Steroid receptors in the human prostate. 2. Some properties of the estrophilic molecule of benign prostatic hypertrophy.

Some physicochemical properties of the estrophilic ‘receptor’ of human benign prostatic hypertrophy were examined by agar gel electrophoresis. 1) Competition analyses revealed the high selectivity of the molecule for the naturally occurring estrogens but not for representatives of other classes of steroid hormones (androgens, corticosteroids, progesterone). This, coupled with the failure of an estrogen ‘receptor’-rich extract to exhibit detectable tissue specific binding of (³H) 5α-dihydrotestosterone suggests that prostatic androgen and estrogen receptors may have separate identities. 2) The molecule proved highly resistant to enzyme attack, a stability conferred by estradiol-17β rather than by the thiol reagent dithiothreitol. Its proteinaceous nature was finally demonstrated when extract was exposed to enzymes at 0°C prior to steroid addition. 3) Initial complex formation between estrogen and its ‘receptor’ protein was rapid and reached a plateau after 4 hours. Binding was greater at 0°C than 37°C.     

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.     

Plasma steroids in benign prostatic hypertrophy and carcinoma of the prostate

Cortisol, 17-hydroxyprogesterone (17-OH-P), progesterone, testosterone, 5 alpha-dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) were determined by RIA after chromatographic separation of steroids on Sephadex LH-20 columns, in 54 hospitalized patients with benign prostatic hyperplasia (BPH) and in 32 hospitalized patients with prostatic carcinoma (PCA) (T34, N01, M01). The patients' values were compared with those of 63 age-matched controls. Increased cortisol and DHT levels, subnormal estrogen and 17-OH-P values and normal progesterone level were found in both benign and malignant groups. Higher mean values for testosterone, and T/DHT ratio and lower mean values for E2/T ratio were found in PCA, as compared with those in BPH. An age invariance of cortisol, testosterone, T/DHT ratio and estradiol was found in both BPH and PCA, instead of the age dependence found in normal subjects. The normal relations between testosterone and its precursor (17-OH-P) and its peripheral metabolite (E2), respectively, and the normal relation between estrone and 17-OH-P were not evident in either BPH or PCA group. The normal direct relation between testosterone and DHT has been found in both patient groups.     

Inhibition by various steroids on dihydrotestosterone binding to androphilic protein in human benign prostatic hypertrophy.

The effect of various steroidal compounds on the binding of the androphilic protein to dihydrotestosterone in the cytosol of tissues of human benign prostatic hypertrophy was examined. Androgens, as well as estrogens and gestagens showed an inhibitory effect on the binding. Non-steroidal anti-androgens were revealed to be weak inhibitors on the binding. Two androphilic proteins were observed in Sephadex G-200 chromatography in fractions eluted at the void volume and in fractions appearing at the site of IgG, and the rate of inhibition on the binding of both fractions by various steroids was compared. The rate of inhibition by various compounds was generally higher in the IgG fraction than in the void volume fraction. When the ligand and inhibitors were incubated with the cytosol prior to fractionation by Sephadex chromatography, rate of inhibition was lower than that obtained when the ligand and inhibitors were reacted with fractions after chromatography. Implications of the difference observed in these two experiments are not clear at this moment. The results obtained by the protamine precipitation experiment were almost the same as those by the experiment using the extract of the unfractionated acetone-dried cytosol, therefore, the protamine procedure does not seem to precipitate the tissue specific androphilic protein specifically.     

Estradiol 17 beta-dehydrogenase and estradiol binding in human mammary tumors.

The metabolism of estradiol was studied in 31 human breast carcinoma in vitro. All 16 estrogen-receptor-poor tumors transformed estradiol to estrone with percent conversions ranging from 11.4 to 95 except for one poorly differentiated tumor where 0.5% conversion to estrone was observed. On the contrary, only 3 out of 15 estrogen-receptor-rich tumors showed higher than 10% conversion of estradiol to estrone (p = 0.001). There is indication that the enzymatic activity in receptor-poor tumors steadily decreases in premenopausal patients as they approach menopausal age, whereas, the activity steadily increases in post-menopausal patients as the duration of menopause lengthens.     

Properties of androphilic proteins in cytosols of human benign prostatic hypertrophy.

Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of 10(9) M-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the 3H-dihydrotestosterone-IgG fraction complex, while binding of 3H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.     

