Characterization of the retinol and retinoic acid binding proteins in the human prostate.

A retinol-binding protein has been detected in the cytosol of human prostates with benign hyperplasia. The binding was of high affinity and specific for retinol (Kd = 35 nM), with other retinoids such as trans-retinoic acid, retinal, and the synthetic analogues, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona tetraenoic acid and p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1-propenyl] benzoic acid, showing little or no competition. The retinol binding, which sedimented as a 2S component on sucrose density gradients, was also unaffected by the addition of excess unlabeled steroid hormones. Furthermore, pretreatment of the cytosol proteins with heat and/or trypsin totally abolished the retinol binding. Parallel experiments with trans-retinoic acid suggest that the hyperplastic prostate possesses a second retinoid-binding site which is specific for retinoic acid and distinct from the retinol-binding component. Experiments with serum from patients with benign prostate hyperplasia revealed no binding at the 2S sedimentation position; this suggests that the retinoid-binding proteins were exclusively associated with prostatic tissue and were not therefore derived from serum.

Evidence that human prostatic 5 alpha-reductase is located exclusively in the nucleus.

The 5 alpha-reductase activities in human prostatic nuclei and microsomes were compared. The activities in both subcellular fractions were identical with respect to pH dependence, heat inactivation and Michaelis constants for NADPH and testosterone. Subcellular distribution studies using DNA and nicotinamide mononucleotide adenyl transferase as nuclear markers showed that the amount of 5 alpha-reductase present in the microsomes was directly proportional to the amount of nuclear contamination. These results indicate that human prostatic tissue contains only one form of 5 alpha-reductase, which is located exclusively in the nucleus. This finding has important implications for the mechanism of steroid action in the prostate.

Cytokine modulation of epidermal growth factor receptor expression on bladder cancer cells is not a major contributor to the antitumour activity of cytokines.

Epidermal growth factor is a potential mitogen for many different human tumours. Its effect is mediated via a bispecific receptor (EGFR), the expression of which correlates well with invasive disease. We investigated the modulation of EGFR by cytokines produced following bacillus Calmette Guerin (BCG)-immunotherapy. Our data demonstrate the IFN gamma, TNF alpha and IL-1 alpha can decrease the expression of EGFR on some bladder tumour cell lines. IFN gamma reduced EGFR expression on two of eight cell lines (RT4, SD). However, IL-1 and TNF did not share this activity. When cells were treated with a combination of all three cytokines, EGFR was decreased on three cell lines (RT4, RT112, SD) and furthermore, the change in the receptor expression was even more marked. Treatment with phorbol ester (thereby activating protein kinase C) resulted in rapid disappearance of the receptor from the cell surface. Interestingly, the decrease of EGFR expression did not require protein synthesis. Although the cytokines studied could down modulate EGFR, this only occurred on three out of eight cell lines; therefore, it is unlikely that the suppression of proliferative activity caused by cytokine-induced decrease of EGFR expression is central to the antitumour action of BCG therapy, but in a proportion of tumours this mechanism may be involved.

Levels of circulating intercellular adhesion molecule-1 in patients with metastatic cancer of the prostate and benign prostatic hyperplasia.

Current reports suggest a role for intercellular adhesion molecule-1 (ICAM-1) in the progression of malignancy. The availability of a new antibody makes it possible to measure circulating ICAM-1 (cICAM-1) in human body fluids including serum; this might help in monitoring tumour burden and in providing additional prognostic information. In this study, serum levels of cICAM-1 were measured by an ELISA assay in patients with benign prostatic hyperplasia (BPH; n = 20) and metastatic cancer of the prostate (CaP; n = 25). Serum ICAM-1 concentrations were also measured in a group of healthy men (n = 8). The mean +/- S.E.M. cICAM-1 level for BPH was 339.52 +/- 15.30 ng/ml compared with 263.55 +/- 18.54 ng/ml for CaP. Even though the difference between the two groups was significant (P < 0.005), there was a marked overlap between the individual values in both groups, thus minimising the prognostic value of these measurements in prostate cancer. Endocrine therapy had no notable effect on the serum levels of cICAM-1. The mean +/- S.E.M. cICAM-1 concentrations in serum from a younger group of healthy volunteers was 204.1 +/- 10.38 ng/ml, and this value was significantly lower than that measured in serum from either BPH or CaP. We also undertook some immunohistochemical studies to examine the distribution of ICAM-1 in prostate tissue. We observed focal epithelial cell membrane staining which was exceedingly patchy in both the BPH and cancer specimens. On the basis of these studies, we suggest that cICAM-1 levels do not provide additional information on patients with metastatic CaP.

