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PubMed1993

Transcriptional repression of the glycoprotein hormone alpha subunit gene by androgen may involve direct binding of androgen receptor to the proximal promoter.

Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.

Androgens↗
PubMed1994

[Androgen receptor and insensitivity to androgens].

Androgen insensitivity syndromes are suspected in XY subjects with normal testosterone secretion presenting with absent or severely impaired androgen dependent sexual differentiation. Such clinical features suggest an abnormality of the androgen receptor, necessary step in the transmission of the hormonal message. The androgen receptor is a member of the steroid/thyroid nuclear receptors superfamily. It is a soluble protein of 919 amino acids, divided in independent functional domains responsible for the various functions of the receptor:hormone and DNA binding, and transcriptional activation. The highest concentration of androgen receptor is found, in both sexes, in tissues resulting from primary or secondary sexual differentiation. Cloning of the androgen receptor and use of molecular biology techniques have led to a new classification of androgen insensitivity syndromes. In complete forms (complete androgen insensitivity:CAI) the phenotype is feminine. In receptor negative CAI (Rc-: complete loss of hormone binding), molecular abnormalities include rare, partial or complete, deletions of the gene, or, more frequently, single point mutations in the hormone binding domain, leading to a functionally inactive receptor. Identification and characterization of these mutations provide valuable information regarding the functional importance of specific amino acids of the androgen receptor. In receptor positive CAI (RC+: conserved hormone binding capacity), abnormalities have been reported in the DNA binding domain (deletion of a zinc finger, single point mutations), but also in the hormone binding domain, thus distinguishing between the hormone binding activity and the transcriptional activation activity of this domain. Partial insensitivity syndromes are characterized by an ambiguous and extremely variable phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed2007

Evaluation of an in vitro model of androgen ablation and identification of the androgen responsive proteome in LNCaP cells.

Proteins responsive to androgen and anti-androgen may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-ablation therapy. These proteins represent potential diagnostic and therapeutic targets for improved management of prostate cancer. We have investigated the effect of androgen (R1881) and anti-androgen (bicalutamide) on the androgen-responsive prostate cancer LNCaP cell line using a quantitative gel-based proteomic approach. Prior to analysis, the in vitro system was evaluated for reproducibility and validated by appropriate molecular responses to treatment. Six replicate samples were independently generated and analysed by 2-D DIGE. According to strict statistical criteria, 197 spots were differentially expressed, of which we have successfully identified 165 spots corresponding to 125 distinct proteins. Following androgen supplementation, 108 spots (68 proteins) were increased and 57 spots (39 proteins) were decreased. Essentially no difference was observed between control and anti-androgen-treated samples, confirming the absence of "off-target" effects of bicalutamide. Identified proteins were involved in diverse processes including the stress response and intracellular signalling. The potential contribution to disease of these processes and identified constituent proteins are discussed. This rigorous, statistically supported study of androgen responses has provided a number of potential candidates for development as diagnostic/prognostic markers and drug targets.

Androgens↗
PubMed2005

Transition of an androgen-dependent human prostate cancer cell line into an androgen-independent subline is associated with increased angiogenesis.

BACKGROUND: Androgen-independent prostate cancer is today an incurable disease, but increased understanding of the mechanisms for the transition into an androgen-independent state may increase the possibilities for more efficient strategies in the future. METHODS: An androgen-independent subline, LNCaP-19, to the androgen-dependent prostate cancer cell line LNCaP was developed in vitro under standard culture conditions. The characteristics of LNCaP-19 regarding androgen responsiveness, PSA, and VEGF secretion was studied in vitro. The growth in vivo and the microvessel density (MVD) of the tumors were studied after inoculation in nude mice. RESULTS: LNCaP-19 grows equally well in dextran-charcoal stripped FBS (DCC-FBS) as in normal FBS, and rapidly gives rise to tumors in both intact and castrated mice, indicating a true androgen-independent growth. The PSA secretion from LNCaP-19 cells was lower than from LNCaP cells, while the VEGF level was comparable to the secretion from LNCaP cells without androgen stimulation. The MVD was increased in the LNCaP-19 tumors, and the vessels also displayed a changed morphology with exclusively small microvessels without lumen. CONCLUSIONS: LNCaP-19 shows characteristics resembling those of androgen-independent prostate cancer. An increased MVD and changed vessel morphology in the tumor, makes it an interesting model system for studies regarding angiogenesis in the context of the acquisition of androgen independence.