Gentamicin level in the prostate and its pharmacokinetics in patients with benign prostatic hypertrophy]

The study involved 15 male patients with periurethral prostatic adenoma without complete anuresis. The patients were given 80 mg of gentamicin intramuscularly one day before surgery and 80 mg in a one-hour infusion immediately before an operation. Gentamicin blood concentrations were measured. Pharmacokinetic parameters were calculated and dosage schemes for each patient basing on the antibiotic blood levels. Gentamicin levels in removed adenomas were also determined. Adenomas weighed between 18.0 and 45.8 grams while gentamicin concentration ranged from 1.31 to 3.8 micrograms/mL. It was found that gentamicin concentration in adenomas depend upon their weight. Moreover, pharmacokinetic parameters of this antibiotic exert negligible effect on its levels in adenoma.     

Binding characteristics of naftopidil and alpha 1-adrenoceptor antagonists to prostatic alpha-adrenoceptors in benign prostatic hypertrophy.

Binding properties of naftopidil and alpha 1-adrenoceptor antagonists to alpha-adrenoceptors in prostates from benign prostatic hypertrophy (BPH) were characterized by radioreceptor assays using [3H]prazosin and [3H]rauwolscine. Specific binding of [3H]prazosin and [3H]rauwolscine in human prostatic membranes was saturable and of high affinity, and it showed a pharmacological specificity which characterized alpha 1 and alpha 2-adrenoceptors, respectively. Naftopidil and several alpha 1 antagonists competed for prostatic [3H]prazosin binding in order: R-(-)-YM-12617 greater than prazosin greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil, and the inhibitory effect (Ki = 11.6 nM) of naftopidil was 10 to 45 times less potent than quinazoline derivatives such as prazosin, bunazosin and terazosin. The potencies of these antagonists in competing for [3H]prazosin binding sites in human prostates correlated well with their pharmacological potencies (pA2). Scatchard analysis indicated that the decrease of prostatic [3H]prazosin binding by naftopidil was due to a marked increase in the Kd value without a change in the Bmax value. The inhibition of prostatic [3H]prazosin binding by naftopidil was reversible. Naftopidil also inhibited prostatic [3H]rauwolscine binding (Ki = 70.0 nM). Thus, it is suggested that naftopidil antagonizes alpha 1-adrenoceptors in human prostates in a competitive and reversible manner.     

Steroid hormone receptors in human malignancy.

Steroid hormones induce responses in target tissues by a mechanism involving the specific initial interaction of hormone with cytoplasmic receptor molecules. These receptors, usually localized in target tissues have high binding affinities and limited binding specificities for biologically active steroids. Examination of human leukemic lymphoblasts has revealed these receptors in some tumor samples. Their presence is well correlated with hormone responsiveness of the tumor $\text{in vitro}$. Similar studies on human breast cancer tumor homogenates has indicated that about $\text{2}\text{3}$ of primary tumors contain estrogen receptor. The absence of receptor predicts a lack of response to hormone therapy almost invariably, while the presence of receptor increases but does not assure that the tumor will be hormone responsive. Recently $\text{in vitro}$ tissue culture systems which mimic the hormone responses observed $\text{in vivo}$ have been developed which should significantly aid in the clarification of the mechanisms whereby steroid hormones stimulate and inhibit growth in target tissues.     

Steroid hormone binding receptors in the rat kidney

Summary The histochemical localization of ⁵-3-HSDH in individual follicles isolated from the adult mouse ovary and in ovulated cumulus cell-oocyte masses recovered from the oviduct was examined using a new embedding technique. The procedure employed involves the histochemical staining of such tissue for ⁵-3-HSDH with subsequent embedding in GMA (glycol methacrylate). This method not only permits the acquisition of sections as thin as 3 m but also preserves the histological detail of the tissue allowing for the specific cellular localization of the enzyme. Results obtained from this technique far surpass those obtained from frozen material. Virgin female mice were injected with PMSG and sacrificed either 10 or 17 h later in order to acquire preovulatory or ovulated oocyte-cumulus cell masses, respectively. The sites of localization of ⁵-3-HSDH corresponded to sites demonstrated by histochemical studies on frozen tissue sections; however, the present study revealed that not all cells of a specific type within the same follicle reacted with the same intensity. Granulosa cells lining the walls of vesicular follicles displayed different degrees of enzyme activity based on their distance from the basement membrane. Intrafollicular transformed cumulus masses and cumulus cells of ovulated masses within the oviduct did not react uniformly in that some were positive for the enzyme and others were not. Such results indicate that not all cells of a given type in the ovary possess similar ⁵-3-HSDH activity at a particular time. Thus, the cells comprising a specific cellular component of the ovary should be treated as individual entities and not as a homogeneous group with respect to their metabolic activities.This project was supported by Grant 1 RO1 OH 00835-01 awarded by the National Institute for Occupational Safety and Health and by a grant from the Edward G. Schlieder Foundation awarded to W.J.S. and NIH Grant 5 RO1 HD08041-03 awarded to A.W.S.     

