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PubMed1993

Immunocytochemical localization of androgen receptors in human skin using monoclonal antibodies against the androgen receptor.

Androgen receptors were localized in cryostat sections of human skin using monoclonal antibodies to the human androgen receptor. Bound antibodies were detected using biotinylated rabbit anti-rat IgG, peroxidase-conjugated streptavidin, and diaminobenzidine as chromogen. In the neonatal foreskin, antibody to androgen receptor bound to keratinocytes in the epidermis and to fibroblasts and vascular endothelial cells in the dermis. Immunohistochemical staining was stronger in nuclei than in cytoplasm. This staining was specific, because there was no significant staining when antibody to the androgen receptor was replaced with IgG from nonimmunized rats or with buffer, or when antibody to androgen receptor was incubated, prior to immunostaining, with a trp E-human androgen-receptor fusion protein used as immunogen. Incubation of androgen receptor antibody with trp E alone did not affect staining. Androgen-receptor antibody also bound to keratinocytes, fibroblasts, and endothelial cells in skin from adult men and women. Skin from the scalp, nose, lip, back, and chest gave positive staining for androgen receptor. Antibody to androgen receptor also bound to the coil and ductal cells of eccrine glands, external root sheath of hair follicles, epithelium in the hair bulb, dermal papilla cells, and sebocytes. There was no significant binding to adipocytes, collagen, or stratum corneum. These results show that androgen receptor is present in cells that are known to be targets for androgens and also in cells in which the biologic effects of androgens are yet to be characterized.

Adult↗
PubMed1999

A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor.

Animals↗
PubMed2001

Comparison of androgen receptor and oestrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testosterone concentrations.

The aims of this study were: (i) to investigate the cellular immunoexpression of androgen receptor and oestrogen receptor beta in the testes of the common marmoset (Callithrix jacchus) during neonatal life compared with their expression at later ages; (ii) to establish whether neonatal marmoset Sertoli cells are targets for androgens or oestrogens or both; and (iii) to investigate the relationship between neonatal plasma testosterone concentrations and androgen receptor immunoexpression by abolishing the neonatal testosterone surge with a potent GnRH antagonist. Androgen receptor and oestrogen receptor beta immunoexpression were evaluated in neonatal animals aged 1-4 days, 4 weeks and 6 weeks, and compared with immunoexpression in animals aged 18-22 weeks (early infancy), 35 weeks (late infancy), 58-62 weeks (late pubertal) and > 100 weeks (adult). Immunoexpression of androgen receptor in the reproductive tract was also evaluated at each age. Sertoli cell immunoexpression of androgen receptor was weak or absent in neonatal animals, but increased substantially in infant animals, reaching adult levels by the end of infancy. In contrast, immunoexpression of androgen receptor during the neonatal period was strong in testicular interstitial cells and very strong in epithelial cell nuclei throughout the reproductive tract, and did not change greatly with age in these cells or tissues. Similarly, immunoexpression of oestrogen receptor beta was prominent in many Sertoli cells and in the germ cells of neonatal animals, and was relatively constant throughout life. Weak immunoexpression of androgen receptor in neonatal Sertoli cells was associated with high plasma testosterone concentrations (2.7-5.5 ng ml(-1)), whereas strong Sertoli cell immunoexpression was associated with baseline (approximately 0.12 ng ml(-1)) testosterone concentrations in infant animals and with > 10 ng ml(-1) in late pubertal and adult animals. Immunoexpression of androgen receptor and oestrogen receptor beta was also evaluated in co-twin males aged 4 and 35 weeks, after treatment from birth to 4 weeks or from week 25 to week 35, respectively, with either vehicle or with GnRH antagonist at a dose known to suppress the neonatal testosterone surge completely. Only GnRH antagonist treatment during weeks 25-35 reduced androgen receptor immunoexpression, whereas immunoexpression of oestrogen receptor beta was unaffected by treatment during either period. On the basis of these findings it is suggested that: (i) neonatal marmoset Sertoli cells may be targets primarily for oestrogens rather than androgens; (ii) androgen receptor expression in the testes of neonatal and infant marmosets is not regulated in a straightforward way by testosterone; and (iii) high neonatal concentrations of plasma testosterone are not absolutely necessary for expression of androgen receptor in marmoset testes at this time.

Aging↗
PubMedDate not supplied

[Androgen-independent prostate carcinoma and androgen-receptor: recent progress in molecular genetics].

