development and androgen receptor expression in the arterial vessel wall.

BACKGROUND: -Recent studies have suggested that testosterone has a protective effect in the arterial vascular system. However, little is known about the molecular aspects of the mechanism(s) involved in these processes. The aim of the present study was to investigate the effect of testosterone on neointimal plaque development and on the expression of the vascular androgen receptor. Methods and Results-Neointimal plaque formation was induced by endothelial denudation in the aortas of male New Zealand White rabbits. Aortic ring segments were cultured for 21 days after endothelial denudation. Testosterone was applied to the culture medium in different doses. Compared with the non-hormone-treated control group, a significant inhibition of neointimal plaque development (expressed as the intima/media ratio) was found at testosterone concentrations of 10 ng/mL (P:=0.037) and 100 ng/mL (P:=0.012; intima/media ratios: median of controls, 0.25; median of 10 ng/mL testosterone group, 0.15; median of 100 ng/mL testosterone group, 0.16). Associated with this inhibitory effect on plaque size was a 50% increase of the amount of androgen receptor mRNA in the arterial segments treated with testosterone. CONCLUSION: -The beneficial effects of testosterone on postinjury plaque development underlines, at least in males, the important role of androgens in the vascular system. As our data suggest, the vascular androgen receptor is probably involved in these processes. Further studies are required to characterize the androgen receptor-dependent pathways in the vascular system.

Response to androgen treatment in a patient with partial androgen insensitivity and a mutation in the deoxyribonucleic acid-binding domain of the androgen receptor.

Supplemental androgen therapy has enhanced virilization in only a few patients with partial androgen insensitivity (PAIS). We herein report on virilization in a patient with PAIS and a point mutation in the DNA-binding domain of the androgen receptor. At the age of 19 yr, the patient sought medical attention because of undervirilization. Endocrine findings were typical for androgen insensitivity, but 5alpha-reductase activity and androgen binding characteristics in fibroblasts cultured from genital skin were normal. In an attempt to improve virilization, high dose testosterone enanthate treatment (250 mg by i.m. injection once a week) was begun. After 3.5 yr of this treatment, marked promotion of virilization was achieved, i.e. lowering of voice, male pattern secondary hair distribution, marked growth of beard and coarse body hair, increase in phallic size, increase in bone mineral density, and decrease in mammary gland size. In addition, serum lipid levels were not affected. To our knowledge this is the first documentation of successful treatment in a patient with PAIS and a point mutation in the DNA-binding domain of the androgen receptor.

Complete androgen insensitivity caused by a new frameshift deletion of two base pairs in exon 1 of the human androgen receptor gene.

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5alpha-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.

Decreased expression of IGF-II and its binding protein, IGF-binding protein-2, in genital skin fibroblasts of patients with complete androgen insensitivity syndrome compared with normally virilized males.

The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 +/- 9.7 vs. 106.9 +/- 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 x 10(-8) M) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.

Structural and developmental analysis of a gene cloned from the early ecdysterone-inducible puff site, I-18C, in Chironomus tentans.

A gene (I-18C) cloned from the early ecdysterone (EDS)-inducible puff site, I-18C, in Chironomus tentans salivary glands, codes by differential processing for at least five transcripts which can be grouped into spliced and unspliced transcripts. The spliced group comprises several RNAs of approx. 4.6 kb, and the unspliced group comprises the 1.8-kb and 6.5-kb RNAs. All these transcripts have a similar or the same transcription start point. The steady-state level of the spliced 4.6-kb RNAs reveals some parallels with EDS concentrations in animals during the various developmental stages analyzed, while the levels of the unspliced 1.8-kb and 6.5-kb RNAs do not correlate with the EDS concentrations.

Inhibitors of chitinases.

In this review we describe inhibition of chitinases from bacteria, fungi, plants and animals by allosamidin and its derivatives, cyclic peptides, styloguanidin and divalent cations. Most information is available for allosamidin, whose important structural features necessary for inhibition are known. At least one N-acetylallosamine sugar must be present, and the spatial arrangement of the allosamizoline moiety are important for inhibition. Less complex compounds are therefore possible as lead structures for the development of agents interfering with chitinase. There is a pronounced species specificity in chitinase inhibition by allosamidin: half-maximal values are often in the range of 0.1-1 microM (e.g. in all arthropods), being lower in nematodes (0.048, 0.0002 microM, respectively) and amoeba (0.002-0.01 microM) and quite divergent in fungi (0.01-70 microM). These differences cannot be caused by the catalytic centers of family 18 and 19 chitinases.

Uptake and retention of moulting hormones by the integument of crayfish in vitro. III. The possible involvement of Na+/K+-ATPase.

The specific uptake of the ecdysteroid [3H]ponasterone A into crayfish hypodermis in vitro is inhibited by increasing concentrations of potassium in the medium. The Na+/K+-ATPase blocker ouabain also decreases uptake of the ecdysteroid. Maximal inhibition of uptake is reached at 5 X 10(-5) M ouabain.