A case for synchronous reduction of testicular androgen, adrenal androgen and prolactin for the treatment of advanced carcinoma of the prostate.

The present study was undertaken mainly to investigate whether prolactin manipulation combined with maximal androgen blockage improves the effectiveness of treatment in advanced prostatic cancer. The efficacy of oral hydrocortisone as an alternative to commercial anti-androgens in reducing the adrenal androgens, and of bromocriptine in reducing the prolactin level were also examined. A consecutive series of 30 patients with untreated and advanced prostatic cancer were entered into a three-arm prospective randomised trial. 10 patients received subcapsular orchiectomy alone (arm 1), another 10 had subcapsular orchiectomy plus flutamide (arm 2), and the remaining 10 had subcapsular orchiectomy plus oral hydrocortisone and bromocriptine (arm 3). Clinical and biochemical parameters, including trans-rectal ultrasound-determined prostatic volumes, hormonal profiles and radionuclide bone scan were evaluated at regular intervals. At 12 months, serum testosterone was reduced by more than 90% in all arms, however, maximum suppression of androstenedione, prolactin, and reduction of prostatic volumes were only observed in arm 3; this was reflected by the significant improvement in clinical response in arm 3 compared with other arms. This study suggests that a combined maximal suppression of androgens and prolactin offers a significant improvement in response over conventional treatments without prolactin suppression in the treatment of advanced prostatic cancer. Importantly, a better clinical outcome in arm 3 was still apparent at the end of 36 months.

The natural history of renal carcinoma.

In summary, some evidence indicating an increase in incidence of renal parenchymal malignancy has been presented. The nature of renal adenoma and its relationship to renal carcinoma remain uncertain, but it is possible that improved computerized tomography will allow in vivo identification of these lesions and initiate a long-term study to provide some clear data on their natural history. The heterogeneity of clinical presentation of this disease has been reviewed and the paraneoplastic syndromes and their importance summarized. Careful clinical and postmortem studies of disease spread, especially lymphatic spread, have been shown to provide useful information to the debate on the role of lymphadenectomy. Many of the unusual aspects of the natural history have been interpreted in terms of the hosts immune response and some data on the complexity and specificity of the host tumor interaction presented. In conclusion, an understanding of the natural history of renal carcinoma forms an important background on which to base clinical management and identifies areas worthy of further investigation in this curious tumor.

Characterization of the prolactin receptor in human prostate.

The uptake of 125I-labelled human prolactin by subcellular fractions obtained from the human hyperplastic prostate was investigated and the specific binding sites were characterized. The binding was both time- and temperature-dependent with maximum specific uptake achieved after incubation for 20 h at 4 degrees C (dissociation constant = 2.4 x 10(-10) mol/l). The present studies also indicate that the bulk of the specific prolactin binding was confined to the 105 000 and 15 000 g membrane fractions whilst the cytosol and nuclear pellet exhibited a lower capacity for the peptide hormone. Attempts to optimize the binding revealed that pretreatment of the subcellular fractions with 4 mol MgCl2/l quadrupled the binding sites; a similar effect was produced after pretreatment with dextran-coated charcoal, but the changes were less pronounced. Furthermore, our studies suggest that the binding was specific for the human prolactin, with other hormones such as ovine prolactin, human LH, human FSH and human GH showing little or no competition. The addition of magnesium and copper ions to the incubation medium also markedly increased the specific binding, whereas calcium, manganese and EDTA inhibited the prolactin uptake. Freezing and storage of tissue at -70 degrees C did not greatly affect the binding sites. The results suggest that we are clearly dealing with a specific prolactin-binding protein.

