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W D Tilley

Publications and source records attributed to W D Tilley.

At least 55 records · Page 3Linked to original sources
PubMed1990

Androgen receptor gene expression in human prostate carcinoma cell lines.

Responses to androgen vary widely among prostate cancers and prostatic carcinoma cell lines. We have explored the basis for this heterogeneity by examining the levels of androgen receptor expression in a prostate carcinoma cell line (LNCaP) that expresses the androgen receptor and two prostate carcinoma cell lines that do not contain detectable androgen receptor. We find that while the LNCaP cell line contains high levels of both the androgen receptor protein and mRNA, the receptor-negative cell lines DU-145 and PC-3 do not express androgen receptor protein as detected by immunoblotting or mRNA as detected by Northern analysis or S1 nuclease protection. These results indicate that the absence of androgen receptor expression in the androgen receptor-negative cell lines is caused by diminished androgen receptor mRNA levels. Genomic Southern analysis indicates that the differences in androgen receptor expression in each of these cell lines is not associated with detectable alterations in the structure of the androgen receptor gene.

Cell Line↗
PubMed1990

Expression of the human androgen receptor gene utilizes a common promoter in diverse human tissues and cell lines.

Androgens mediate their effects through an intracellular receptor that is a member of the steroid/thyroid hormone family of receptors. The expression of this protein is tightly regulated in different tissues and among cell types within a single tissue. To define the mechanisms controlling the expression of the androgen receptor, we have isolated and characterized the promoter of the androgen receptor gene in the human prostate cell line LNCaP. The major site of transcription initiation is approximately 1.1 kilobases upstream of the initiator methionine of the androgen receptor protein. The promoter region lacks typical "TATA" and "CAAT" sequence motifs but lies in a GC-rich region and contains a putative Sp1 binding site characteristic of a "housekeeping" promoter and a 44-base segment composed of alternating adenosine and guanosine residues. S1 nuclease protection analyses indicate that the same promoter is employed both in human tissues (prostate, testes), in genital skin fibroblasts, in T47D and MCF-7 breast cancer cells, and in LNCaP prostate cancer cells.

Base Sequence↗
PubMed1990

A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance.

Mutations of the androgen receptor that impair the action of 5 alpha-dihydrotestosterone and testosterone result in abnormal male sexual development. The definition of the organization of the androgen receptor gene has permitted us to examine its structure in nine patients with androgen resistance that exhibit absent 5 alpha-dihydrotestosterone binding in cultured fibroblasts (receptor-negative androgen resistance). Using labeled probes specific for each individual coding exon, we find no gross rearrangements, insertions, or deletions of the androgen receptor gene in these patients. To analyze the genetic defect in these receptor-negative patients, we used the polymerase chain reaction to amplify each individual exon of the androgen receptor gene in nine affected patients. In all patients, the size of each amplified exon segment was identical to that in normal individuals. The nucleotide sequence of the entire coding region of the androgen receptor was determined in one of these patients. A single nucleotide substitution was identified that results in a premature termination codon in exon 6 at amino acid 794. S1 nuclease protection assays demonstrated that normal levels of androgen receptor mRNA are present in skin fibroblasts of this patient. Transfection of a mutated androgen receptor cDNA containing a termination codon at position 794 into eukaryotic cells resulted in formation of a normal amount of receptor protein, as indicated by immunoblotting, but the expressed protein does not bind 5 alpha-dihydrotestosterone. These findings suggest that the presence of a premature termination codon at amino acid 794 of the androgen receptor is the cause of androgen resistance in this patient.

Adolescent↗
PubMed1990

Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate.

