Biomedical subjects

W D Tilley

Publications and source records attributed to W D Tilley.

At least 37 records · Page 2Linked to original sources

PubMed1996

Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence.

Progression to androgen-independent growth of human prostate cancers may be mediated by alterations in the structure and/or expression of the androgen receptor (AR) gene. To date, mutations in the AR gene have largely been identified in hormone refractory tumors. In this study, single-strand conformational polymorphism analysis and DNA sequencing of the entire AR gene coding region was performed on 25 primary prostate tumors sampled prior to initiation of hormonal (i.e. , androgen ablation) therapy. Base changes leading to amino acid substitutions in the AR were identified in 11 (44%) tumors. The presence of AR amino acid substitutions was associated with decreased immunohistochemical staining for AR in tumor cells and the rapid failure of subsequent hormonal therapies. Single-strand conformational polymorphism analysis of exons 2, 3, and 8 of the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) gene in the same samples revealed no bandshifts, suggesting that the high frequency of AR gene mutations detected was not a consequence of generalized genetic instability. These data indicate that AR gene mutations occur commonly in advanced prostate cancers prior to endocrine treatment of disease and may contribute to altered androgen responsiveness of the tumors.

Androgens↗

PubMed1995

Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles.

Basic fibroblast growth factor (bFGF, FGF2) controls cell proliferation and differentiation in many organs and tissues. In the ovary, cells proliferate and differentiate during folliculogenesis and during formation of the corpus luteum. While previous studies have inferred a role for bFGF in these processes, the precise contribution of bFGF to follicular activation or recruitment has not been established. For this reason, bFGF was immunolocalized in bovine follicles, using anti-bFGF immunoglobulin specific for the 1-24-amino acid terminus of the 18-kDa peptide. Basic FGF was immunolocalized to the cytoplasm of oocytes from bovine primordial and primary follicles. Strong immunostaining was also observed in corpora lutea, the ovarian surface epithelium, and smooth muscle cells surrounding blood vessels, while substantial levels of immunostaining were also present in cells of the theca interna. In most of the healthy antral follicles examined, the three or so layers of granulosa cells which were closest to the basement membrane were also stained, with greatest levels of staining at the most basal region of each cell. Atretic antral follicles had significant and uniform levels of immunostaining throughout the theca interna and the membrana granulosa. Immunostaining as described above was reduced to background levels when the primary specific immunoglobulin was preabsorbed with a 350 molar excess of peptide comprising the NH2-terminal 24 amino acids of bFGF. Based upon our previous observations and those reported here, we propose that basic fibroblast growth factor is synthesized by immature oocytes, especially those from primordial and primary follicles, and that bFGF has a potential role in activating follicle growth via stimulation of granulosa cell proliferation and follicular basement membrane synthesis.

Animals↗

PubMed1995

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

Androgen-Binding Protein↗

PubMed1995

Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3.

Progression to hormone-refractory disease is a common outcome of human prostate cancer. In this study, we have investigated the basis of androgen insensitivity in the human prostate cancer cell line, PC-3, which was derived from a bone metastasis of a hormone-refractory prostate cancer. PC-3 cells with virtually undetectable (PC-3AR-) or low (PC-3AR+) levels of androgen receptor (AR) RNA expression were examined. RNase protection assays demonstrated that the level of AR RNA in PC-3AR+ cells was similar to that in a normal androgen-responsive genital skin fibroblast cell strain. Quantitative immunocytochemical staining of AR in PC-3AR+ cells using antibodies directed against the amino and carboxyl termini of the receptor revealed staining in 30% of cells with either antibody. Furthermore, the level of AR staining in PC-3AR+ cells was higher than in the androgen-responsive breast cancer cell lines ZR-75-1, T47-D, and MCF-7. Despite the expression of AR RNA and protein, PC-3AR+ cell proliferation was unaffected by the presence of 0.1-10 nM mibolerone. Scatchard analysis demonstrated a complete absence of specific [3H]dihydrotestosterone ([3H]DHT) binding to PC-3AR+ cytosolic extracts, which could not be explained by structural alterations in the AR gene. The sizes of individual AR exons amplified from genomic DNA derived from PC-3AR+ cells were identical to those amplified from normal human cells. Furthermore, sequence analysis did not reveal a mutation in the DNA- or hormone-binding domains of the AR gene in PC-3AR+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗

PubMed1995

Androgens induce divergent proliferative responses in human breast cancer cell lines.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

Androgen Receptor Antagonists↗

PubMed1995

Differential expression of apolipoprotein-D and prostate specific antigen in benign and malignant prostate tissues.