Interaction between prolactin and zinc in the human prostate gland.

The interaction between prolactin and zinc was examined in vitro in the human prostate gland. The results indicated that prolactin did not modulate the acute uptake of zinc into benign prostatic hypertrophy tissue whereas zinc, in contrast, increased the uptake of prolactin into the prostate gland. Our study further showed that the augmented uptake of prolactin by zinc was partly due to an increase in the non-specific binding properties of the peptide hormone. We were also able to demonstrate that the specific binding of 125I-labelled human prolactin to the receptor was reduced in the presence of zinc by a competitive mechanism.

5alpha-dihydrotestosterone stimulation of human prostate in organ culture.

Explants of human benign hypertrophied prostate were cultured for 8 days. The effect of 5alpha-dihydrotestosterone on the morphology, DNA and glucose utilization of 14 cases was investigated. In four cases proliferation was clearly more extensive in the dihydrotestosterone-treated explants than in the controls. In six explants, there were no morphological differences between those cultured with or without dihydrotestosterone and in half of these, proliferation was present. Four explants were either necrotic or did not contain epithelial cells. The mean DNA content of the treated explants was higher than that of the controls (P smaller than 0.01). There was no significant difference in the rate of glucose utilization by the explants treated with dihydrotestosterone, and by the controls.

Nuclear retinoic acid binding protein in human prostate adenomas.

A retinoic acid binding protein has been detected in salt extracts of nuclei obtained from human prostate adenoma. The binding was characterized by competition experiments, temperature/time studies and saturation analysis. Substantial binding was only observed after sonication of nuclei and charcoal-pretreatment of a salt extract. The binding of radiolabelled all-trans-retinoic acid was displaced by all-trans-retinoic acid, retinol and to a lesser extent retinal and two synthetic retinoids, RO 10-1670 and RO 13-7410. Testosterone and dihydrotestosterone, at a 100-fold excess, had little effect on the binding. The association between retinoic acid and nuclear protein was both temperature and time dependent. At 37 degrees C, equilibrium was rapidly reached (30 min) whereas at 4 and 25 degrees C, ligand binding occurred at a slower rate. Saturation analysis performed under steady-state conditions yielded a dissociation constant of 15 +/- 2 nmol/l. Metabolism studies failed to show conversion of either radiolabelled all-trans-retinol or [3H]retinoic acid in vitro; these data suggest that both acid and alcohol forms of vitamin A are recognized by the extracted nuclear protein. The effect of three enzyme inhibitors on [3H]retinoic acid binding was studied. Binding was unaltered in the presence of aprotinin and phenylmethylsulphonyl fluoride but sodium molybdate (10 mmol/l) increased binding by 18%. The presence of a specific retinoid binding protein in prostate nuclei suggests that retinoids may play some role in the function of the gland.

Androgen metabolism in the epithelial and stromal components of the human hyperplastic prostate.

The reduced and oxidized metabolites of testosterone and dihydrotestosterone were measured in the stromal and epithelial components of 23 human hyperplastic prostates. Our studies indicate differences in the hormonal metabolic patterns of the stroma and epithelium of the resected specimens when compared with tissues obtained retropubically. Testosterone 5 alpha-reductase was evenly distributed between the two components of the specimens obtained retropubically whereas the 3 alpha (beta)-hydroxysteroid dehydrogenase was predominantly located in the stroma. The measurements on the resected specimens suggest, on the other hand, that the bulk of the 5 alpha reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase activities were confined to the stroma although these activities were considerably lower than those measured in the corresponding components of the retropublically obtained specimens. The conversion of testosterone to androstenedione was negligible in all the samples analysed. We therefore conclude that the stroma is the main site for the transformation of dihydrotestosterone to the androstanediol epimers and that the asymmetric distribution of the 3 alpha (beta)-hydroxysteroid dehydrogenase may be instrumental in the development of hyperplasia in the prostate gland. Furthermore, the results of this study indicate that electroresection impairs the enzymatic activities of the tissue.