Androgens↗
PubMed1989

Hypogastric ganglion perinatal development: evidence for androgen specificity via androgen receptors.

The hypogastric ganglion (HG) has previously been shown to be sensitive to both the organizational and activational influences of testosterone. The current investigations examined whether testosterone exerts similar effects prenatally, whether these events are specifically controlled by androgen, and whether androgens might directly masculinize the HG. Prenatal treatment with an anti-androgen, flutamide, resulted in significant decreases in the adult levels of tyrosine hydroxylase (TH) activity, an index of postsynaptic noradrenergic ontogeny, and choline acetyltransferase (ChAT) activity, a marker for presynaptic terminal formation. In addition, testosterone propionate and dihydrotestosterone benzoate reversed the effects of neonatal castration on the development of TH and ChAT activities. In contrast estradiol benzoate was unable to restore enzyme activities. To determine whether the above observations might be produced by direct effects on the HG, androgen cytosol receptor characteristics were studied. Competition and saturation analyses demonstrate that the affinity and specificity of the androgen cytosol receptor in the HG are similar to that displayed in the pituitary, which has previously been shown to contain androgen receptors. These results suggest that the adult levels of TH and ChAT activities are organized during prenatal and early postnatal development. In addition, the organization of the HG appears to be androgen specific. The presence of cytosol androgen receptors suggests that the organizational effects of androgens are possibly induced by a direct mechanism.

Adrenergic Fibers↗
PubMed1995

Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues.

Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system.

Adolescent↗
PubMed1997

Androgen induction of the SVS family related protein MSVSP99: identification of a functional androgen response element.

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is specifically expressed in the mouse seminal vesicle under androgenic control. To study hormonal regulation, fragments of the 5'-flanking region, extending from -2365 to +16 were linked to the chloramphenicol acetyl transferase (CAT) gene and cotransfected with an androgen receptor expression vector into CV-1 cells. A minimal region (-387 to +16) was sufficient for full androgen induction. Further deletion, up to nt-261, almost completely abolished androgen inducibility. DNase I footprinting and band-shift assays, using the DNA binding domain of the androgen receptor (AR-DBD), revealed three AR binding sites: two putative androgen response elements (AREs) occurring at positions -361 (AREd) and -208 (AREp), and an androgen receptor binding region (ARBR) located between positions -317 and -293. Transient transfection assays revealed that site-directed mutation in AREp abolished androgen induced expression, whereas mutation in AREd or in ARBR had no effect. The results demonstrate that AREp is a functional sequence that must cooperate with additional cis-acting elements, located between -387 and -261, for androgen induction of the MSVSP99 gene.

Animals↗
PubMed2001

Novel therapeutic strategy for advanced prostate cancer using antisense oligodeoxynucleotides targeting anti-apoptotic genes upregulated after androgen withdrawal to delay androgen-independent progression and enhance chemosensitivity.

Progression to androgen-independence remains the main obstacle to improving survival for patients with advanced prostate cancer. In this review, findings are summarized that have recently been demonstrated to establish novel therapeutic strategy targeting several genes playing functionally important roles after androgen withdrawal and during androgen-independent progression. The authors initially characterized changes in gene expression after androgen withdrawal in the androgen-dependent Shionogi and LNCaP tumor models using cDNA arrays. Based on these results, they focused on genes highly upregulated after androgen ablation (i.e. bcl-2, bcl-xL, TR.PM-2, IGFBP-5), which have anti-apoptotic or mitogenic activities, and thereby confer a resistance to androgen withdrawal as well as cytotoxic chemotherapy. The authors further demonstrated the efficacy of an antisense oligodeoxynucleotide (ODN) strategy for patients with advanced prostate cancer through the inhibition of target gene expression, resulting in a delay in the progression to androgen-independence by enhancing apoptotic cell death induced by androgen ablation and chemotherapy. The authors also showed the effectiveness of combined antisense ODN therapy and cytotoxic chemotherapy by achieving additive or synergistic effects. These findings provide a basic significance for the design of clinical studies using antisense ODN either alone or in combination with chemotherapeutic agents in patients with advanced prostate cancer.

