Androgen receptors containing expanded polyglutamine tracts exhibit progressive toxicity when stably expressed in the neuroblastoma cell line, SH-SY 5Y.

The pathogenesis of X-linked spinal and bulbar muscular atrophy (SBMA) has been traced to an expansion of repeated glutamine (Gln) residues within the amino terminus of the human androgen receptor (AR). To examine the mechanisms by which these expanded repeat ARs (Exp-ARs) are toxic to neurons, we have established and characterized a cell culture model by stably transfecting SH-SY 5Y neuroblastoma cells with cDNAs containing either normal AR (81 series; 23 Glns) or Exp-AR (902 series; 56 Glns). At a low passage number, no differences in cell morphology, growth properties, or susceptibility to toxic insults were observed between clones expressing normal AR or Exp-AR. Initially, both types of cultures were found to express similar levels of specific hormone binding in monolayer binding assays. Immunohistochemical studies demonstrated the vast majority of both the normal AR and Exp-AR were localized to the nucleus in the absence and presence of androgen. As the 902 series of clones were propagated, the Exp-AR content in the cells appeared to decline progressively. However, this decrease actually reflects a gradual disappearance of the Exp-AR cell population. No such selection occurred during the propagation of cells expressing the normal AR. This selection against cells expressing physiological levels of Exp-AR occurs in the absence of intracellular aggregates and suggests that mechanisms other than those involving the formation of aggregates underlie the observed toxicity of Exp-ARs.

Factors that mediate and modulate androgen action.

Androgens mediate a wide range of processes during embryogenesis and in the adult. In mammals, although a number of steroids can be shown to exert androgenic effects using in vitro and in vivo assays, testosterone and its 5alpha reduced metabolite, 5alpha-dihydrotestosterone (DHT) are considered to represent the principal physiologic androgens. Furthermore, although the effects that androgens exert differ widely among different tissues and cell types, genetic and biochemical data suggest that these effects are mediated via the protein products of a single androgen receptor gene, which is encoded on the X-chromosome in mammals.

Virilization of the female spotted hyena cannot be explained by alterations in the amino acid sequence of the androgen receptor (AR).

The external genitalia of the female spotted hyena are male in character, consistent with virilization by androgens during embryogenesis that results in the fusion of the vaginal labia to form a pseudo scrotum and enlargement of the clitoris to form a phallus. Explanations advanced to account for these anatomic differences have centered on the production or metabolism of androgens in utero or on abnormalities of the androgen receptor (such as a constitutively active AR). The structure of the spotted hyena AR was examined at the level of genomic DNA and cDNA. Southern analysis detected two Eco RI endonuclease cleavage fragments (4.4 and 4.7 kb) that encode the bulk of the AR hormone-binding domain. Isolation of the smaller fragment from a size fractionated genomic library revealed that it contained exons 6, 7 and 8. The remaining portions of the coding sequence were cloned by RT-PCR and RACE analyses. The spotted hyena cDNA sequence predicts protein 912 amino acids in length, which is most closely related to the sequence of the dog AR. Although a number of differences in the predicted amino acid sequence are identified, particularly within the amino terminus, only single amino acid substitutions are present in the DNA- and ligand-binding domains compared to the human AR. In transfection assays, the spotted hyena AR does not exhibit constitutive activity and responds normally to a range of androgenic and non-androgenic ligands. These findings suggest that the structural changes in the AR do not account for the abnormal virilization in the female spotted hyena. These results serve to focus attention on processes proximal (an abnormality of hormone formation in situ) or distal (activation by other mechanisms of processes normally regulated by androgen) to the AR as the cause of the virilization.

Expression of the human androgen receptor in eukaryotic cells using a recombinant adenovirus vector yields high levels of the soluble, functional receptor protein.