Serum of patients with prostatic cancer or benign prostatic hypertrophy contains nonpolar testosterone.

We have previously described a nonpolar form of radioimmunoassayable serum testosterone (NPT) not measured by available antitestosterone antibodies. It is detected by mild alkaline hydrolysis of the petroleum ether extract of serum and subsequent radioimmunoassay. The properties of NPT are consistent with that of a fatty acid ester of testosterone or dihydrotestosterone. The serum of young males contains 1 to 3 ng/ml NPT, but it is not detected in female serum. We measured serum testosterone and NPT levels in 36 men between 58 and 87 years of age. Seventeen subjects with advanced prostatic cancer (NPT 1.70 +/- 1.44 ng/ml) were compared with a control group consisting of six patients with benign prostatic hypertrophy (BPH) and 13 patients with no prostatic disease (NPT 0.72 +/- 0.46, P = 0.017). There was no significant difference between BPH patients and patients with no prostatic disease; the results were pooled. The concentration of NPT in prostatic cancer patients but not in controls was inversely correlated with that of testosterone. Immunoassayable testosterone was present in the serum of two orchiectomized patients and, therefore, cannot derive solely from the testes.     

Clinical experience with alpha-adrenoceptor antagonists in benign prostatic hypertrophy

The symptomatic treatment of benign prostatic hyperplasia with alpha-adrenoceptor antagonists is based on the concept of a dynamic component in prostatic obstruction, dependent on the sympathetically controlled tone of smooth muscle in the prostate and its capsule. Blockade of the alpha adrenoceptors minimizes this tone, hence the degree of obstruction, and also relieves the irritative symptoms. A review of the published clinical experience reveals convincing evidence of a beneficial action on the irritative symptoms of diurnal and nocturnal frequency, and on the objective parameters of uroflow, residual urine, urethral closure pressure, and cystometrographic evidence of detrusor instability. It is also of value in the prophylaxis or treatment of certain cases of acute retention in these patients. The greatest amount of experience has been with phenoxybenzamine, which appears to be the most effective drug in practice, but other drugs are being assessed for this purpose. It is possible that a combined alpha 1 and alpha 2 antagonist may be more effective than a pure alpha 1 antagonist.     

Steroid receptors in cns: Estradiol(ER) and Progesterone(PR) receptors in human spinal cord tumors

To investigate the role of steroid hormones in the occurrence and growth of human spinal cord tumors and to study the biochemical basis of the sex related difference in the incidence of the various spinal oncotypes, ER and PR have been measured in 24 human spinal cord tumors (12 neurinomas, 6 meningiomas, 5 ependymomas and 1 astrocytoma) by means of sodium thiocyanate exchange assay at low temperature and with dextran-coated charcoal method and R5020 for PR. Cytoplasmic and nuclear receptors have been evaluated. Neurinomas are the oncotypes which contained both ER (usually in the nuclear fraction) and PR: estradiol binding has been found only in one meningioma; ependymomas contained cytoplasmic ER and the astrocytoma had nuclear ER and cytoplasmic PR. The dissociation constant is in favour of a high affinity binding (Kind = 0.15 × 10⁻⁹ M) for ER_c; K_d = 3.85 × 10⁻⁹ M for ERn; K_d = 8.07 × 10⁻⁹ M for PRc). The overall incidence of steroid receptors in the spinal oncotypes studied is higher in male than in female patients (63.64 vs 46.15% for ER and 100 vs 71.43% for PR). Further studies on a greater number of patients will help to define the correlation between different spinal oncotypes and steroid receptors as well as the possibility of an endocrine therapy as adjuvant treatment for selected patients.     

Polymorphisms of MLH1 in benign prostatic hyperplasia and sporadic prostate cancer

Mismatch repair is one of several DNA repair pathways of which defects may lead to cancer. We hypothesize that polymorphisms of the MLH1 gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. The genetic distribution of MLH1 polymorphisms that lead to amino acid changes at codons 132, 219, 384, and 723 were analyzed in BPH and sporadic prostate cancer patients, and compared to healthy controls from an Asian population. These experiments demonstrate a protective role for the codon 384 variant allele against prostate cancer (P = 0.031) but not BPH when compared to normal controls and furthermore, an inverse association was observed with stage (P = 0.074) and grade (P = 0.056) of cancer. This is the first report that demonstrates a protective effect for the race-related MLH1 polymorphism at codon 384 against prostate cancer and these results are important in understanding their role in this disease.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.