Prostate cancer is an androgen-dependent tumor which presents an androgen-independent regrowth after clinical regression in response to antiandrogen treatment. Four hypotheses have been developed to understand how androgen signal transduction pathway mediate androgen-independent tumor progression: over expression of the wild-type androgen-receptor gene, androgen-receptor gene mutation, excessive recruitment of transcriptional co-activator ARA-70 and a cross-talk between the androgen-receptor and the growth factor receptor pathways. In this work, C. Sawyers's group elegantly demonstrates, in LAPC-4 androgen-independent prostate cancer sublines, that forced hyperexpression of HER-2/Neu receptor tyrosine kinase allowed androgen-independent growth, that HER-2/Neu activated the androgen-receptor pathway in the absence of androgens and synergized with low levels of androgen to superactivate the pathway. These important data could have therapeutic implications for the management of androgen-independent prostate cancer.

Disease Progression↗
PubMed2000

Androgen binding profiles of two distinct nuclear androgen receptors in Atlantic croaker (Micropogonias undulatus).

In the present study, the binding affinities of 28 androgens for two nuclear androgen receptors (AR), termed AR1 and AR2, in Atlantic croaker (Micropogonias undulatus) brain and ovarian tissues, respectively, were determined using competitive binding assays. The 5alpha-reduction of steroids, in general, increased the metabolite's binding affinity for AR2 while decreasing it for AR1. In addition, few androgens bound to AR1 with high affinity and modifications to the basic 3-ketone,4-ene,17beta-hydroxy structure of testosterone usually reduced its binding affinity for AR1. However, androgens with ketone groups at the 3- and 17-position bound with high affinity to AR1 provided that the androgen had either a 5alpha-reduced A-ring or a third ketone group at the 11-position. This suggests that there may be several high affinity conformations that AR1 can occupy depending upon whether an androgen possesses a ketone or a hydroxyl group at the 17-position. The binding of androgens to AR2 showed a more predictable pattern, 5alpha-reduced steroids bound better than 4-ene steroids and any changes to the basic 3-keto,17-hydroxy motif of 5alpha-dihydrotestosterone lowered the binding affinity of a steroid. However, these structural changes often caused only minor decreases in binding affinity, such that AR2 has a broader affinity for androgens and a greater affinity than AR1 for structurally diverse androgens. Widely different androgen binding affinities of AR1 and AR2 suggest that these two nuclear androgen receptors may mediate the physiological actions of different androgens in teleosts.

Androgen Antagonists↗
PubMed1984

Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat.

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.

Androgen-Binding Protein↗
PubMed1996

Androgen-like and anti-androgen-like effects of antiprogestins in human mammary cancer cells.

In addition to their antiprogestational activity, the antiprogestins RU486, ZK98.299 and ZK98.734 possess varying antiglucocorticoid as well as androgen-like or antiandrogen-like properties in human mammary cancer cells. The human mammary cancer cell line MFM-223, which contains only androgen receptors, was used as a model to investigate androgen receptor mediated effects of these antiprogestins. Proliferation of MFM-223 cells is inhibited by androgens and does not respond to oestrogens, progestins and glucocorticoids. As shown in proliferation assays, ZK98.734 was a strong inhibitor of cell proliferation. This effect was antagonised by the antiandrogen hydroxyflutamide. ZK98.734 was found to displace [3H]R1881 from the androgen receptor in MFM-223 cells, substantiating the involvement of the androgen receptor. The antiprogestin ZK98.299 failed to influence the proliferation of MFM-223 cells. ZK98.299 did not bind to the androgen receptor and was devoid of androgenic or antiandrogenic activity. RU486 bound to the androgen receptor. It was a weak inhibitor of MFM-223 cell proliferation, but the inhibition of proliferation by RU486 was not antagonised by hydroxyflutamide. This effect was probably not mediated by the androgen receptor. RU486 had antiandrogenic activity in this cell line, as it antagonised the inhibitory effect of dihydrotestosterone at a 100-molar excess. These results were confirmed by transfection experiments with an MMTV-CAT construct in the same cell line, demonstrating the biological function of the ZK98.734-androgen receptor complex. ZK98.299 and RU486 were not able to induce CAT activity. The different androgenic or antiandrogenic properties of the antiprogestins investigated should be considered when selecting antiprogestational properties of the antiprogestins investigated should be considered when selecting antiprogestational compounds for clinical applications, as a partial androgenic activity may be of benefit in breast cancer but can have undesired side-effects in other diseases.