The androgen receptor (AR) and closely related members of the steroid receptor family have proven difficult to obtain in native form in large quantities. In the case of the human AR (hAR), high-level expression in prokaryotic or non-mammalian cells leads to the synthesis of a high proportion of non-binding, insoluble, or degraded forms of the receptor protein. To circumvent these difficulties, we have constructed a recombinant adenovirus that directs the expression of hAR under the control of a potent, constitutive promoter. Infection of eukaryotic cells with this recombinant virus leads to the synthesis of large quantities of the intact AR. In contrast to expression methods designed to direct the full-length AR in bacteria, yeast, and insect cells, AR expressed in mammalian cells using this adenoviral vector accumulates at high levels, retains many properties of the native AR, and is not rapidly proteolyzed. This method will prove useful for large-scale preparations of hAR for use in functional and structural studies.

Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.

BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.

Complexities of androgen action.

Androgens mediate a wide range of processes during embryogenesis and in the adult. In mammals, the principal androgens are testosterone and its 5alpha-reduced metabolite, 5alpha-dihydrotestosterone (DHT). Although these androgenic hormones are diverse in character, it is believed that their effects are mediated via the protein products of a single androgen receptor gene encoded on the X-chromosome. A great deal of information has now accumulated pertaining to the mechanisms by which nuclear receptors, such as the androgen receptor, modulate the activity of responsive genes. The studies have demonstrated the participation of a number of ancillary proteins in modulating activation or repression by nuclear receptors. In addition to studies focused on the mechanisms of nuclear receptor function, additional work has illuminated the mechanism by which androgens are metabolized in selected tissues. This information provides a perspective on the number of levels of complexity by which differential gene regulation by androgens may occur in different tissues and in different cell types.

The androgen receptor (AR) in syndromes of androgen insensitivity and in prostate cancer.

The actions of androgens, principally testosterone and 5alpha-dihydrotestosterone, are mediated by a specific receptor protein, the androgen receptor (AR), which is encoded by a single-copy gene located on the human X-chromosome. This receptor protein is a prototypical member of the nuclear receptor family and modulates a range of processes during embryogenesis and in the adult. During embryogenesis, normal AR function is critical to the development of the male phenotype and defects of the AR cause a range of phenotypic abnormalities of male sexual development. Complete loss of AR function has been traced to a number of distinct types of genetic events, including abnormalities of mRNA splicing, the introduction of premature termination codons, and amino acid substitution mutations. An interesting subset of mutations is that in which the AR is completely undetectable using sensitive immunoassays. In all instances, these functional abnormalities are associated with a phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, partial defects of AR function are almost invariably caused by amino acid substitutions within the DNA- and hormone-binding domains of the receptor protein. Such partial defects of receptor function may be caused by changes in either receptor function or receptor abundance. The alterations of AR function and expression that have been characterized in clinical prostatic cancers and in prostate cancer cell lines differ in several important respects. A number of studies have documented the emergence of considerable heterogeneity of AR expression at early stages in the development of prostate cancer. Despite these early changes of AR expression, a substantial body of information suggests that the AR is expressed in advanced forms of prostate cancer, in some cases as the result of amplification events. While infrequent in localized tumors, mutations of the AR have been identified in a number of advanced prostatic cancers and in some instances appear to alter the ligand specificity of the AR. Finally, it appears that other signaling pathways can act to influence AR function.

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

Aromatase messenger RNA is derived from the proximal promoter of the aromatase gene in Leydig, Sertoli, and germ cells of the rat testis.

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-alpha) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.

Irradiation selectively inhibits expression from the androgen-dependent Pem homeobox gene promoter in sertoli cells.

How radiation blocks spermatogenesis in certain strains of rats, such as LBNF(1), is not known. Because the block depends on androgen, we propose that androgen affects Sertoli cell function in irradiated LBNF(1) rats, resulting in the failure of spermatogonial differentiation. To begin to identify genes that may participate in this irradiation-induced blockade of spermatogenesis, we investigated the expression of several Sertoli genes in response to irradiation. The expression of the PEM: homeobox gene from its androgen-dependent Sertoli-specific proximal promoter (Pp) was dramatically reduced more than 100-fold in response to irradiation. In contrast, most other genes and gene products reported to be localized to the Sertoli cell, including FSH receptor (FSHR), androgen receptor (AR), SGP1, and the transcription factor CREB, did not exhibit significant changes in expression, whereas transferrin messenger RNA (mRNA) expression dramatically increased in response to irradiation. Irradiation also decreased Pp-driven PEM: mRNA levels in mouse testes (approximately 10-fold), although higher doses of irradiation than in rats were required to inhibit PEM: gene expression in testes of mice, consistent with their greater radioresistance. The decrease in Pem gene expression in mouse testis was also selective, as the expression of CREB, GATA-1, and SGP1 were little affected by irradiation. We conclude that the dramatic irradiation-triggered reduction of Pem expression in Sertoli cells is a conserved response that may be a marker for functional changes in response to irradiation.