We have developed polyclonal antibodies to two synthetic peptides corresponding to the amino-(N-)terminal or carboxyl-(C-)terminal segments of the human androgen receptor (hAR) protein, as deduced from the nucleic acid sequence of the androgen receptor cDNA. Immunoreactive antisera were identified by solid phase enzyme-linked immunosorbent assay and purified by peptide affinity chromatography. Specific immunoreactivity with the hAR was confirmed by immunoblotting, using both a fusion protein produced in E. coli that contains the C-terminal 880-amino acid sequence of hAR and the full-length receptor protein produced in COS cells after transfection with a plasmid containing the entire hAR-coding region. Immunohistological evaluation of rat and human prostatic tissue using anti-C-terminal or anti-N-terminal antibodies demonstrated similar patterns of specific staining of the nuclei of epithelial and stromal cells. Castration resulted in a decrease in the amount of nuclear AR detected in the rat prostate after a short time of exposure to anti-C-terminal antibodies (less than 4 h), but did not alter the level of specific staining obtained with anti-N-terminal antibodies. This decrease in nuclear staining using anti-C-terminal antibodies could be reversed by treating castrated animals with dihydrotestosterone. When longer times of exposure to the primary antibodies were used, high levels of nuclear staining were obtained with both types of antibodies in prostate specimens from castrate as well as as intact rats. This immunohistochemical staining pattern contrasts with receptor measurements in rat prostate homogenates that indicate the partition of AR binding into the low salt (cytosolic) fraction in the castrate animal and into the high salt (nuclear) fraction in the intact animal. Our results suggest that the AR is predominantly a nuclear protein even in the absence of ligand and that dihydrotestosterone serves to tighten its association with the nucleus. These data also suggest that the immunoreactivity of anti-C-terminal antibodies is influenced by the presence of dihydrotestosterone, presumably via an alteration in the physical state of the receptor protein.

Amino Acid Sequence↗
PubMed1990

Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance.

We have isolated and characterized the gene encoding the human androgen receptor. The coding sequence is divided into eight coding exons and spans a minimum of 54 kilobases. The positions of the exon boundaries are highly conserved when compared to the location of the exon boundaries of the chicken progesterone and human estrogen receptor genes. Definition of the intron/exon boundaries has permitted the synthesis of specific oligonucleotides for use in the amplification of segments of the androgen receptor gene from samples of total genomic DNA. This technique allows the analysis of all segments of the androgen receptor gene except a small region of exon 1 that encodes the glycine homopolymeric segment. Using these methods we have analyzed samples of DNA prepared from a patient with complete androgen resistance and have detected a single nucleotide substitution at nucleotide 1924 in exon 3 of the androgen receptor gene that results in the conversion of a lysine codon into a premature termination codon at amino acid position 588. The introduction of a termination codon into the sequence of the normal androgen receptor cDNA at this position leads to a decrease in the amount of mRNA encoding the human androgen receptor and the synthesis of a truncated receptor protein that is unable to bind ligand and is unable to activate the long terminal repeat of the mouse mammary tumor virus in cotransfection assays.

Amino Acid Sequence↗
PubMed1989

Effect of pubertal development on estrogen receptor levels and stromal morphology in the guinea pig prostate.

