Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression.

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.

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.

The exon-intron organization of the prohormone convertase PC2 gene from the insect Lucilia cuprina.

Prohormone or proprotein convertases are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues. Among the different members of this prohormone convertase family, the prohormone convertase 2 (PC2) is almost exclusively expressed in endocrine and neuroendocrine tissues and plays an important role in the endoproteolytic processing of prohormones. Here we describe the exon-intron organization of the PC2 gene from the insect Lucilia cuprina by characterization of PCR-amplified genomic DNA fragments. The insect PC2 gene contains 12 exons with an estimated size of over 14.5 kb. The exon sizes range from 38 bp to > 448 bp. All identified intron-exon boundaries are consistent with the GT-AG-rule. A comparison of the genomic structures of the thus far known prohormone convertase genes with that of the insect PC2 gene revealed a conservation of the positions of most introns interrupting the exons coding for the amino-terminal and catalytic domains. This conservation is consistent with the suggestion of a common evolutionary origin for the prohormone convertase gene family.

Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone.

Crustacean hyperglycemic hormone (CHH) from Carcinus maenas, a 72 amino acid neuropeptide, originates in neurosecretory perikarya in the eyestalk ganglia. Poly (A)RNA was isolated from these perikarya and a cDNA library was prepared. Screening of 20,000 clones with a 26-mer oligonucleotide, corresponding to a partial sequence of CHH, yielded one positive clone with an insert of approximately 2,000 bp, which contained the complete coding sequence for a pre-pro CHH. This precursor consists of a putative 26 amino acid signal sequence, a 38 amino acid peptide of unknown function (Peptide C), and the CHH sequence at the carboxyl end. The CHH-sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by the tetrapeptide Gly-Arg-Lys-Lys which is followed by the stop codon.

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.

Molecular cloning of crustacean putative molt-inhibiting hormone (MIH) precursor.

A cDNA encoding the complete precursor of the putative molt-inhibiting hormone (MIH) of the shore crab, Carcinus maenas, was isolated and sequenced. The precursor consists of a putative 35 amino acid signal peptide and the 78 amino acid mature MIH. The deduced MIH amino acid sequence is in complete agreement with the sequence previously determined by Edman degradation. In situ hybridization revealed MIH-expression in a subpopulation of large neurosecretory perikarya of the medulla terminalis X-organ in the eyestalk.

Structure and localization of mRNA encoding a pigment dispersing hormone (PDH) in the eyestalk of the crayfish Orconectes limosus.

The pigment-dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light-adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, we cloned and sequenced cDNA encoding PDH in the crayfish Orconectes limosus. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor-related peptide (PPRP) and the highly conserved PDH peptide at the carboxy-terminal end. In situ hybridization in combination with immunocytochemistry revealed four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stained both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stained with the PDH antiserum, suggesting the presence of a PDH-like peptide in the LG.

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.

Isolation and characterization of insect PC2-like prohormone convertase cDNA.

Endoproteolytic processing of large precursor molecules at basic amino acid residues plays an important role in the maturation of many hormones, neuropeptides and other regulatory proteins. Enzymes performing these reactions are designated as prohormone or proprotein convertases and belong to the subtilisin family of serine proteases. The screening of a larval cDNA library of the sheep blowfly Lucilia cuprina resulted in the isolation of two cDNAs encoding a PC2-like prohormone convertase. The predicted 675 amino acid preproprotein (LcuPC2) exhibits its highest identity to invertebrate and vertebrate prohormone convertase 2 homologues, and a noticeably lower identity to the so far known insect furin-like prohormone convertases of Drosophila melanogaster and Aedes aegypti. In Northern blot experiments a signal at 2.5 kb could be detected.

Molecular cloning of crustacean pigment dispersing hormone precursor.

The cDNA encoding the precursor of the pigment dispersing hormone (PDH) of the shore crab, Carcinus maenas, was isolated and sequenced. The precursor consists of a putative 22 amino acid signal peptide, a putative 33 residue peptide of unknown function, and the 18 amino acid mature PDH, followed by a Gly residue which serves as a possible amide donor. The deduced mature PDH amino acid sequence is identical to those of Uca pugilator and Cancer magister, previously determined by Edman degradation.

A cDNA encoding a chitinase from the epithelial cell line of chironomus tentans (Insecta, diptera) and its functional expression.

A cDNA coding for chitinase was isolated from Chironomus cells, which possesses conserved regions I and II characteristic for family 18 chitinases, a C-terminus enriched in Glu and Pro without the typical "PEST-region," putative glycosylation sites, a reduced number of C-terminal cysteines, and no typical chitin binding domain. Northern blots revealed one specific signal with an apparent size of 2.3 kb. The cDNA was expressed in the baculovirus/Spodoptera system as a His-tag fusion protein, which was secreted as a functionally active enzyme into the medium and could be separated from endogenous viral and Spodoptera-specific chitinases.

Molecular cloning of crustacean red pigment concentrating hormone precursor.