Localization of epidermal growth factor receptors in the human prostate by biochemical and immunocytochemical methods.

The receptor for epidermal growth factor (EGF) was characterized in the particulate fraction from human benign prostatic hyperplasia (BPH) and was present in 85% of tissues analysed. The uptake of 125I-labelled EGF by BPH was dependent on both time and temperature, with maximum specific uptake achieved after incubation for 90 min at 37 degrees C. Binding characteristics revealed two classes of binding sites of higher (mean dissociation constant (Kd) +/- S.D. = 0.8 +/- 0.2 nmol/l) and lower (Kd = 7.6 +/- 2.8 nmol/l) affinities. Competition studies demonstrated the specificity of the receptor assay since the binding of labelled EGF was abolished with excess unlabelled EGF but not with excess unlabelled human GH, human insulin, venom nerve growth factor, human FSH, human LH and human prolactin. There was a complex biphasic relationship between specific binding and protein concentration in the range 0.1-8 mg/ml. Subcellular fractionation of BPH homogenates demonstrated that the bulk of the specific binding was confined to the 800 g (crude heavy pellet) and 15,000 g (mitochondrial pellet) fractions. The 105,000 g (microsomal pellet) and the 105,000 g (cytosol fraction) exhibited low and variable binding capacities for the growth factor. The presence of EGF receptor was also confirmed by immunocytochemical staining of frozen sections from BPH using monoclonal antibody specific for EGF receptors. A positive correlation between 125I-labelled EGF binding and the intensity of staining was found. The presence of a specific EGF-binding receptor protein in human BPH tissues suggests that EGF may play a role in the pathogenesis of human BPH.

Identification of an androgen receptor in the cytosol of the female Mastomys prostate.

An in vivo and in vitro study was carried out on the prostate from the female Praomys (Mastomys) natalensis to identify and characterize the binding of androgens within the cytoplasm. The labelled cytosol was prepared and subjected to gel exclusion chromatography and density gradient centrifugation. A macromolecular protein associated with the radioactivity was isolated on Sephadex G-200. Subsequent analysis of the steroid receptor complex showed that the major part of the radioactive steroid (64 percent) was dihydrotestosterone. This binding was inhibited by unlabelled testosterone and could not be demonstrated in liver cytosol. Characterization of this dihydrotestosterone receptor complex revealed a sedimentation coefficient of 4.6 s in the presence of a high salt solution (0.4 M KCl). The complex aggregated in the absence of 0.4 M KCl and sedimented preferentially from 5.6-7.4 s together with polydisperse aggregates of higher sedimentation coefficients. The use of this animal as an experimental model for hormonal studies on the prostate is suggested.

Clinical evaluation of the urinary sediment after renal allotransplantation.

The detection of lymphoid cells by routine examination of the urine after renal allotransplantation has proved to be a useful early indication of rejection. In a study of 36 rejection episodes, 20 (56%) were associated with a significant number of lymphocytes in the urine. The incidence was much higher when rejection occurred during the first month after operation (76%); lymphocytes were rarely found when rejection occurred after three months. The appearance of lymphocytes in the urine was of particular value for detecting rejection in patients with prolonged oliguria after transplantation.

Induction of ICAM 1 expression on bladder tumours by BCG immunotherapy.