PURPOSE: To investigate Apolipoprotein-D (Apo-D) and prostate specific antigen (PSA) immunohistochemical staining of nonmalignant and malignant human prostate tissues. MATERIALS AND METHODS: Apolipoprotein-D and PSA immunoreactivity were evaluated by video image analysis in nonmalignant prostates and in 30 stage D2 prostate cancers. RESULTS: Apolipoprotein-D was detected in all 30 tumors, and the level of staining was elevated in comparison to age-matched nonmalignant prostates (p < 0.05). In contrast, the level of PSA staining in tumors was less than that detected in nonmalignant prostates. CONCLUSIONS: Apolipoprotein-D is expressed in normal human prostate. Elevated Apo-D staining is associated with advanced prostate cancer.

Adolescent↗

PubMed1995

Medroxyprogesterone acetate therapy in advanced breast cancer: the predictive value of androgen receptor expression.

PURPOSE: To determine the predictive value of androgen receptor (AR) levels in primary tumors of women who undergo medroxyprogesterone acetate (MPA) therapy for advanced breast cancer after relapse on tamoxifen adjuvant therapy. METHODS: Between 1984 and 1987 at Flinders Medical Centre, South Australia, 136 postmenopausal women received adjuvant tamoxifen therapy for lymph node-positive breast cancer. Estrogen receptor (ER), progesterone receptor (PgR), and AR levels, tumor size, and degree of axillary node involvement were determined at the time of diagnosis. The median follow-up period was 81 months; 89 women developed metastatic disease, 83 of whom subsequently received MPA (500 mg/d). The objective response rate ([RR] ie, complete response [CR] and partial response [PR]) and progression-free interval (PFI) were assessed in response to MPA therapy. Associations between RR, PFI, and primary tumor characteristics including ER, PgR, and AR levels were examined using the Mann-Whitney U test, Kaplan-Meier product-limit estimator, and Cox proportional hazards regression, as appropriate. RESULTS: Thirty-two of 83 patients (38.6%) responded to MPA. RR was significantly associated with the presence of AR (P < .001), but not with other primary tumor characteristics or duration of tamoxifen therapy. After initiation of MPA treatment, PFI increased with increasing concentration of AR in the primary tumor. CONCLUSION: Response to MPA after adjuvant tamoxifen treatment for lymph node-positive breast cancer was positively associated with AR level in the primary tumor. This finding suggests that MPA action in breast cancer may be mediated in part by the AR.

Adult↗

PubMed1994

Detection of discrete androgen receptor epitopes in prostate cancer by immunostaining: measurement by color video image analysis.

To determine whether multiple features of immunohistochemical staining of the androgen receptor (AR) in prostate cancer could reliably predict androgen dependence, tumor biopsy specimens from 30 patients (stages A-D2) were stained using anti-peptide antibodies to the amino- and carboxyl-terminal of the AR. Measurements were made of the mean area and total amount (i.e., integrated optical density) of AR staining in at least 20 fields per section using a color video image analysis system, and the mean intensity of AR staining per cell and the percentage of AR positive tumor cells were derived. Video image analysis measurement identified quantitative differences in AR staining between the two antibodies, suggesting that this approach may provide a means of identifying receptor variants in prostate tumors. The AR staining measurements were analyzed by discriminant function analysis to assign individual cases to good and poor clinical outcome groups. AR staining features measured with a single antibody (e.g., amino-terminal) were sufficient to predict outcome following hormonal therapy in stage D2 patients (predictive value, 1.0), whereas all features of AR staining measured with both antibodies were required for the entire patient group (predictive value, 0.97). The principal discriminant in both patient groups contributing to the correct assignment of outcome was the mean intensity of AR staining per cell. These findings suggest that AR staining features measured by video image analysis have the potential to predict outcome in prostate cancer.