Uptake and retention of moulting hormones by the integument of crayfishes in vitro. II. Influence of metabolic inhibitors and sulphydryl group inhibitors.

Antimycin A and 2,4-dinitrophenol reduced both the initial uptake and the total uptake of ecdysone into crayfish hypodermis in vitro. Antimycin A did not reduce the retention of moulting hormone, in contrast to 2,4-dinitrophenol. The inhibitory effect of antimycin A could be completely overcome. The membrane-mediated ecdysteroid uptake was sensitive to the sulphydryl group inhibitors N-ethylmaleinimide (NEM) and p-chloromercuriphenylsulphonate (PCMPS). Inhibition of steroid uptake by PCMPS was completely reversed by dithiothreitol.

Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus.

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.

Uptake and retention of moulting hormones by the integument of crayfishes in vitro: I. Influence of temperature, hormone concentration and hormone structure.

Uptake kinetics of [3H]ecdysone into crayfish hypodermis in vitro reveals three different components: a very rapid (within 1 min), highly temperature-dependent (Q10 = 3) and high uptake rate, a second much lower uptake which is only slightly temperature-dependent (Q10 = 1-1.3), and a plateau which is reached after about 20 min at 20 degrees C. Preincubation of tissue with unlabelled hormone reduces the initial uptake rate to a value characteristic for the second, less steep uptake phase. The activation energy for the initial uptake is 45 kJ/mole. At equilibrium the hormone is 4-fold enriched as compared to the medium, the delta G for this enrichment being 3 kJ/mole. The efflux is much lower than the influx and is linear. There is no linear relation between external hormone concentration and initial uptake rate. The initial uptake can be significantly reduced by an excess of diverse ecdysteroids with moulting hormone activity but not by hydrocortisone.

An androgen receptor mutation in the direct vicinity of the proposed C-terminal alpha-helix of the ligand binding domain containing the AF-2 transcriptional activating function core is associated with complete androgen insensitivity.

Subjects with androgen insensitivity syndromes (AIS) are characterized by a 46, XY karyotype, presence of testes, normal or elevated androgen levels in blood, and impairment of the usual response to androgens associated with various aberrations of male differentiation and virilization ranging from slightly undervirilized men to phenotypic females. Here we describe a novel proline to serine mutation in codon 892 (exon 8) of the androgen receptor in a patient with complete androgen insensitivity. The mutation is located in the direct vicinity of the proposed C-terminal alpha-helix of the ligand binding domain containing the AF-2 transcriptional activating function core. Investigation of androgen binding in cultured testicular fibroblasts of the patient revealed a reduced AR binding capacity (11 fmol/mg protein) and a highly elevated Kd value (3.1 nM) in comparison to control genital skin fibroblasts. Cotransfection studies with an androgen-responsive reporter gene revealed a diminished transactivation property of the mutant androgen receptor.

Ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta) are phosphoproteins and are regulated differently by molting hormone.

Three different isotypes of the ecdysteroid receptor (cEcR) (66, 68 and 70 kDa) and several molecular variants of the dimerization partner "ultraspiracle" (cUSP) (58-77 kDa) can be separated electrophoretically in homogenates of the epithelial cell line from Chironomus tentans. After phosphatase treatment the bands with the lowest electrophoretic mobility disappear in both cases. Phosphorylation occurs exclusively at ser/thr in EcR and USP. Binding studies with 3H-ponasterone A using 0.4 M NaCl extracts revealed two classes of high-affinity binding (KD1 = 0.47 and KD2 = 7.2 nM) competable either with 20-OH-ecdysone or muristerone A. At least KD2 and Bmax2 are unchanged after dephosphorylation. In hormonally naive cells a considerable part of EcR and USP is already present in nuclei. The phosphorylation pattern of both transcription factors is the same in cytosol and nuclear fractions. Incubation with 20-OH-ecdysone (1 microM, up to 4 days) does not alter the extent and mode of phosphorylation of EcR, although EcR concentration increases. In contrast USP concentration remains constant but phosphorylation is enhanced.

Uptake of corticosterone into isolated rat liver cells: possible involvement of Na+/K(+)-ATPase.

Isolated rat hepatocytes possess a saturable glucocorticoid uptake system with high affinity (Kd value = 2.8 +/- 0.7 x 10(-8) M; 318,000 +/- 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30-40% if the cells are incubated simultaneously with [3H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na+/K(+)-ATPase-inhibitors, ouabain and quercetine. These Na+/K(+)-ATPase-blockers exert half-maximal inhibition at 3 x 10(-7) and 3 x 10(-6) M, respectively. A slight increase in K+ concentration and a corresponding decrease in Na+ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5 alpha- and 5 beta-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.

Electron microscopic demonstration of glucocorticoid recognition sites on isolated rat hepatocytes.