AIMS: To determine the expression of intercellular adhesion molecule 1 and 2 (ICAM 1 and 2) in transitional cell carcinoma cells before and after immunotherapy with Calmette-Guérin bacillus (BCG). METHODS: Frozen sections from 22 untreated bladder carcinomas were immunohistochemically examined with monoclonal antibodies to ICAM 1 and 2. Urinary cytospin slides were made for six patients for each of the six clinical instillations which constitute a therapeutic course. These slides were also stained for ICAM 1 and for leucocyte function associated antigen 1 (LFA 1). RESULTS: Bladder cancer cells did not essentially express either ICAM 1 or 2, but cells in the stromal areas surrounding tumour expressed both these antigens. After repeated instillations of BCG organisms ICAM 1 positive normal and neoplastic epithelial cells were observed in the urine. Cells obtained from the first three instillations expressed lower densities of ICAM 1 than those from the later instillations. Many neutrophils expressing LFA-1 and some lymphocytes were also noted in the cytospin slides and some of these were conjugated to tumour cells expressing ICAM 1. Six months after treatment a single maintenance dose of BCG induced ICAM 1 expression. CONCLUSION: Untreated superficial bladder carcinoma cells do not express ICAM 1 or 2, but these important immunological molecules were expressed in the stromal areas of tissue. Importantly, neoplastic cells in the urine expressed ICAM 1 after immunotherapy. This molecule can render bladder tumour cells vulnerable to non-antigen specific cytotoxicity mediated by activated lymphocytes.

Identifying leucocytes and leucocyte subpopulations in semen using monoclonal antibody probes.

An indirect immunoperoxidase technique employing specific monoclonal antibodies has been used to identify leucocyte subpopulations in cytocentrifuge smears of washed human ejaculate. Cells reacting with the pan antihuman leucocyte monoclonal antibody (HLe-1) were demonstrated in 63/67 specimens from subfertile patients with a mean count of 14.5 +/- 17.1 leucocytes per HPF (X 320). Cells with similar reactivity were observed in all specimens examined from 10 fertile men with a mean count of 41.6 +/- 26.3 leucocytes per HPF (X 320). Leu-T4+ cells (T-lymphocytes) were demonstrated in only 13/63 of the subfertile group with a mean count of 4.46 +/- 3.3 T-lymphocytes per HPF (X 320). Studies with the anti-leu 2a antibody revealed that these leu-4+ cells were mainly of the suppressor/cytotoxic phenotype. In contrast, no leu-4+ cells were detected in the control group. No leu-12+ cells (B-lymphocytes) were detected in any of the 80 specimens examined.

A radiofrequency method of thermal tissue ablation for benign prostatic hyperplasia.

OBJECTIVES: To evaluate the safety, feasibility, and efficacy of transurethral ablative prostatectomy (TURAPY), a radiofrequency method of thermal tissue ablation of benign prostatic hyperplasia (BPH). METHODS: Twenty men, ages 55 to 81 years (mean, 67), with symptomatic BPH and with peak flow rate 12 mL/s or less, were treated with the TURAPY device (Direx Medical Systems). A 2 cm long heating element and 2 thermoprobes (for simultaneous prostatic temperature monitoring) are mounted on a Foley-like catheter and used for TURAPY treatment administration. This TURAPY catheter was modified by placing an extra set of thermoprobes in the sphincter region to allow sphincteric temperature monitoring. The treatment was administered at a maximum temperature of 70 degrees to 75 degrees C for 1 hour under local anesthesia as a day case. RESULTS: All 20 patients completed the treatment. The maximum recorded rectal temperature was 39.5 degrees C. The maximum sphincter temperature was not allowed to exceed 42 degrees C. The post-treatment morbidity included dysuria and minor urethral bleeding in 12 patients. Three patients developed urinary infection requiring antibiotic treatment. All 20 patients were followed up 3 months after treatment. The mean International Prostate Symptom Score (IPSS) improved from 19.4 to 6.2 (68%), the mean peak flow rate increased from 7.9 to 12.9 mL/s (63%), and the mean postvoid residue decreased from 222 to 81 mL (64%). Overall, 80% of patients exhibited at least a 50% improvement in either the IPSS or the peak flow rate. There was a mean reduction in prostatic volume measured by transrectal ultrasound of 14 mL (29% reduction). The subjective and objective improvements, in 8 patients followed up 6 months after treatment, have been maintained. There was extensive coagulative heat necrosis of periurethral tissue with sparing of subcapsular tissue in prostate biopsy specimens taken from 1 patient 5 days after treatment. There was endoscopic and sonographic evidence of canalization of the obstructed prostatic urethra 3 months after treatment. CONCLUSIONS: Treatment with the TURAPY device was found to be safe, feasible, and effective in improving both subjective and objective measurements of benign prostatic obstruction in this pilot study on 20 patients.