Aged↗

PubMed1994

Glycosaminoglycans of guinea pig prostate fibromuscular stroma: influence of estrogen and androgen on levels and location of chondroitin sulfate.

The effects of aging and hormone manipulation on the glycosaminoglycan (GAG) content of prostatic stroma in guinea pigs were investigated. Total GAG and individual GAG classes (chondroitin, dermatan, and heparan sulfates, and hyaluronic acid) were measured biochemically in stromal extracts. Chondroitin sulfate was also measured and localized by video image analysis of immunocytochemically-stained tissue sections. The weight and total GAG (uronic acid) content of prostatic stroma increased between the ages of 2 weeks and 2 years by 7-8-fold and 4-5-fold respectively. GAG concentration per unit weight of stroma declined 4-fold during puberty and remained essentially unchanged thereafter. Similar results were obtained for each of the GAG classes. The decreases in GAG concentration were associated with a 3-fold increase in the size of the smooth muscle cells of the prostatic stroma during puberty. Hormonal control of GAG deposition in the prostatic stroma was investigated by steroid replacement in prepubertally-castrated animals. Administration of dihydrotestosterone (DHT) to castrate animals for 6 weeks resulted in significantly reduced concentrations of stromal uronic acid, compared with untreated castrate animals (P < 0.05). The GAG levels post-DHT treatment were similar to those observed after pubertal development in sham-operated control animals. Estradiol treatment had the opposite effect to that of DHT, resulting in a significantly increased concentration of uronic acid compared with castrate animals (P < 0.05). These steroid-induced changes in stromal GAG deposition were mostly contributed to by chondroitin and dermatan sulfates. Combined treatment with DHT and estradiol resulted in stromal uronic acid concentrations similar to those of animals receiving DHT alone, indicating that the effect of DHT on stromal GAG deposition is dominant over the effects of estradiol. Morphometric measurement, using computer-assisted video image analysis of a chondroitin sulfate epitope in prostatic sections stained with a monoclonal antibody (6C3), supported the biochemical data. Stereometric profiles across several sectioned glands demonstrated that chondroitin sulfate was confined to the periacinar basement membranes of the prostatic stroma in all groups except the estradiol-treated castrate animals, where the immunostaining extended from the periacinar basement membrane throughout the fibromuscular stroma. Treatment of castrate animals with estradiol alone also induced a physicochemical change in the chondroitin sulfate molecule, resulting in reduced electrophoretic mobility. In summary, this study identifies changes in the quantity, structure, and localization of chondroitin sulfate in the prostatic stroma of estradiol-treated guinea pigs. Furthermore, estradiol and DHT have opposing effects on the level of chondroitin and dermatan sulfate expression in the prostatic stroma.

Aging↗

PubMed1994

MDA-MB-453, an androgen-responsive human breast carcinoma cell line with high level androgen receptor expression.

The role of androgens and the androgen receptor (AR) in the development and progression of breast cancer is poorly understood. To further define a potential model for androgen action in breast cancer, MDA-MB-453 cells, which express AR in the absence of oestrogen receptors and progesterone receptors, were further characterised in terms of AR expression and androgen responsiveness. High level expression of AR was confirmed by northern blot analysis, radioligand binding and immunocytochemistry, and could not be accounted for by AR gene amplification. Three endogenous androgen-responsive genes (fatty acid synthetase, gross cystic disease fluid protein of 15 kDa and prolactin receptor) and a transfected reporter gene, containing an androgen-responsive element, were induced following androgen administration. A synthetic androgen, mibolerone, induced moderate (27% above control) stimulation of MDA-MB-453 cell proliferation, which was abrogated by the simultaneous administration of the synthetic androgen antagonist, anandron, demonstrating that the effect was AR-mediated. In summary, MDA-MB-453 cells express high levels of functional AR, and thus provide a valuable in vitro model for further studies on androgen regulation of gene expression, and perhaps cell proliferation in breast cancer.