Using an immunocytochemical assay (ERICA) with a monoclonal antibody (H222Sp gamma) to the human estrogen receptor, we have demonstrated a stromal localization of the estrogen receptor in the dorsolateral prostate of the guinea pig. Specific staining of estrogen receptor in the guinea pig prostate was confined to the nuclei of periacinar and interacinar stromal cells. In comparison with prepubertal tissues, estrogen receptor staining intensity was markedly reduced in postpubertal prostatic tissues. No immunoreactive estrogen receptor was detected in the acinar epithelial cells irrespective of the developmental stage of the guinea pig prostate. Electron microscopic examination of the guinea pig prostate showed that the stromal component consists predominantly of smooth muscle cells, which, during pubertal development, undergo marked cytological changes and increase in size. These changes in the prostatic stroma were associated with a greater than fivefold reduction in levels of cytosolic and nuclear estrogen receptor determined by either a radioligand binding assay or an enzyme immunoassay (EREIA) and expressed relative to soluble protein. Morphometric analysis of the prostatic stromal cell density (SCD: nuclei/mm2 interacinar stroma), which is inversely proportional to stromal cell size, indicated that the SCD decreased approximately threefold during pubertal development. Furthermore, cytosolic estrogen receptor levels in mechanically separated prostatic stromal fractions were found to vary concordantly with the SCD during pubertal development. To determine whether estrogen influences normal development of the guinea pig prostate, the effect of various hormonal manipulations on stromal development was examined. Castration of prepubertal animals prevented the threefold decrease in SCD that is characteristic of pubertal development. Treatment of prepubertal castrates with estradiol and 5 alpha-dihydrotestosterone (DHT) in combination over a period equivalent to the transpubertal growth phase resulted in a stromal cell density similar to that seen in prostatic sections from intact postpubertal animals. In contrast, treatment of prepubertal castrates with either estradiol or DHT alone resulted in a prostatic stromal cell density intermediate between that observed in intact prepubertal and postpubertal animals. These findings suggest that both estrogen and androgen are required for the normal development of the guinea pig prostatic stroma.

Animals↗
PubMed1989

Development and characterization of primary cultures of smooth muscle cells from the fibromuscular stroma of the guinea pig prostate.

Primary cultures of smooth muscle cells (SMCs) were obtained by a two-step enzymatic digestion of guinea pig prostatic stroma. Ultrastructural morphology and growth characteristics of these cells conformed to those reported for SMCs isolated from vascular and visceral tissue sources. Electron microscopic examination indicated that the cells assumed modified myofibroblastoid features in culture. Microfilaments with associated dense bodies were markedly depleted in cultured smooth muscle cells, in comparison with those of the parent tissue. Cultured cells also possessed increased content of rough endoplasmic reticulum indicating the increased secretory or protein-synthetic capacity of the cells. Immunoperoxidase staining for cytoskeletal markers using monoclonal antibodies to desmin and vimentin supported the ultrastructural observations, suggesting a decline in desmin-staining intermediate filaments during "modulation" to the myofibroblastoid form. Despite this depletion of smooth muscle-specific differentiation markers and reversion to more general mesenchymal properties, the cells retained the ability to contract on challenge with norepinephrine, and grew in the characteristic "hill and valley" pattern on attaining confluence. Inasmuch as the estrogen and androgen receptor expression of the parent stromal tissue is also retained, these primary cell cultures should provide a useful model to study regulation of prostatic development.

Androgens↗
PubMed1989

Risk factors for breast cancer by oestrogen receptor status: a population-based case-control study.

Data from a population-based case-control study conducted in Adelaide, South Australia, and involving 451 case-control pairs, were analysed to determine whether the associations of menstrual, reproductive, dietary and other factors with risk of breast cancer differed by oestrogen receptor (ER) status. Data on ER status were available for 380 cases. The proportion of tumours which were ER+ increased with age, and there was a higher proportion of ER+ tumours in post-menopausal than in premenopausal women. Both oral contraceptive use (P = 0.055) and cigarette smoking (P = 0.047) were associated with increased (unadjusted) risk of ER- cancer, while having little association with risk of ER+ cancer. Most dietary factors had little association with risk of either cancer type, the main exception being the reduction in risk of ER- breast cancer with increasing beta-carotene intake (P for trend = 0.017). In general, however, links with the factors examined were not strong enough to suggest different causal pathways for ER- and ER+ breast cancer.

Adult↗
PubMed1989

Immunocytochemical assay for oestrogen receptor in fine needle aspirates of breast cancer by video image analysis.