RFG (ARA70, ELE1) interacts with the human androgen receptor in a ligand-dependent fashion, but functions only weakly as a coactivator in cotransfection assays.

Abnormalities of the human androgen receptor (hAR) cause a range of clinical defects in male development. A large proportion of these mutations are single amino acid substitutions in the hormone-binding domain (HBD) that alter AR function by interfering with the capacity of the AR to bind androgen or to form stable hormone-receptor complexes. Prior studies have suggested that the formation of such stable, active hormone-receptor complexes is a crucial step in the modulation of genes by the AR. It is presumed that these hormone-receptor complexes interact with other proteins to participate in the formation of active transcription complexes at the initiation sites of androgen-responsive genes. Using a yeast two-hybrid screening method, we isolated a partial cDNA encoding the carboxy terminus of a protein that interacts with the hAR-HBD (amino acid residues 623-917) in a ligand-dependent fashion in a yeast two-hybrid assay. Sequence analysis of this clone revealed that it encoded a portion of a protein that had been previously characterized as RFG (RET Fused Gene). Using glutathione-S-transferase (GST) fusions of the hAR HBD and immunoprecipitation of the in vitro translated proteins, we have demonstrated that this interaction can be reproduced in vitro. To determine the capacity of this protein to modulate the activity of the AR in transfection assays, we expressed full-length RFG in the CV1 and DU145 cell lines, in combination with an AR expression vector and model androgen-responsive genes [mouse mammary tumor virus (MMTV) and PRE2-tk luciferase]. Our results demonstrate that RFG alters the induction of these reporter genes very weakly (no greater than 2-fold compared with transfections without the RFG expression plasmid). Thus, while our findings are in agreement with published reports which indicate that RFG interacts with AR-HBD in a ligand-dependent fashion, in our assays RFG does not exert major effects on the activity of the hAR in response to androgen or to other steroid hormones.

Molecular defects of the androgen receptor.

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these--the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy--leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.

Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment.

In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.

A steroidogenic factor-1-binding site and cyclic adenosine 3',5'-monophosphate response element-like elements are required for the activity of the rat aromatase promoter in rat Leydig tumor cell lines.

Although transcription initiation within CYP19 (cytochrome P450 aromatase) occurs immediately 5' to the initiator methionine (proximal promoter) in two rat Leydig tumor cell lines (R2C and H540) that express high aromatase activity and in rat ovary, the patterns of aromatase expression in the two cell types are distinctive. To define mechanisms controlling different patterns of expression of the rat aromatase proximal promoter, we performed transient transfection and gel mobility shift assays. Transfection experiments using different sized promoter fragments fused to a reporter gene were used to identify regions that are functionally important for transcriptional regulation in steroidogenic cell lines [R2C, H540, and Y1 (mouse adrenocortical cells that express low aromatase activity)]. These experiments indicate that the cAMP response element (CRE) at -231 and the steroidogenic factor-1 (SF1) motif are both required for expression of the reporter gene in each steroidogenic cell line and that the CRE at -169 is similarly required in R2C cells. Gel mobility shift assays confirm binding of nuclear proteins from the steroidogenic cell lines to the SF1 motif and to CRE (-231). Leydig tumor cells also contain nuclear proteins that bind to the CRE (-169), but nuclear extracts from R2C cells produce a uniquely shifted band compared with H540 cells. These results suggest that differences in proteins that bind to distinct elements within the rat aromatase promoter may be responsible for different patterns and levels of aromatase expression in these steroidogenic cell lines.