Blotting, Southern↗

PubMed1994

Effects of oestradiol-17 beta and 5 alpha-dihydrotestosterone on guinea-pig prostate smooth muscle cell proliferation and steroid receptor expression in vitro.

Smooth muscle cells (SMCs) are the major cellular component of the prostatic stroma. The aim of this study was to examine the effects of oestradiol-17 beta (OE2) and 5 alpha-dihydrotestosterone (DHT) on the proliferation of guinea-pig prostate SMCs in vitro. OE2 stimulated SMC DNA synthesis at all concentrations examined. At a plating density of 3.0 x 10(4) cells/cm2, maximal incorporation of [3H]thymidine (136% of control) was observed after 36 h of treatment with 1 nmol OE2/l. At the same plating density, DHT had an inhibitory effect on SMC DNA synthesis, with maximal effects (73% of control) being observed 24 h after treatment with 1 nmol DHT/l. These effects of OE2 and DHT were prevented by co-incubation with specific steroid receptor antagonists. At a threefold lower plating density (1.0 x 10(4) cells/cm2), the maximal stimulatory and inhibitory effects of OE2 and DHT were delayed by approximately 24 and 12 h respectively. At the lower plating density, a biphasic effect of DHT was observed on DNA synthesis; DHT was both inhibitory and stimulatory. Maximal inhibition (71% of control) and maximal stimulation (168% of control) were observed after 36 and 134 h treatment with DHT respectively. At the lower plating density, longer term treatment of SMC cultures with OE2 and DHT also resulted in an increase in cell number. After 7 days of treatment with OE2 and DHT, cell number increased by 13% and 12% respectively. When OE2 and DHT were added in combination, the short-term inhibitory effect of DHT on SMC DNA synthesis was dominant over the stimulatory effect of OE2. Treatment with DHT for 24 h significantly inhibited OE2-induced stimulation of [3H]thymidine incorporation, irrespective of the prior duration of OE2 treatment. At the lower plating density, OE2 also decreased oestrogen receptor (ER) mRNA levels to 38% of control levels after 24 h of treatment. ER mRNA levels remained repressed until 72 h after treatment with OE2, and returned to control values following 96 h of treatment. Both the androgen-induced inhibition and stimulation of DNA synthesis observed following treatment of SMCs with 1 nmol DHT/l were associated with a reduction in androgen receptor (AR) mRNA levels. At an intermediate time (i.e. 48 h after commencement of treatment with DHT) AR mRNA levels were increased more than twofold over control levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Age-related changes in guinea pig prostatic stroma.

BACKGROUND: The histology of benign disease of the human prostate (benign prostatic hyperplasia) is heterogeneous. No other species demonstrates the same complexity, and current animal models do not appear to fully encompass the stromal and epithelial developmental changes involved in the human disease. This study describes age-related changes in the prostatic smooth muscle stroma of guinea pigs and humans, which may be pertinent to some aspects of the disease process in humans. EXPERIMENTAL DESIGN: Histologic and ultrastructural changes were examined and measured in the prostates of guinea pigs during aging (2 weeks to 31 months). Similar measurements were also made in human prostatic tissues during aging and the development of benign prostatic pathology. RESULTS: Morphometric analyses of prostates in guinea pigs and men demonstrated similar changes in stromal volume with age. The stromal volume proportion of the prostate in both species decreases at puberty due to the expansion of the epithelial cell compartment, and is followed by a progressive increase during adulthood until a maximum stromal content of approximately 75% of total tissue volume is reached at age 2 years in guinea pigs, and at age 70 years in men. The pathognomic feature of nodularity and the dramatic increase in gland size observed during the late stages of human benign prostatic disease did not occur in the guinea pig prostate. Ultrastructural analysis of guinea pig prostatic smooth muscle cells identified a progressive hypertrophy (approximately 10-fold) from prepuberty through to old age. Two-thirds of smooth muscle cells in the prostatic stroma of aging individuals of both species demonstrated perinuclear organelles (rough endoplasmic reticulum and free ribosomes) that were not present in younger individuals. CONCLUSIONS: The prominent histologic features of the guinea pig prostate during aging are increased stromal mass, significant stromal fibrosis, and occasional prostatitis. These features are frequently observed in men with clinical benign prostatic hyperplasia. The age-related increases in prostatic smooth muscle cell size and content of perinuclear organelles in the guinea pig suggest a re-activation of cellular synthetic activity. The similarity in some features of the prostatic smooth muscle stroma between aging men and guinea pigs suggests that there may be common pathophysiologic processes. We conclude that the guinea pig could be a useful model for examination of the age-related hypertrophy of the smooth muscle cell and the processes inducing reversion to a more synthetic smooth muscle cell phenotype.