Assessment of heterogeneity in oestrogen receptor (ER) expression aims to improve prediction of prognosis and treatment assignment in breast cancer. Current assessments are performed manually and are subjective. Automated image analysis as described here objectively quantitates ER in breast cancer nuclei obtained by needle aspiration. ER was visualised by ERICA with diaminobenzidine (DAB) substrate. Various indices of ER positivity were derived from the integrated density and average density measurements of nuclear DAB. Each index was compensated for background staining by non-specific antibody binding and endogenous peroxidase activity. Total nuclear ER content (integrated optical density of stain) was strongly associated with the biopsy ER concentration determined by saturation analysis of radioligand binding (DCC), P less than 0.005. Nuclear ER concentration by image analysis (mean optical density of stain) was not associated with the DCC measurement of ER concentration, P greater than 0.05. This was attributed to technical artefacts of cytocentrifugation. Using threshold values of 5% positive cells and 10 fmol mg-1 concordance of assignment of ER status by image analysis with the DCC assay was 91%, sensitivity was 89% and specificity 100%. It was concluded that image analysis is an appropriate, easy and economic method for determining the nuclear ER status of aspirated cancer cells. Image analysis has the potential to become a powerful diagnostic tool in the assessment of hormone receptor status of breast cancer patients.

Biopsy, Needle↗
PubMed1989

Characterization and expression of a cDNA encoding the human androgen receptor.

We report the isolation of a cDNA that encodes the complete human androgen receptor. The cDNA predicts a protein of 917 amino acids with a molecular weight of 98,918. Introduction of the cDNA into heterologous mammalian cells caused expression of high levels of a protein that binds dihydrotestosterone with the affinity, specificity, and sedimentation properties characteristic of the native human androgen receptor. Comparisons with the amino acid sequence of previously cloned steroid hormone receptors revealed a high degree of sequence conservation with the progesterone, glucocorticoid, and mineralocorticoid receptors in the putative hormone and DNA-binding domain regions.

Amino Acid Sequence↗
PubMed1989

Steroid hormone and epidermal growth factor receptors in meningiomas.

A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

Adult↗
PubMed1987

Effects of ageing and hormonal manipulations on the level of oestrogen receptors in the guinea-pig prostate.

Cytosolic oestrogen receptor levels in guinea-pig prostate tissue were found to decrease with increasing age, irrespective of whether the binding was expressed relative to cytosolic protein or cellular DNA. This decrease in oestrogen receptor levels was also observed using enriched fibromuscular stromal tissue prepared by mechanical fractionation of the prostate. The most pronounced change in cytosolic oestrogen receptor levels (from 133 to 35 fmol/mg protein) occurred at the onset of puberty. The pubertal decrease in receptor levels could not be attributed to an increase in the level of proteolytic activity in prostatic cytosol fractions derived from mature animals, a change in the affinity of the receptor for oestradiol or an increase in oestrogen receptor levels in salt-extracted nuclear fractions. Administration of tamoxifen (1 mg/day) to intact guinea-pigs throughout the transpubertal growth phase did not influence the age-related decrease in cytosolic and nuclear oestrogen receptor levels. In contrast, the decrease in oestrogen receptor levels was prevented by castration. Administration of dihydrotestosterone (DHT; 1 mg/day) to intact prepubertal animals for 4 days before study resulted in diminished cytosolic oestrogen receptor levels; this effect of DHT was blocked by the non-steroidal antiandrogen flutamide (2 mg/day). Furthermore, elimination of testicular hormones by castration during the late-pubertal growth phase resulted in a greater than twofold increase in prostatic oestrogen receptor levels. Collectively, these observations suggest an age-related decrease in oestrogen receptor levels in the guinea-pig prostate which, in part, may be due to increased testicular function at puberty.

Aging↗
PubMed1986

Relationship between ploidy and steroid hormone receptors in primary invasive breast cancer.