Androgens↗
PubMed1994

Both Sertoli and peritubular cells respond to androgens with increased expression of an androgen response element reporter.

To explore the ability of androgens to affect gene expression in Sertoli and peritubular cells in vitro, constitutively expressed pSV2-Luc and a regulated reporter containing an androgen response element (MMTV-Luc) were transiently transfected into these cells. Reporter expression was markedly affected by cell density and maturational age. Mibolerone-stimulated MMTV-Luc expression increased in Sertoli cells with animal age between 15 and 25 days, consistent with the developmental increase in androgen receptor concentration, but was not markedly age-dependent in peritubular cells. The antiandrogen hydroxyflutamide inhibited stimulation of MMTV-Luc expression by 1 and 10 nM testosterone in both Sertoli and peritubular cells, consistent with an androgen receptor-mediated event. In contrast, dexamethasone at 1 and 10 nM elicited no effect in Sertoli cells, but stimulated MMTV-Luc expression in peritubular cells. In this study, the androgen receptor/glucocorticoid receptor ratio was 2.4 and 0.06 in Sertoli and peritubular cells, respectively. Cotransfection of Sertoli and peritubular cells with a plasmid expressing the rat androgen receptor further increased the androgen-stimulated expression of MMTV-Luc. These data demonstrate the functionality of the androgen receptors in both Sertoli and peritubular cells in culture. Receptor expression appears to be a limiting factor in the response. The data are consistent with potential roles for both Sertoli and peritubular cells in androgen-mediated transcriptional events in the testis.

Age Factors↗
PubMed2005

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes--androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5alpha-reduced androgens.

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16alphaOH-DHEA, 7alphaOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16alphaOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16alpha-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5alpha-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5alpha-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16alphaOH-testosterone. 5alpha-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5alpha-dihydro-ASD, 5alpha-dihydrotestosterone, and negligible quantities of E1 and E2. 5alpha-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5alpha-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16alpha-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1981

Identification of an 8S androgen receptor-promoting factor that converts the 4.5S form of the androgen receptor to 8S.

A protein has been identified that reconstitutes the 4.5S androgen receptor to the classical 8S form on sucrose gradients of low ionic strength (25 mM KCl and 50 mM Tris). Rat prostate Dunning tumor (R3327) cytosol labeled with [3H]dihydrotestosterone was chromatographed on phosphocellulose to separate the 4.5S receptor from this protein, which we refer to as 8S androgen receptor-promoting factor. The 8S promoting factor has the following physiocochemical properties: heat labile (60 C; 30 min), Stokes radius of 58 degrees A, molecular weight of 170,000 or more, precipitates in 40% saturated (NH4)2SO4, elutes from DEAE-Sepharose in 0.1 M KCl, and elutes from phosphocellulose in 0.1 M KCl. The reconstituted 8S receptor complex is similar to the native 8S receptor in that it is labile to heat and physiological salt concentrations, has a Stokes radius of 91 degrees A, and has a molecular weight of approximately 326,000. The 8S promoting factor is present in mature male rat serum, but is undetectable in sera of male rats 16 days of age or younger. The factor appears to be produced by androgen-responsive cells, since it was found in all tissues of the 15-day-old male rat known to contain androgen receptor. Spleen was found to lack both the 8S promoting factor and the androgen receptor. The 8S promoting factor was detected in serum of female rats and in hypophysectomized (44 days) or castrated (2 or 4 weeks) mature male rats. Salt extracts of purified nuclei from the androgen-dependent Dunning tumor also contain the factor. It is suggested that a specific interaction between the two intracellular proteins, 8S androgen receptor-promoting factor and the androgen receptor, may modulate the androgen responsiveness of target cells.

Adenocarcinoma↗
PubMed2006

Endocrine differentiation of fetal ovaries and testes of the spotted hyena (Crocuta crocuta): timing of androgen-independent versus androgen-driven genital development.