Androgen Antagonists↗
PubMed2000

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study.

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

Androgens↗
PubMed1993

Progression of androgen-sensitive mouse tumor (Shionogi carcinoma 115) to androgen-insensitive tumor after long-term removal of testosterone.

Shionogi Carcinoma 115 (SC115) is an androgen-sensitive transplantable mouse tumor. To study the mode of progression from androgen-sensitive to -insensitive tumor, cloned SC115 cells were serially cultured without androgen. Shortly after withdrawal of androgen, SC115 cells showed markedly decreased growth, but growth resumed gradually with loss of response to androgen and the cells 60 weeks after androgen removal [A(-)60 cells] grew faster than SC115 cells cultured in the presence of androgen. A(-)60 cells showed malignant phenotype with morphological changes and tumorigenicity in male and female mice. Although mRNA and binding capacity of androgen receptor were maintained, the cells after removal of androgen rapidly lost expression of mouse mammary tumor virus-related gene and the loss was irreversible in A(-)60 cells. The stimulating effect of basic fibroblast growth factor (bFGF) temporarily decreased, then recovered to the initial level after long-term androgen removal. This fluctuation of response to bFGF was accompanied with changes in the number of bFGF receptors and amount of bFGF-like substance(s) secreted. The substance(s) seemed to be an FGF-like growth factor different from known factors. It was concluded that progression of SC115 cells to androgen-insensitive ones under an androgen-deprived condition proceeded with adaptation by means of increases in production of an FGF-like growth factor and in binding capacity to this factor.

Androgens↗
PubMed1992

Point mutations detected in the androgen receptor gene of three men with partial androgen insensitivity syndrome.

OBJECTIVE: Determine the sequence of the androgen receptor gene in men with impaired responsiveness to androgens in order to identify the molecular basis of their under-virilization. DESIGN: Blood samples were used as the source of genomic DNA. Portions of the androgen receptor gene were amplified by polymerase chain reaction and sequenced. PATIENTS: Samples were obtained from three patients and five normal fertile controls. Patients were all 46 XY and were undervirilized with ambiguous external genitalia, gynaecomastia and infertility. MEASUREMENTS: Total cellular DNA was purified from peripheral blood leucocytes. Pairs of oligonucleotide primers designed to flank the individual exons of the androgen receptor gene were synthesized. The specific regions of the androgen receptor were amplified from the samples of cellular DNA by polymerase chain reaction. Amplified DNA was purified, sequenced and compared to the published sequence. RESULTS: In all three patients point mutations in the androgen receptor gene were detected but no defects were detected in samples from normal controls. In two of the patients, an identical single nucleotide change from G to T was detected. This nucleotide was within the codon for amino acid 866 and would change it from valine to leucine. Amino acid 866 is found within an area of the steroid binding domain thought to be involved in receptor dimerization. Within the repetitive sequence of exon I patient 1 had 21 glutamine residues and patient 2 had 25. In the third patient a single change of G to A would result in incorporation of lysine in place of a conserved arginine residue at position 607 within the second zinc finger of the DNA binding domain. The sequence of the androgen receptor gene of the mother of the third patient revealed her to be heterozygous for the same defect. CONCLUSION: Patients 1 and 2 are unrelated although they have an identical point mutation in their androgen receptor gene. A patient with complete androgen insensitivity syndrome has been reported to have a defect at the same position causing the amino acid substitution of methionine for valine. Therefore we confirm that the nature of the amino acid change in the peptide sequence of the androgen receptor as well as its location within the protein, can have a profound effect on the phenotypic severity of androgen resistance. Studies on mutated receptors from individuals with a wide range of degrees of androgen resistance may enable us to construct a map of the key amino acids in the different domains of the protein.

Adult↗
PubMed1999

Methylcobalamin decreases mRNA levels of androgen-induced growth factor in androgen-dependent Shionogi carcinoma 115 cells.