Ultrastructural evidence is presented for the presence of membrane-bound glucocorticoid recognition and binding sites. Corticosterone was derivatized at 3 different positions and coupled covalently to bovine serum albumin (BSA). All three derivatives competed for binding of [3H]corticosterone by isolated rat hepatocytes. The most effective competitor, corticosterone-succinate-BSA (CSB), was adsorbed onto colloidal gold particles (CSB-gold, 17 +/- 3 nm dia). When isolated rat hepatocytes or mouse pituitary tumor cells (AtT 20) are incubated with CSB-gold, specific binding in the microvilli-rich region of these cells is seen. This binding of CSB-gold is reduced by about 50% in the presence of unlabelled CSB or corticosterone.

Interactions between steroid hormones and the nervous system.

After a short description of the endocrine and nervous system and their similarities and differences, interactions between both parts are mainly dealt with in this brief review. This is especially exemplified by the modulation of receptors for neurotransmitters, of ion channels and other membrane components by neurosteroids and steroid hormones and the physiological significance of this regulation. Both classes of hormones can act via nongenomic and genomic actions. As biological responses to neurosteroids and steroid hormones in the nervous system exocytosis of peptide hormones and neurosteroids, regulation of neurotransmitter release, sexual brain development and male behavior and cell death are described. Neurotransmitters and their receptors are also modulated in their expression and biological activity by steroid hormones and neurosteroids in non-neuronal tissues, as demonstrated for GABA-receptors in the uterus and the embryonic cholinergic system. Because of the close links between endocrine and nervous system agents toxic for one component may also interfere with the other one.

Derivatives of the insect moulting hormone for affinity chromatography, and their biological activities.

Several derivatives of the arthropod moulting hormone have been synthesized which were coupled to AH Sepharose 4B yielding about 2 mumole ligand per g wet gel. As an indication of the suitability of the ligands for biological work the puff inducing capacity of their methyl esters was tested. The methyl ester of inokosterone-C-26-carboxylic acid possesses the highest biological activity; lower activities were obtained with the esters of ecdysterone-C-6-CM-oxime and ecdysterone hemisuccinates. Therefore, inokosterone-C-26-carbocylic acid should be a useful ligand for affinity chromatography of ecdysone recptors from insect tissues.

Ecdysteroid resistant subclones of the epithelial cell line from Chironomus tentans (Insecta, Diptera). I. Selection and characterization of resistant clones.

Chironomus tentans cells were cultured in the presence of gradually increasing concentrations of 20-OH-ecdysone or a nonsteroidal molting hormone agonist, the benzoylhydrazine RH 5992, for a period of about 2 yr. From these cultures, subclones were selected, which are resistant to up to 25 microM 20-OH-ecdysone according to morphological (changes in cell shape and cell arrangement) and physiological criteria (acetylcholinesterase induction, secretion of chitinolytic enzymes, thymidine incorporation). Some subclones, selected in the presence of 20-OH-ecdysone, are resistant only to molting hormone, but still respond to RH 5992 morphologically and biochemically, whereas subclones selected in the presence of the benzoylhydrazine showed no reaction neither to 20-OH-ecdysone nor to the hormone agonist. Hormone resistance is stable; 3 mo. after hormone withdrawal, resistant clones still do not respond to renewed exposure to 20-OH-ecdysone or RH 5992, respectively. Because in all resistant subclones tested so far all hormonally regulated responses known from sensitive cells were no longer detectable, it is assumed that the hormone signaling pathway itself is interrupted. Possible mechanisms of hormone resistance were discussed.

Immunohistochemical localization of ecdysteroid receptor and ultraspiracle in the epithelial cell line from Chironomus tentans (Insecta, Diptera).

Ecdysteroid receptor (EcR) and its heterodimerization partner, ultraspiracle (USP), were demonstrated in the epithelial cell line from Chironomus tentans by immunohistochemistry. In untreated cells both proteins are present in nuclei as well as in granular compartments of the cytosol. At 1 day after addition of 1-microM 20-OH-ecdysone (20E) total immunofluorescence had increased in the nuclei, whereas the cytoplasmic staining had disappeared. At the 2nd and 3rd days all cells within a vesicle appear identical according to morphological criteria, but the EcR and USP immunoreactivity becomes restricted into patches of neighbouring cells. The hormonally induced changes in the pattern of localization of functional ecdysteroid receptor, the heterodimer of EcR and USP, are discussed in relation to similar effects of 20E on acetylcholinesterase and muscarinic acetylcholine receptor distribution in this cell line.

Action of brassinosteroids on the epithelial cell line from Chironomus tentans.

The two brassinosteroids, 22 S,23 S-homobrassinolide and 22 S,23 S-homocastasterone are weak competitors of the binding of [3H]ponasterone A to the intracellular ecdysteroid receptor from the epithelial cell line from Chironomus tentans. The relative affinities to the ecdysteroid receptor are 0.001 for both brassinosteroids as compared to 20-OH-ecdysone and 0.1 in comparison to ecdysone. Both substances exert morphological effects similar to those observed with 20-OH-ecdysone. Like moulting hormones both brassinosteroids inhibit chitin synthesis. However, these effects were observed only at rather high concentrations (10(-5) to 10(-4) M) which were cytotoxic for 22 S,23 S-homobrassinolide.