Immunocompetent cells in human testis in health and disease.

The authors have investigated lymphocyte subpopulations and macrophages in normal human testes and the testes of patients under investigation and treatment for subfertility. Specific monoclonal antibodies were used in an indirect immunoperoxidase technique. In normal tissues, T lymphocytes (Leu 4-positive cells) were present in the rete testis with a preponderance of cells of the suppressor/cytotoxic phenotype. In contrast, no lymphocytes were detected within the peripheral portions of the testis. Cells reacting with the anti-Leu M3 monoclonal antibody, which defines monocytes/macrophages, were detected in appreciable numbers in peripheral testis with a specific location around the seminiferous tubules. HLA-DR-positive cells (human leukocyte antigens--class II [DR] determinants of the major histocompatibility complex) also were identified and showed a similar pattern of distribution to that of the Leu-M3 positive cells. While no lymphocytes were seen in the normal peripheral testis, T lymphocytes were detected in testicular biopsies from subfertile patients. Suppressor/cytotoxic T cells (Leu 2a-positive) predominated in patients with oligozoospermia and obstructive azoospermia while T cells of the helper/inducer phenotype predominated in patients with unilateral testicular obstruction and in postvasectomy patients. Sperm antibody measurements correlated with these findings.

Follow-up of prostate carcinoma with serial bone scanning using cyclotron-produced 18-fluorine.

Seventy-four patients with carcinoma of the prostate were studied annually by combined radiological and 18F scintigraphy over a 5-year period. Of the patients who have no radiological evidence of bone metastases, 25% have a positive 18F bone scan. Follow-up of these patients has shown that scan abnormalities perceded x-ray changes from between 1 to 4 years. False negative scans were not seen with 18F which allows for greater accuracy in the detection of skeletal metastases. Teh accurate staging of carcinoma of the prostate cannot be made without bone scanning. Preliminary results with 111In bleomycin as an adjunct to 18F have shown this to be a useful radio-pharmaceutical to distinguish metastases from benign lesions, and further studies are warranted.

Intracellular localisation of 5alpha-dihydrotestosterone in human benign prostatic hypertrophy.

When slices of benign hypertrophied human prostate and abdominal muscle were incubated with either [3H]testosterone or 5alpha-dihydro[3H]testosterone, the uptake of radioactivity by prostatic tissue was significantly higher than that of the muscle (P less than 0.01). The uptake of labelled androgen by prostatic tissue could be significantly reduced by adding the unlabelled steroid to the incubation medium. After the incubation of prostatic tissue with 5alpha-dihydro[3H]testosterone, the amount of the radioactivity taken up by the whole homogenate and the nuclear preparation of the prostatic tissue were measured. DNA content of the nuclei and the whole homogenate was also estimated. The mean+/-S.E.M. of 5alpha-dihydrotestosterone associated with the nuclei was 65+/-4.4%, ranging from (52.2-79.8%). The activity of acid phosphatase was measured in 30 samples of prostatic tissue. The mean +/- S.E.M. was 20.7+/-1.5 U/g tissue (9.8+/-0.9 U/mg DNA). The correlation between the activity of this enzyme and the uptake of androgen by prostatic tissue is evaluated.