Female spotted hyenas (Crocuta crocuta) have an erectile peniform clitoris and a pseudoscrotum but no external vagina, all established by day 35 of a 110-day gestation. Recent studies indicate that these events are androgen-independent, although androgen secretion by fetal ovaries and testis was hypothesized previously to induce phallic development in both sexes. We present the first data relating to the capacity of the ovaries and testes of the spotted hyena to synthesize androgens at different stages of fetal life. Specifically, spotted hyena fetal gonads were examined by immunohistochemistry at GD 30, 45, 48, 65, and 95 for androgen-synthesizing enzymes, as related to the morphological development. Enzymes included 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5, 3beta-hydroxysteroid dehydrogenase (3betaHSD), and cholesterol side-chain cleavage cytochrome P450 (P450scc). Anti-Müllerian-hormone (AMH) expression was also examined. AMH was strongly expressed in fetal Sertoli cells from GD 30 and after. P450c17 expression was detected in Leydig cells of developing testes and surprisingly in Müllerian duct epithelium. Fetal ovaries began to organize and differentiate by GD 45, and medullary cells expressed P450c17, cytochrome b5, 3betaHSD, and P450scc. The findings support the hypothesis that external genital morphology is probably androgen-independent initially, but that fetal testicular androgens modify the secondary, male-specific phallic form and accessory organs. Fetal ovaries appear to develop substantial androgen-synthesizing capacity but not until phallic differentiation is complete, i.e. after GD 45 based on circulating androstenedione concentrations. During late gestation, fetal ovaries and testes synthesize androgens, possibly organizing the neural substrates of aggressive behaviors observed at birth in spotted hyenas. These data provide an endocrine rationale for sexual dimorphisms in phallic structure and reveal a potential source of androgenic support for neonatal aggression in female and male C. crocuta.

Androgens↗
PubMed1990

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor.

Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein. All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues.

Animals↗
PubMed2001

Interleukin-6 induces androgen responsiveness in prostate cancer cells through up-regulation of androgen receptor expression.

Interleukin-6 (IL-6) induces prostate cancer (CaP) cell proliferation in vitro. Several lines of evidence suggest that IL-6 may promote CaP progression through induction of an androgen response. In this work, we explored whether IL-6 induces androgen responsiveness through modulation of androgen receptor (AR) expression. We found that in the absence of androgen, IL-6 increased prostate-specific antigen (PSA) mRNA levels and activated several androgen-responsive promoters, but not the non-androgen responsive promoters in LNCaP cells. Bicalutamide, an antiandrogen, abolished the IL-6 effect and IL-6 could not activate the PSA and murine mammary tumor virus reporters in AR-negative DU-145 and PC3 cells. These data indicate the IL-6 induces an androgen response in CaP cells through the AR. Pretreatment of LNCaP cells with SB202190, PD98059, or tyrphostin AG879 [p38 mitogen-activated protein kinase (MAPK), MAP/extracellular signal-regulated protein kinase kinase 1/2, and ErbB2 MAPK inhibitors, respectively) but not wortmannin (PI3-kinase inhibitor) blocked IL-6-mediated induction of the PSA promoter, which demonstrates that IL-6 activity is dependent on a MAPK pathway. Finally, IL-6 activated the AR gene promoter, resulting in increased AR mRNA and protein levels in LNCaP cells. These results demonstrate that IL-6 induces AR expression and are the first report of cytokine-mediated induction of the AR promoter. Taken together, our results suggest that IL-6 induces AR activity through both increasing AR gene expression and activating the AR in the absence of androgen in CaP cells. These results provide a mechanism through which IL-6 may contribute to the development of androgen-independent CaP.

Anilides↗
PubMed1980

Effect of androgen depletion on postoperative regrowth of androgen-dependent and -independent Shionogi carcinomas in mice.

About 350 castrated male DS mice given testosterone propionate (TP, 100 micrograms/mouse/day) were grafted with androgen-dependent SC115 tumor or androgen-independent tumors (NHD with positive receptor; NHF with negative receptor). After 20 or 15 days, most of the developed tumors were excised and the mice were divided into 3 groups. TP injection was continued in one group and TP was stopped immediately or 20 days after the excision in the other groups. The cumulative 120-day mortality after transplantation of SC115 tumor into mice was significantly lower in the immediate androgen removal group (10%, 6/59) than in the androgen-injected group (59%, 51/87) or the delayed androgen removal group (42%, 19/45). The cumulative mortalities in mice with androgen-independent NHD and NHF tumors in the immediate androgen removal groups (97 and 91%, respectively) were similar to those in the androgen-injected groups (90 and 95%, respectively). All non-operated and TP-injected mice grafted with SC115 and androgen-independent tumor died due to the tumors within 50 and within 30 days after the transplantation, respectively. These findings suggest the usefulness of adjuvant endocrine therapy for preventing the recurrence of hormone-dependent tumors.