Functional activities of the A and B forms of the human androgen receptor in response to androgen receptor agonists and antagonists.

The androgen receptor (AR) is present in many cells in two forms. The B form migrates with an apparent mass of 110 kDa and constitutes more than 80% of the immunoreactive receptor in most cell types. The A form of the AR migrates with an apparent mass of 87 kDa, appears to derive from internal translation initiation at methionine-188 in the AR open-reading frame, and usually constitutes 20% or less of the immunoreactive AR present. Previous experiments designed to examine the functional capacity of the A and B forms of the AR have been hampered by marked differences in the expression levels of the two isoforms, as the nucleotide sequence surrounding the codon encoding methionine-188 causes it to be used inefficiently as a translation initiation site. To circumvent this, we altered the nucleotide sequence surrounding methionine-188 to render it more similar to that surrounding the codon encoding methionine-1. Transfection of a cDNA containing these changes resulted in similar levels of expression of A and B forms of the AR as assessed by immunoblot assays using antibodies directed at an epitope preserved in both. Functional activities of these cDNAs were assessed using cotransfection assays that employed two model androgen-responsive genes (MMTV-luciferase and PRE2-tk-luciferase) in response to mibolerone, a potent androgen agonist, in three different cell lines. These studies demonstrated subtle differences in the activities of the A and B isoforms, which depended on the promoter and cell context. Additional studies failed to reveal any major differences in the responses of the AR-A and AR-B isoforms to a variety of androgen agonists and antagonists, suggesting that the previously reported functional defect of the AR-A is due principally to its level of expression. When assays of AR function are performed under conditions in which levels of expression of the two isoforms are equivalent, the AR-A and AR-B possess similar functional activities.

Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA) is a rare form of motor neuron degeneration linked to a CAG repeat expansion in the first exon of the androgen receptor gene coding for a polyglutamine tract. In order to investigate the properties of the SBMA androgen receptor in neuronal cells, cDNAs coding for a wild-type (19 CAG repeats) and a SBMA mutant androgen receptor (52 CAG repeats) were transfected into mouse neuroblastoma NB2a/d1 cells. The full length androgen receptor proteins, of 110-112 kDa and 114-116 kDa for the wild-type and mutant protein, respectively, were detected by Western blotting in transfected cells. In addition, the presence of an expanded polyglutamine tract in the SBMA androgen receptor appears to enhance the production of C-terminally truncated fragments of the protein. A 74 kDa fragment was particularly prominent in cells expressing the SBMA androgen receptor. From its size, it can be deduced that the 74 kDa fragment lacks the hormone binding domain but retains the DNA binding domain. The 74 kDa fragment may therefore be toxic to motor neurons by initiating the transcription of specific genes in the absence of hormonal control. Immunofluorescence microscopy on transfected NB2a/d1 cells showed that, after hormone activation, the wild-type androgen receptor translocated to the nucleus whereas the SBMA androgen receptor was mainly localized in the cytoplasm in the form of dense aggregates with very little androgen receptor protein in the nucleus. This could explain the reduction in transcriptional activity of the SBMA mutant as compared with wild-type androgen receptor.

Development and characterization of a mouse prostate adenocarcinoma cell line: ductal formation determined by extracellular matrix.

BACKGROUND: Tumor vaccines show promise as a new approach for treating cancer. We have developed a murine prostate cancer cell line which can be used to study growth factor and extracellular matrix regulation of prostate differentiation and will be useful for generating tumor vaccines using the C3(1)/TAG transgenic model of prostate cancer. METHODS: Pr-14 cells were established in defined growth media (GM) and grown in GM, GM + 2% fetal bovine serum (FBS) or DMEM + 10% FBS on plastic, collagen, or Matrigel. Immunofluorescence and Western blot analyses were performed using antibodies to cytokeratin, vimentin, SV40 large T-antigen, and androgen receptor (AR). RESULTS: Pr-14 cells are cytokeratin-positive, vimentin-negative, and express SV40 large T-antigen. These cells are tumorigenic when injected into athymic nude mice and appear to be androgen-independent. Pr-14 cell lines are nontumorigenic when injected into syngeneic FVB/N mice, but form tumors in transgenic TAG-expressing FVB/N mice. Cell growth and morphology are dependent on media composition which determines whether ductal or acinar structures form when grown on Matrigel. CONCLUSIONS: The mouse prostate adenocarcinoma cell line, Pr-14, undergoes alterations in the state of differentiation dependent upon serum concentration when grown on Matrigel. The Pr-14 cell line is a useful reagent to study prostate cell/extracellular matrix interactions, and for immunotherapy and cancer vaccine studies in C3(1)/TAG transgenic mice.