Adolescent↗

PubMed1992

Regulation of androgen receptor gene expression by steroids and retinoic acid in human breast-cancer cells.

Although the androgen receptor (AR) has been detected by ligand-binding assays, there is little known about the expression and regulation of the AR gene in human breast-cancer cells. AR mRNA, measured by Northern analysis in 18 cell lines, was found to be expressed predominantly in oestrogen- and progesterone-receptor-positive (ER+, PR+) lines as a single species of approximately 10.5 kb but was also comparatively abundant in I ER- and PR-negative cell line, MDA-MB-453. Dexamethasone (Dex), Organon 2058 (Org 2058), dihydrotestosterone (DHT), and all-trans-retinoic acid (RA) down-regulated AR mRNA levels in T-47D (ER+, PR+) cells 6 hr after treatment, whereas oestradiol (E2) had no effect. In MDA-MB-453 (ER-, PR-) cells, regulation of AR mRNA by RA differed from the other cell lines: RA increased the level of AR mRNA. DHT-binding assays indicated a corresponding increase in AR protein. Transfection of the androgen-responsive mouse mammary tumour virus long-terminal repeat (MMTV LTR) linked to a chloramphenicol acetyltransferase (CAT) reporter gene was used to examine the effect of altered AR levels on androgen action. The increased level of AR following RA pre-treatment in MDA-MB-453 cells resulted in enhanced induction of CAT activity by DHT and, conversely, a decrease in the level of AR following RA pretreatment in T-47D cells resulted in reduced induction of CAT activity by DHT. These data demonstrate that AR is expressed predominantly in ER+ and PR+ cell lines and its expression is regulated by ligands also known to regulate ER or PR, including progestins and retinoids. Androgen responsiveness measured by a transfected reporter gene was altered according to the extent of up- or down-regulation of AR expression, demonstrating that responsiveness is dependent on receptor concentration.

Breast Neoplasms↗

PubMed1992

Molecular basis of androgen resistance.

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Androgens↗

PubMed1991

Inhibition of T47D human breast cancer cell growth by the synthetic progestin R5020: effects of serum, estradiol, insulin, and EGF.

The mechanism of the antiproliferative effects of progestins on human breast cancer cells is not known. In view of the ability of estrogen to stimulate human breast cancer cell production of peptide growth factors, and since previous studies have suggested that the inhibitory action of progestins is dependent on estrogen-stimulated growth, the present study examined the interaction of growth factors and the synthetic progestin R5020 on the proliferation of T47D human breast cancer cells. In this study, the concentrations of estradiol, insulin, and EGF for optimal stimulation of T47D cell growth in 3% dextran-charcoal treated fetal bovine serum (DCC-FBS) were determined to be 1 nM, 100 nM, and 1 nM, respectively. Furthermore, incubation with these optimal concentrations of estradiol, insulin, and EGF in various combinations produced additive effects on T47D cell proliferation, suggesting that these agents act, at least in part, by different mechanisms. In contrast, in a chemically defined medium (DM), both estradiol and EGF were unable to stimulate T47D cell proliferation. In the case of estradiol, the inability to demonstrate stimulation of T47D cell growth in DM was not due to down-regulation of the estrogen receptor. R5020 inhibited the growth of T47D cells, although its effect was more marked in the presence of 3% DCC-FBS than in DM. Stimulation of T47D cell growth by either estradiol or insulin in 3% DCC-FBS was effectively inhibited by R5020. In contrast, growth of T47D cells stimulated by EGF in the absence of estradiol was not markedly inhibited by R5020, the growth being comparable to that of untreated control cells. These findings suggest that the inhibitory effect of R5020 on T47D cell proliferation is dominant over the action of some, but not all, breast cancer mitogens.

