Binding of permixon,a new treatment for prostatic benign hyperplasia,to the cytosolic androgen receptor in the rat prostate

The benign hyperplasia of the prostate is a manifestation of aging, involving the accumulation, within the gland, of dihydrotestosterone, the probable mediator of the hyperplasia. Binding studies were performed on the cytosolic androgenic receptor of the rat prostate using [³H]methyltrienolone as a ligand. The binding of [³H]methyltrienolone at 5 nM, was inhibited by various drugs, such as methyltrienolone and cyproterone acetate. Permixon, a liposterolic extract of the plant, Serenoa Repens B, inhibits competitively the binding to the cytosolic receptor of the rat prostate. Various vegetable and mineral oils, the plant steroid: β sitosterol and the antiprostatic drug: Tadenan, were all found to be inactive. The antiprostatic activity of Permixon shown in animal studies and controlled clinical trials, may thus result from a direct action at the cytosolic receptor.     

Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor

The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5'-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes.     

Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: can estrogen treatment increase androgen receptor content?

The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (3H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15 degrees C rather than at 0 degrees C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.     

A new, non-perturbing, sampling procedure in tracer exchange measurements

An isotope procedure for the tracing of ion fluxes and rate constants in intact plants is presented and applied to 42K-labelled potassium fluxes in cells of intact barley (Hordeum vulgare L.) roots. This procedure differs from conventional tracer efflux protocols in that tracer accrual in the external solution bathing the labelled roots is continually monitored by solution subsampling, whereas conventional protocols involve monitoring the specific-activity decline in a sequence of eluates that wash out tracer released by roots. The new technique minimizes physical disturbance to the plant system, while permitting excellent time resolution of efflux kinetics. In the high-affinity transport (HATS) range, the flux and exchange parameters determined using this method showed close agreement with those found using a conventional protocol. However, in the low-affinity transport (LATS) range, substantially higher influx and efflux were seen than are normally observed with conventional tracer techniques. It is shown that this difference is attributable to the greater disturbance-sensitivity of LATS transport, and conclude that the measurement of fluxes is much more difficult in this transport range than in the disturbance-resistant HATS range.     

A microassay for the measurement of androgen receptors in human prostatic tissue.

A microassay utilizing R 1881 (methyltrienolone) has been developed for the measurement of androgen receptor sites in the cytosol and nuclear extract of human prostatic tissue. Binding of R 1881 to the progesterone binding molecule in cytosol was eliminated by the addition of triamcinolone acetonide. Utilizing a six tube, single point assay, the number of binding sites estimated in nuclear extract averaged 95% of the number measured by a full 7 point Scatchard analysis; the number estimated by the microassay in cytosol averaged 91%. When the single point assay was applied to needle biopsy specimens (200 mg of tissue), the estimated number of binding sites in nuclei averageed 83% of the number measured in bulk tissue (2 grams) utilizing a 7 point Scatchard analysis; the number in cytosol estimated by the microassay on needle biopsy specimens averaged 73%. It is hoped that this technique may be useful in correlating receptor content with hormonal responsiveness in men with metastatic carcinoma of the prostate.     

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.     

A procedure for quantitation of total oxidized uranium for bioremediation studies