Assessment of androgen receptor function in genital skin fibroblasts using a recombinant adenovirus to deliver an androgen-responsive reporter gene.

Mutations of the androgen receptor (AR) cause defects in virilization and can result in a spectrum of phenotypic abnormalities of male sexual development that includes patients with a completely female phenotype (complete testicular feminization) and individuals with less severe defects of virilization, such as Reifenstein syndrome. These phenotypes are not specific for mutations of the AR gene, however, and defects in other genes can also result in similar abnormalities of male development. For this reason, the diagnosis of an AR defect is laborious and requires data from endocrine studies, the family history, and in vitro binding experiments. To assist in the evaluation of patients with possible AR defects, we previously employed the use of a recombinant adenovirus to deliver an androgen-responsive gene into fibroblast cultures to assay AR function in normal subjects and patients with complete forms of androgen resistance. Although these studies demonstrated measurable differences between these two groups of subjects, we did not assay samples from patients with partial defects of androgen action. In the current study, we have modified this method to examine AR function in three groups of patients with known or suspected defects of AR function: patients with Reifenstein syndrome, patients with spinobulbar muscular atrophy, and patients with severe forms of isolated hypospadias. When assayed using this method, the AR function of patients with Reifenstein syndrome was intermediate between that of normal control subjects and that of patients with complete testicular feminization. Using the parameters established by the aforementioned experiments, we found that defective AR function can be detected in fibroblasts established from patients with spinobulbar muscular atrophy and in some patients with severe forms of isolated hypospadias, including two with a normal AR gene sequence. These results suggest that this method may have some utility in screening samples to detect defects of AR function, particularly when viewed in the context of other AR assays results.

Definition of the elements required for the activity of the rat aromatase promoter in steroidogenic cell lines.

The rate limiting step in estrogen biosynthesis is catalysed by an enzyme complex that includes the aromatase cytochrome P450 (CYP19), and regulation of the synthesis of this steroidogenic P450 is the level at which estrogen synthesis is controlled. In the rat, initiation of aromatase mRNA transcription occurs immediately 5' to the initiator methionine (proximal promoter) in the rat ovary and in two rat Leydig tumor cell lines that express high levels of aromatase (R2C and H540). Although the same site of transcription initiation is employed in both the Leydig tumor cells and in granulosa cells, the patterns of aromatase expression are distinctive. To define the mechanisms controlling aromatase expression in these model cell lines, we have studied the proximal promoter of the rat aromatase using transfection and gel mobility shift assays. These experiments indicate that the SF-1 motif is required for the expression of the reporter gene in each steroidogenic cell line, and that different combinations of CRE-like elements (particularly those located at -169 and -231) are required for full promoter activity in each steroidogenic cell line. In keeping with the results of the functional assays, we were able to demonstrate the binding of nuclear proteins from each of the Leydig tumor cells to the SF-1 motif in mobility shift assays. Nuclear proteins which bind to the CRE-like elements at -169 and -231 were detected in each of the steroidogenic cell lines, but different complexes were observed using extracts from the Leydig tumor cell lines compared to those visualized using extracts prepared from Y1 cells, an adrenocortical tumor cell line that expresses low levels of aromatase activity. Our findings suggest that, in these model steroidogenic cell lines, differences in the proteins that bind to different combinations of elements within the rat aromatase promoter are responsible for the different patterns and levels of aromatase expression which are observed.