Androgens↗
PubMed1995

Human androgen insensitivity due to point mutations encoding amino acid substitutions in the androgen receptor steroid-binding domain.

Mutations of the human androgen receptor gene were identified in five subjects from four families with androgen insensitivity syndrome. Individual exons of the androgen receptor gene were amplified by the polymerase chain reaction from genomic DNA and screened for sequence-dependent differences in their melting characteristics by denaturing gradient gel electrophoresis. DNA fragments from exons with altered mobility were sequenced. Four different single nucleotide base substitutions were found within exons 5, 6, and 7 encoding the steroid-binding domain of the androgen receptor. In one subject with ambiguous genitalia, amino acid residue 763 was changed from tyrosine to cysteine (TAC-->TGC; Y763C). Four subjects, including two siblings, had complete androgen insensitivity. In one subject, residue 779 was changed from arginine to tryptophan (CGC-->TGG; R779W), another subject (M807V) had a substitution of valine (GTG) for methionine (ATG) residue at position 807, and the two siblings (R855C) had a mutation in residue 855 changing arginine (CGC) to cysteine (TGC). Binding of the synthetic androgen ligand, methyltrienolone (R1881), by the mutant receptor Y763C was decreased by 54% compared to the normal receptor. Transcriptional activation of a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene by AR mutant Y763C was negligible at 0.1 nM R1881 and only 55% at 10 nM R1881 when compared to the maximal response with the normal AR, as assessed by CAT activity. Mutant M807V retained only 22% of normal R1881 binding and mutant R855C was unable to bind the steroid. In accordance with the steroid binding, transcriptional activation of MMTV-CAT by M807V rose to only 26% of control in the presence of 10 nM R1881, a concentration at which R855C remained functionally inactive. In summary, missense mutations within the exons of the androgen receptor gene encoding the steroid-binding domain of the receptor are common causes of both partial and complete forms of androgen insensitivity syndrome.

Adolescent↗
PubMed1999

Phosphorylation/dephosphorylation of androgen receptor as a determinant of androgen agonistic or antagonistic activity.

Protein phosphorylation/dephosphorylation is an important posttranslational modification that plays a critical role in signal transduction. The androgen receptor (AR) is under such control. We demonstrate that androgen receptor phosphorylation determines whether or not AR ligands perform as agonists or antagonists in LNCaP cells. Androgen receptor ligands (such as dihydrotestosterone and beta-estradiol) stimulate receptor expression and phosphorylation and, as a result, they act as agonists or partial agonists. In contrast, agents such as bicalutamide and estramustine inhibit the receptor phosphorylation and act as antagonists. This model is supported by gene expression and transactivation assays. Significant increases in levels of both mRNA and protein of prostate-specific antigen (PSA), a natural AR target gene, occur following the treatment of LNCaP cells with DHT, beta-estradiol, or hydroxyflutamide. In contrast, exposure of LNCaP cells to bicalutamide or estramustine results in a sharp decrease of PSA expression. Agonistic or antagonistic effect of these compounds on PSA expression parallels the level of phosphorylated, but not dephosphorylated androgen receptors. These agonistic or antagonistic effects are also observed in HeLa cells transfected with wild-type AR expression plasmid (pAR0) and AR-driven luciferase expression plasmid GRE-tk-LUC in the presence of different groups of AR blockers. Our data indicate that the functional status of androgen receptors is strongly correlated with the phosphorylation status of the receptors, and that the phosphorylated androgen receptor is the form of the receptor transcriptionally active in regulation. Thus the androgen receptor phosphorylation/dephosphorylation may serve as a new molecular target for screening androgen antagonists for the treatment of prostate cancer.

Androgen Antagonists↗
PubMed1999

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.

Adult↗