Methylcobalamin (MeCbl) is an important enzyme cofactor required for methionine synthase activity. It also inhibits, in a dose-dependent manner, the proliferation of an androgen-dependent cell line, SC-3, derived from an androgen-dependent mouse mammary tumor (Shionogi carcinoma 115). In SC-3 cells, androgen induces the production of androgen-induced growth factor (AIGF), an autocrine growth factor increasing the proliferation of SC-3 cells. MeCbl treatment suppressed the androgen-induced, AIGF-mediated growth of SC-3 cells, as well as the androgen-induced increase of AIGF mRNA. In SC-3 cells, androgen receptors linked with androgen form complexes that tightly bind DNA and act as transcription factors in the nucleus to regulate the expression of specific genes such as AIGF. The number and dissociation constants of androgen receptors in control and MeCbl-treated SC-3 cells were the same. Similarly, the extent of binding of normal androgen receptors in nuclei from control and MeCbl-treated cells was virtually identical. The androgen receptors from control and MeCbl-treated cells showed similar capacities for conversion to a form that tightly binds to DNA on heat activation. These results suggest that the reduction of AIGF mRNA, subsequent to the nuclear binding of androgen receptors, may be a partial cause of the growth-inhibitory activity of MeCbl in SC-3 cells.

Androgens↗
PubMed1998

Regional expression of transforming growth factor-alpha in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement.

The prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-alpha (TGFalpha), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFalpha in the rat ventral prostate during postnatal development to adulthood. TGFalpha-immunopositive cells were located throughout the ductal epithelium from postnatal days 5-20. By day 45 and thereafter, regional variation in TGFalpha expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFalpha in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFalpha staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFalpha in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFalpha expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFalpha in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFalpha is not regulated by androgen in this region of the ventral prostate. In summary, TGFalpha expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFalpha may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.

Aging↗
PubMed1994

Characterization of mutant androgen receptors causing partial androgen insensitivity syndrome.

The androgen insensitivity syndrome (AIS) is an X-linked disorder caused by mutations of the androgen receptor (AR) gene resulting in a spectrum of sex phenotypes that ranges from complete female (complete AIS) to nearly complete male (partial AIS). Using the polymerase chain reaction and denaturing gradient gel electrophoresis, we have analyzed the AR gene in three 46,XY individuals with partial AIS. In one subject whose androgen insensitivity was manifest at birth by clitoromegaly, posterior labial fusion, and a urogenital sinus, androgen-binding affinity in genital skin fibroblasts was similar to that of the control. In this subject, a mutation was identified in exon C encoding the second zinc finger of the androgen receptor. The mutation converted a leucine residue at position 616 to arginine, causing greatly reduced binding of receptor to an androgen-response element DNA sequence. However, the mutant AR retained a low level of transcriptional activity at physiological androgen concentrations in keeping with the subject's phenotype of partial AIS. In the second subject, who also had an ambiguous external genital phenotype, a single base mutation was identified in exon G, converting arginine at position 840 to histidine. Androgen-binding affinity in genital skin fibroblasts of this subject was 7-fold lower than control, and the mutant receptor had reduced transcriptional activity. In the third subject, who had a female phenotype with normal pubic hair reflecting a low degree of androgen responsiveness, the valine residue at position 889 was replaced by methionine. This mutant receptor had apparent normal androgen-binding affinity but reduced androgen-binding capacity when examined by expression of the recreated mutant AR in COS 7 cells. These results demonstrate the clinical, functional, and molecular heterogeneity in the syndrome of partial androgen insensitivity.

Adolescent↗
PubMed1994

An androgen receptor mutation causing androgen resistance in undervirilized male syndrome.

The molecular basis of androgen resistance was investigated in a patient with undervirilized male syndrome. Binding studies of the androgen receptors in the patient's genital skin fibroblasts revealed a normal binding capacity of 5 alpha-dihydrotestosterone, although the affinity to androgen was slightly lower than the normal control value. The androgen binding of the patient's receptor showed a moderate thermal instability when the assay temperature was raised from 30 to 41 C. Nucleotide sequencing analysis of the androgen receptor gene revealed a single nucleotide substitution in exon F, resulting in an amino acid alteration from leucine (CTC) to phenylalanine (TTC) at position 789 within the steroid-binding domain of androgen receptor. When expressed in COS-7 cells, the mutant androgen receptor harboring phenylalanine at position 789 showed thermolabile androgen-binding properties similar to those observed in the patient's genital skin fibroblasts. Cotransfection experiments with an androgen-inducible reporter gene demonstrated a decreased transactivational capability of the mutant receptor. These results indicate that this point mutation modified the receptor function and caused androgen resistance in this patient. This mutation caused the mildest form of all androgen insensitivity syndromes ever examined for mutations in the androgen receptor gene.