A procedure was developed for the quantitation of complexed U(VI) during studies on U(VI) bioremediation. These studies typically involve conversion of soluble or complexed U(VI) (oxidized) to U(IV) (the reduced form which is much less soluble). Since U(VI) freely exchanges between material adsorbed to the solid phase and the dissolved phase, uranium bioremediation experiments require a mass balance of U in both its soluble and adsorbed forms as well as in the reduced sediment bound phase. We set out to optimize a procedure for extraction and quantitation of sediment bound U(VI). Various extractant volumes to sediment ratios were tested and it was found that between 1:1 to 8:1 ratios (v/w) there was a steady increase in U(VI) recovered, but no change with further increases in v/w ratio.Various strengths of NaHCO(3), Na-EDTA, and Na-citrate were used to evaluate complexed U(VI) recovery, while the efficiency of a single versus repeated extraction steps was compared with synthesized uranyl-phosphate and uranyl-hydroxide. Total recovery with 1 M NaHCO(3) was 95.7% and 97.9% from uranyl-phosphate and uranyl-hydroxide, respectively, compared to 80.7% and 89.9% using 450 mM NaHCO(3). Performing the procedure once yielded an efficiency of 81.1% and 92.3% for uranyl-phosphate and uranyl-hydroxide, respectively, as compared to three times. All other extractants yielded 7.9-82.0% in both experiments.Biologically reduced U(IV) was treated either alone or mixed with uncontaminated sediment slurries to ensure that the procedure was not interfering with subsequent U(IV) quantitation. While U(VI) was recovered, it represented 0.07% of the total uranium alone or 7.8% when mixed with sediments. Total uranium recovered did not change.The procedure was then used to monitor changes in complexed U(VI) levels during uranium-reduction in pure culture and sediments. There was no appreciable complexed U(VI) concentration in pure culture. In sediments however, once soluble U(VI) levels and reduction rates decreased, complexed U(VI) levels began to decrease while U(IV) levels continued to increase. This indicated that once soluble U(VI) was nearly exhausted, sorbed U(VI) became bioavailable and was reduced microbiologically.Typically, uranium is quantified in two steps, soluble U(VI) and U(IV). However, the present study shows that after successive washings with water to remove soluble U(VI), a significant pool of oxidized uranium remains which may be mistakenly quantified as U(IV). This procedure can be used to quantified this pool, does not interfere with U(IV) quantitation, and has an overall efficiency of 95.8%.     

The role of androgen in determining differentiation and regulation of androgen receptor expression in the human prostatic epithelium transient amplifying population

The original article to which this erratum refers was published in J Cell Phys (2007) 212:572–578. J. Cell. Physiol. 215: 283–284, 2008. © 2007 Wiley‐Liss, Inc.     

The role of androgen in determining differentiation and regulation of androgen receptor expression in the human prostatic epithelium transient amplifying population

Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.     

A mini-column method for routine measurement of human prostatic androgen receptors

The complex heterogeneous nature of the human prostate gland is such that it is advisable to know the histological characteristics of each sample used for androgen receptor (AR) measurement. Adequate size of sample for AR determination is thus a problem if specimens provided during routine transurethral prostatectomy are to be used for both estimation of AR and histological examination. We present a simple method suitable for these small specimens in which [3H]R 1881 bound to AR is separated from free steroid on mini-columns of controlled-pore glass beads. Data obtained indicate a single class of binding sites of high affinity and low capacity with steroid specificity typical of an androgen receptor. The assay is suitable for samples as small as 20 mg wet weight and is linear using 25-125 microliter cytosol (correlation coefficient 0.995). Intra-assay variation is 6.8% and interassay variation 25.8% (n = 22) over 4 months. A single saturating concentration of steroid measures 97% of AR calculated by Scatchard analysis. Inclusion of high salt (0.4 M KNO3) and 10 mM dithiothreitol in incubation buffer at pH 8.4 are essential; inclusion of 10 mM sodium molybdate in the homogenisation buffer improves measurement. A comparison of AR measured in histologically similar samples obtained by a transurethral resectoscope (TUR) and a cold punch resectoscope (CPR) taken in juxtaposition demonstrated no difference in receptor content. Although carcinomatous samples contained significantly higher receptors levels than benign samples, no differences were observed between TUR and CPR specimens.     

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.     

A consensus DNA-binding site for the androgen receptor.

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.     

Optimal radioligand exchange conditions for measurement of occupied androgen receptor sites in rat ventral prostate