Animals↗

PubMed1991

Androgen resistance caused by mutations in the androgen receptor gene.

Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein assayed in cultured fibroblasts and in broken cell assays. In some patients these abnormalities are associated with absent ligand binding, in other qualitative or quantitative abnormalities of ligand binding are present, and in some no abnormality of ligand binding is detected. Analysis of the androgen gene structure in such patients has permitted identification of the causative mutation in many families. Although results of these studies often reinforce concepts established by in vitro mutagenesis studies of other steroid receptors, some mutations have provided unusual insight into the structural organization of the androgen receptor molecule.

Androgens↗

PubMed1991

Molecular basis of androgen resistance in a family with a qualitative abnormality of the androgen receptor and responsive to high-dose androgen therapy.

We have examined the nature of the mutant androgen receptor in a family with a severe defect in virilization associated with a qualitative defect in receptor function. The androgen receptor gene in this family contains two structural alterations: a single nucleotide substitution at position 2444 in exon 5 (adenosine----guanosine) that converts tyrosine 761 to a cysteine residue and a shortened glutamine homopolymeric segment in exon 1 that encodes 12 rather than the usual 20-22 glutamines. A family study was performed using polymerase chain reaction amplification of the glutamine-rich segment, and it was shown that the sister of the proband does not carry the mutant allele. The effects of these two mutations on the function of the androgen receptor were studied by introducing the changes, individually and in combination, into cDNAs encoding the normal human androgen receptor and analyzing the receptor protein produced after transfection of the cDNAs into eukaryotic cells. The presence of a cysteine residue at position 761 causes rapid dissociation of dihydrotestosterone from the receptor protein. Marked thermolability of the transfected receptor protein, however, was demonstrable only upon introduction of an androgen receptor cDNA containing both the partial deletion of the glutamine homopolymeric segment and a cysteine residue at position 761. Likewise, the ability of the receptor to stimulate a reporter gene is strikingly diminished only when both alterations are present, suggesting that the shortened glutamine homopolymeric segment amplifies the impairment of receptor function caused by the tyrosine to cysteine substitution.

Base Sequence↗

PubMed1991

Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg----Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function.

Analysis of the nucleotide sequence of the coding segment of the androgen receptor gene in a patient (N105) with the receptor-negative form of complete testicular feminization has revealed a single substitution (CGC----TGC) at nucleotide 2476. This alteration results in the conversion of an arginine at amino acid 772 to a cysteine. Introduction of this mutation into an androgen receptor cDNA and transfection of the mutant cDNA into COS cells result in the production of a receptor protein with an alteration in the apparent Kd of ligand binding (3 nM) compared to that of the normal androgen receptor (0.5 nM). The mutant receptor protein predicted for patient N105 also demonstrates thermal instability of ligand binding that is not associated with quantitative or qualitative changes in the immunoreactive androgen receptor protein. When assayed in cotransfection experiments using a mouse mammary tumor virus-chloramphenicol acetyl transferase reporter system, the N105 receptor protein appears to be about a tenth as active as the control receptor. These functional characteristics do not appear sufficient to account for the phenotype of complete testicular feminization and do not explain the profound deficiency of androgen receptor in cultured skin fibroblasts. Quantitative S1 nuclease protection assays reveal that the level of androgen receptor mRNA in fibroblasts from patient N105 is markedly reduced. These results suggest that the phenotype in patient N105 is due to two effects of the nucleotide substitution at residue 2476: the replacement of a crucial amino acid (772) in the hormone-binding domain that impairs the function of any receptor molecules formed and a decrease in the level of androgen receptor mRNA.

Androgen-Insensitivity Syndrome↗