Adult↗
PubMed1993

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Androgens↗
PubMed1993

Single nucleotide substitution of the androgen receptor gene in a case with receptor-positive androgen insensitivity syndrome (complete form).

Complete androgen insensitivity syndrome is caused by X chromosome linked disorder resulting in a target organ insensitivity to androgen. Two variants have been described in this syndrome. In the first, the binding of [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) to the androgen receptor is undetectable (receptor-negative), whereas in the second variant normal levels of androgen receptor are detectable but the binding of [3H] dihydrotestosterone to the androgen receptor is significantly thermolabile under certain conditions (receptor-positive). In receptor-negative cases, genetic disorders of the androgen receptor gene have been demonstrated. On the other hand, the genetic disorder of androgen receptor in receptor-positive cases is little known. In this study, the gene structure of androgen receptor in a receptor-positive case using a polymerase chain reaction technique is studied in the fibroblasts cultured from genital skin. The results demonstrate that the substitution of nucleotide (guanine-->cytosine) in exon G of the androgen receptor causes the replacement of an amino acid in position 820 (glycine-->alanine) which occurs in the hormone-binding domain of the androgen receptor. The substitution of nucleotide may explain the thermolability of the androgen receptor in a case with receptor-positive androgen insensitivity syndrome.

Adult↗
PubMed1992

Cultured dermal papilla cells from androgen-dependent human hair follicles (e.g. beard) contain more androgen receptors than those from non-balding areas of scalp.

Androgens stimulate hair growth in many areas, e.g. the beard; they also induce regression and balding on the scalp with increasing age in genetically disposed individuals. The cause(s) of this biological conundrum is unknown but age-related; androgen-potentiated changes also occur in the prostate. The mesenchyme-derived dermal papilla situated at the base of the hair follicle is thought to play an important role in regulating the growth and development of the follicular epithelium. Since androgens probably act on the hair follicle via the dermal papilla, cultures of dermal papilla cells from human hair follicles with differing responses to androgens in vivo have been established and their ability to bind androgens assessed. Receptor binding was assayed by saturation analysis (0.05-10 nmol/l) using the synthetic non-metabolizable androgen, [3H]mibolerone. Shionogi 115 cells were also assayed as a positive control. Specific high-affinity low-capacity androgen receptors were identified in 12 dermal papilla primary cell lines with similar characteristics to established androgen receptors. Cells from androgen-sensitive follicles (beard, scrotum and pubis) contained higher levels of androgen receptors than those derived from relatively androgen-insensitive non-balding scalp follicles whether the receptor content was calculated in relation to cell number, protein or DNA content of the cells. These results support the hypothesis that androgens act on hair follicles via the dermal papilla in vivo and demonstrate that dermal papilla cells exhibit an altered phenotype in culture which depends on the body site from which they were derived.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed2001

Neuronal size in the spinal nucleus of the bulbocavernosus: direct modulation by androgen in rats with mosaic androgen insensitivity.

The motoneurons of the spinal nucleus of the bulbocavernosus (SNB) and its target muscles, the bulbocavernosus and levator ani, form a sexually dimorphic circuit that is developmentally dependent on androgen exposure and exhibits numerous structural and functional changes in response to androgen exposure in adulthood. Castration of male adult rats causes shrinkage of SNB somata, and testosterone replacement reverses this effect, but the site at which androgen is acting to cause this change is undetermined. We exploited the X-chromosome residency of the androgen receptor (AR) gene to generate androgenized female rats that were heterozygous for the testicular feminization mutant (tfm) AR mutation and that, as a consequence of ontogenetic random X-inactivation, expressed a blend of androgen-sensitive wild-type cells and tfm-affected androgen-insensitive cells in the SNB. Chronic testosterone treatment of adult mosaics increased soma sizes only in androgen-competent wild-type SNB cells. The size of tfm-affected SNB somata in the same animals did not differ from the size of either the wild-type or tfm-affected SNB neurons in control mosaics that did not receive androgen treatment in adulthood. Because the muscle targets of the SNB are known to be uniformly androgen-sensitive in tfm mosaics, this mosaic analysis provides unambiguous evidence that androgenic effects on motoneuron soma size are mediated locally in the SNB. It is possible that the neuronal AR plays a permissive role in coordinating the actions of androgen.

Androgen-Insensitivity Syndrome↗