This study was undertaken to define optimal conditions for exchange of ³H-R-1881 with endogenous hormone bound to androgen receptor (AR) sites in homogenates of rat ventral prostate (RVP) of mature animals. To minimize inactivation of AR binding sites under exchange conditions, extracellular proteases present in RVP were removed by mincing and washing tissue fragments in a Ca²⁺-free cell culture medium (J-MEM) containing 1% casein, prior to homogenization in a TEDG buffer (50 mm Tris-maleate buffer, pH 7.4; 1.5 mm EDTA; 2.0 mm DTT; and 10% ($\text{v}\text{v}$) glycerol) containing 0.5 mm phenylmercuric sulfonyl fluoride (PMSF) and 1.0 mm sodium azide. Na₂MoO₄ (final concentration, 20 mm) was added to homogenate fractions which then were incubated at 0–4 °C for 1–5 days with a saturating concentration of ³H-R-1881 (20 nm) in the absence and presence of 2 μm radioinert R-1881. Heparin (200 μg/ml) was added to the incubation medium to “solubilize” nuclear chromatin. Free and bound R-1881 were separated by a hydroxylapatite (HAP) batch procedure. Using these conditions, it has been found that (i) incubation periods of 72–96 h at 0–4 °C were required to achieve maximal specific exchange binding of ³H-R-1881 (B_max) in total homogenates from normal intact rats. Heparin addition (200 μg/ml) did not change B_max and had little or no effect on the rate of exchange. Mean B_max was 6.7 ± 1.6 (SD) pmol ³H-R-1881/mg DNA. R-1881 exchange at 24 h of incubation was only about 40% of B_max. Nonspecific binding, a small fraction (<10%) of B_max, was near maximal at 2 h. Incubation at 15 °C gave similar R-1881 exchange values to those obtained at 0 °C during the first 24 h, but at 48 h and thereafter R-1881 binding markedly decreased. These B_max values in total homogenates of normal intact RVP are about 2.5 times greater than the AR values obtained in 1-day castrated rats, when compared on a DNA basis, (ii) Addition of gelatin (0.25%) to inhibit seminin activity had no effect on B_max. Deletion of either MoO₄^2? or PMSF from the standard exchange medium reduced B_max values ~20%; if both PMSF and MoO₄^2? were deleted, B_max was reduced to a greater extent (~35%). B_max was reduced (40%) when homogenates were prepared without preliminary J-MEM:casein pretreatment and incubated in standard exchange medium with PMSF and MoO₄^2?. (iii) Despite AR stabilization by MoO₄^2? and inhibition of protease activities during exchange incubation, AR inactivation increased exponentially, so that the maximal ³H-R-1881 binding value achieved at 96 h was estimated to represent about 50% of the AR sites originally present. (iv) The binding sites in total homogenates occupied by ³H-R-1881 at 24, 72, and 96 h of exchange exhibited steroid specificity characteristics of AR, as determined by competition studies with a wide variety of steroid hormones and analogues. Scatchard plots of ³H-R-1881 exchange binding in total homogenates of normal intact RVP incubated for 72 or 96 h indicated a single class of affinity sites with apparent K_d of 5 to 6 nm. (v) Sucrose density gradient centrifugation of homogenates incubated for 72 or 96 h showed that the specific ³H-R-1881 binding sites were distributed in two broad peaks associated with low-molecular-weight components. One with S value ~3.5 may be “activated” AR; the other near the top of the gradient (S < 1.6) may include meroreceptor forms of AR.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

A comparison of estrogen and androgen receptor levels in human prostatic tissue from patients with non-metastatic and metastatic carcinoma and benign prostatic hyperplasia

Estrogen receptors (ER, N = 72) and androgen receptors (AR, N = 33) were determined by high pressure liquid chromatography (HPLC) in 72 human prostatic tissues obtained at prostatectomy, and exploratory statistical analyses of the resulting data were performed. To facilitate use of these data as well as other pertinent information from the patient charts, a program for a comparatively large data base was implemented on a Wang minicomputer. The median values of cytosolic AR in the four cancer stages examined were statistically different from each other (P = 0.01), with AR increasing from stages A through D. Even though ER differences between the four stages were not significant (P = 0.13), there was a trend, in the data examined, for median ER values to decrease with stages B through D. On the other hand, median BPH values for both ER and AR were found to lie mid-scale compared with the respective cancer stages, leading to the conclusion that receptor measurements probably cannot distinguish between CA and BPH in human prostatic tissue, at least as measured by competitive binding techniques.     

A protein in rat prostatic interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostatechromatin(residual proteins)contain proteins able tointeract specifically with androgen-receptor complex andis,therefore,a part of the acceptor complex.Amongresidual proteins,a 97 KDa protein has been found whichbinds signifieantly to a genomic fragment containingan androgen-regulated gene coding for a 22 KDa protein.The biological significance of this binding in androgenaction need to be further studied. A mini-plasmid clone containing 22 KDa proteincoding sequence was cloned into Charon 4A genomiclibrary from which a 5.7 Kb genomic fragment wasisolated,identified by hybridization with a 5' and a 3'cDNA probes,and shown to contain the 5' flankingsequence.Restriction enzyme treatment of this fragmentyielded a 4.7 Kb restriction fragment representingthe 5' upstream region and a 1.0 Kb containing part ofthe coding sequence.Deletion studies indicated that the97 KDa protein bound only to a subclone of about 300 bpsegment.Furthermore,gel shifting experiment supportedits DNA-prptein binding.