The relationship between ploidy, as measured by flow cytometry, and the presence of oestrogen and progesterone receptors was investigated in 145 primary invasive breast cancers. The tumours were considered as an integral group, and as subgroups of lobular and ductal carcinomas. An association was found between the presence of aneuploid stemlines and an absence of oestrogen receptors (ER), for the total tumour population (P less than 0.02), and for the ductal carcinoma group (P less than 0.05). An association between aneuploidy and an absence of progesterone receptors (PR) was observed for the total tumour group (P less than 0.05). Evaluation of a combined oestrogen and progesterone receptor status indicated that the association between aneuploidy and an absence of both receptors was highly significant. The probability of such an association was P less than 0.001 for the total tumour population, and P less than 0.01 for the ductal tumour group. Assessment of progesterone receptor expression by breast cancers containing oestrogen receptors indicated that aneuploid tumours were as likely to express PR as were diploid tumours. Hence, the biological activity of oestrogen receptors appears unmodified by the presence of aneuploid nuclei.

Aneuploidy↗
PubMed1985

Specific binding of oestradiol to guinea-pig prostate cytosol and nuclear fractions.

A high affinity (Kd approximately 0.15 nM), saturable oestradiol binding site, which is specific for natural and synthetic oestrogens has been identified in guinea-pig prostate cytosol fractions. The binding site is protein in nature (heat- and protease-sensitive) and has a sedimentation coefficient of approx. 8S on glycerol gradients. A high affinity (Kd approximately 0.16 nM), saturable oestradiol binding site was also identified in salt-extracted (0.5 M KC1) nuclear fractions. The optimum incubation conditions for measuring the cytosolic and nuclear oestradiol binding sites were determined to be 20 h at 4 degrees C. Saturation analysis studies revealed that following oestrogen treatment of intact animals, approx. 80% of the specific oestradiol binding sites in prostatic cytosol fractions were transferred into the nucleus. The presence of a specific oestradiol binding protein with characteristics of an oestrogen receptor in the guinea-pig prostate, is consistent with oestrogen having biological activity in this tissue. In view of the abundance of stroma in the prostate of this species, and the consistent finding that the stroma of male accessory sex tissues is oestrogen sensitive, the guinea-pig may be an appropriate experimental animal for further investigating the role of oestrogen in the growth and development of the prostate.

Animals↗
PubMed1985

Distribution of oestrogen and androgen receptors between the stroma and epithelium of the guinea-pig prostate.

A procedure is described for separating guinea-pig prostatic tissue into viable epithelial and stromal fractions. Epithelial cells were isolated using 0.1% protease, but this method resulted in significant damage to the stromal cells. However, using a mechanical tissue fractionation technique, a viable stromal matrix consisting predominantly of confluent sheets of smooth muscle cells and intervening collagen fibres was obtained. Although this method selectively spilled-out the epithelial cells, the majority were non-viable and therefore not suitable for receptor studies. Electron microscopy confirmed that cell architecture and organelle morphology were well preserved in both the enzymatic epithelial preparation and the mechanically prepared stroma. Saturation analysis studies indicated that the concentration of high affinity (Kd approximately 0.15 nM) oestrogen receptors was approx. 10-times greater in the separated stroma than in the epithelial fraction. In contrast, the concentration of androgen receptors (Kd approximately 2.2nM) was almost 2-fold greater in the epithelial than in the stromal fraction. These findings suggest that oestrogen, either independently or in association with androgen, may play a role in the growth and development of the stromal component of the guinea-pig prostate.

Animals↗
PubMed1978

Effect of snap freezing, dithiothreitol and storage on estimations of estrogen receptor sites.

Estrogen receptor assays were performed on human myometrium in order to determine optimal conditions for certain steps of the assay. The findings indicate that: (1) tissue should be snap-frozen in liquid nitrogen prior to assay, (2) dithiothreitol does not increase measurable binding site numbers, (3) estrogen receptors in the cytosol fraction are unstable and should be stored in excess of 1 day; (4) if storage prior to receptor assay is unavoidable, tissue should be stored at -20 degrees C or -70 degrees C for no longer than 1 week.

Cytosol↗