A cDNA encoding the precursor of the red pigment concentrating hormone (RPCH) of the shore crab, Carcinus maenas, was isolated and sequenced. The predicted preprohormone sequence consists of a putative 25 amino acid signal peptide, the eight amino acid RPCH sequence followed by a Gly as amide donor, a dibasic processing site and a 74 residue peptide of unknown function, referred to as RPCH-precursor-related peptide (RPRP). In in situ hybridisation experiments, the RPCH gene transcript could be localised in two clusters of neurons in the medulla terminalis of the eyestalk.

Point mutations of the human parathyroid calcium receptor gene are not responsible for non-suppressible renal hyperparathyroidism.

The calcium-dependent secretion of parathyroid hormone (PTH) is mediated through an extracellular G protein-coupled calcium receptor (CaR). Inactivating point mutations of this receptor have been found in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. These diseases feature a decreased calcium sensitivity of the parathyroid glands, resulting in a rightward shift of the Ca2(+)-PTH relationship. Severe non-suppressible renal hyperparathyroidism (rHPT) is often characterized by similar setpoint shifts to the right. Thus, point mutations of the CaR gene could contribute to non-suppressible rHPT. We examined genomic DNA of hyperplastic or mainly nodular tissues of 39 parathyroids from 25 rHPT-patients with resistance to calcitriol therapy. Amplification of the six exons of the CaR gene was followed by single-strand conformation polymorphism (SSCP) analysis. DNA sequencing was performed where band shifts were observed. No point mutations in the coding sequence of the CaR gene were detected using the PCR-SSCP strategy. Point mutations in the coding regions of the CaR gene probably play no role in the evolution of renal HPT and are not responsible for the calcitriol resistance of PTH secretion.

Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene.

OBJECTIVE: Androgen insensitivity syndromes (AIS) in subjects with 46, XY karyotype and normal or even elevated androgen blood levels are characterized by various aberrations in male differentiation and virilization. AIS is often accompanied by a broad spectrum of abnormal binding characteristics of the androgen receptor (AR). In order to investigate the correlation between the degree of virilization defect and the type of androgen binding abnormalities and/or the nature of the mutation in the AR gene, we determined androgen binding characteristics of the AR protein and the sequence of the AR gene in clinically and biochemically well characterized patients with various degrees of androgen resistance. DESIGN AND PATIENTS: The activity of 5 alpha-reductase and the binding of androgen to its receptor (KD-values, Bmax, thermolability) were determined in genital skin fibroblasts from 20 patients with various degrees of defects in virilization (2 CAIS, complete AIS; 18 PAIS, partial AIS patients). The AR gene of these 20 subjects was characterized by PCR-SSCP analysis. In case of aberrant electrophoretic mobility the corresponding exon was sequenced. RESULTS: The 2 patients with CAIS and 7 with PAIS showed a mutation in the AR gene. In two, the mutation was in the DNA binding domain, and in all others in the ligand binding domain. In 11 patients with severe virilization defects no abnormal behaviour was detected in the PCR-SSCP. Transcriptional activation studies of two mutant ARs revealed that an approximately tenfold higher androgen concentration (methyltrienolone) is necessary to achieve maximal response as compared to the wild type AR. CONCLUSIONS: There is no obvious relation between the degree of androgen resistance and the binding parameters of the AR and/or the nature of mutation in the AR gene. Androgen insensitivity syndrome can occur despite normal androgen binding and presumably non-mutated AR genes. Even if there is abnormal binding of androgen and/or a mutation in the AR gene there is no clear-cut relationship between these parameters and the degree of virilization defects. Thus, in a proportion of patients, neither the determination of binding parameters of the AR nor the detection of mutations in the AR gene are sufficient to understand the mechanisms underlying the androgen insensitivity syndrome.

Genital skin fibroblasts (GF) of patients with androgen insensitivity syndrome express higher insulin-like growth factor binding protein (IGFBP)-2, -3 and -5 than GF of normally virilized males.

BACKGROUND: We investigated the effects of androgens, estradiol (E2) and insulin-like growth factor (IGF)-I on IGF-II, insulin-like growth factor binding protein (IGFBP)-2, -3 and -5 and mRNA in genital fibroblasts (GF) from patients with complete androgen insensitivity (CAIS) and normally virilized males (C). METHODS: Proteins were measured by specific RIA and Western ligand blot, and specific mRNA levels by RT-PCR normalized by GAPDH levels. RESULTS: Secretion of IGF-II was lowered in CAIS (p<0.001) GF and by testosterone + IGF-I in C GF. Secretion of IGFBP-2 was higher (p<0.001) in CAIS GF and IGFBP-2 mRNA levels were increased by E2 in C GF (p<0.05). E2 stimulated IGFBP-2, -3 and -5 expression in CAIS GF. CAIS GF also secreted more IGFBP-3 (p<0.001) and accumulated 3-5 times more IGFBP-5 mRNA than C GF (p<0.001). CONCLUSION: In contrast to C GF, the availability of IGF-II in CAIS GF is apparently decreased by two facts: by the decreased expression and by increased expression of IGFBP-2, -3 and -5. Furthermore, E2 and IGF-I modulate the expression of IGF-II and IGFBP in GF. This may play a role in the failure to develop male external genitals in CAIS patients.