Diverse secretory patterns of clusterin by epididymis and prostate/seminal vesicles undergoing cell regression after orchiectomy.

Nucleotide sequence analysis of the complimentary DNAs (cDNA) and N-terminal amino acid sequence analysis have shown that clusterin is equivalent to sulfated glycoprotein-2 (SGP-2), testosterone-repressed prostate protein-2 (TRPP-2), and androgen-repressed protein (ARP) in the rat, as well as serum/seminal plasma protein, SP-40,40, in the human. In view of its widespread presence in various species, a specific RIA was established to quantify the tissue distribution of this protein. Rat clusterin is present in almost all organ tissues examined, including testis, epididymis, serum, liver, prostate, seminal vesicles, and uterus. Displacement curves generated using cytosols prepared from these organs were parallel to those obtained using purified rat clusterin and crude Sertoli cell-enriched culture medium. Immunoreactive clusterin was also visualized in these organ extracts by immunoblots. Studies on the tissue distribution of immunoreactive clusterin using RIA revealed that the concentration of clusterin in the epididymis of adult rats was 6- and 10-fold higher than that in the serum and testis, respectively and is 50- to 100-fold higher in the liver, spleen, kidney, brain, ventral prostate, seminal vesicles, and uterus. A study of the distribution of clusterin in various compartments of the epididymis indicated its concentration in the caput epididymis was almost 3-fold higher than that in the corpus and cauda epididymis. After orchiectomy, the concentrations of clusterin in the ventral prostate and seminal vesicles increased as much as 100- and 10-fold and peaked at day 4 after surgery, respectively; daily injection of dihydrotestosterone (DHT) beginning at day 3 after orchiectomy reduced the concentrations of clusterin and restored them to a normal level. A different pattern was noted in the epididymis after orchiectomy; the concentration of clusterin in the caput epididymis decreased with time; however, daily injection of DHT beginning at day 3 increased the caput epididymal clusterin concentration and restored it to a normal level. The concentration of clusterin was not altered in the corpus or cauda epididymis after castration and/or DHT administration. Also, the serum and liver clusterin levels did not change with time after orchiectomy. These observations suggest that clusterin will be a valuable marker to monitor the diverse effects of androgen withdrawal in the male reproductive tract. We conclude that clusterin may be a multifunctional protein in view of its broad tissue distribution and association with numerous physiological and pathological conditions.

Different carcinogenic responses in a variety of organs, including the prostate, of five different rat strains given 3,2'-dimethyl-4-aminobiphenyl.

Tumorigenic response in the prostate of F344, ACI, Lewis, CD and Wistar rat strains to 3,2'-dimethyl-4-aminobiphenyl (DMAB) was examined in relation to development of other types of tumors. Rats of each strain aged 6 weeks were divided into two groups receiving DMAB s.c. at a dose of 50 mg/kg body wt once every other week for 10 times, with or without 1 week dietary ethynyl estradiol (EE) pretreatment. The experiment was terminated at week 60, carcinomas of the ventral prostate, all of microscopic size, being respectively found in 50, 17, 21, 15 and 0% of F344, ACI, Lewis, CD and Wistar strain animals treated with EE plus DMAB. The tumor yield correlated well with DMAB-DNA adduct formation. One invasive adenocarcinoma also developed in the periurethral part (occupying both of lateral and dorsal areas) of the prostate. The final survival rates were 46, 24, 65, 4 and 0% in F344, ACI, Lewis, CD and Wistar rats respectively. DMAB administration without EE pretreatment resulted in similar incidences of prostate tumors and mortalities. Tumors arose in greater than 14 different sites with strain dependency, lesions predominating in the skin/subcutis of ACI and F344, preputial gland of F344, urinary bladder of ACI, and mammary glands of CD rats respectively. Consideration of mortality and the relative incidence of prostate cancer and other types of tumors indicates the F344 rat strain to be the most appropriate for investigation of DMAB prostate carcinogenesis.

Expression of Zn-alpha 2-glycoprotein and PSP-94 in prostatic adenocarcinoma. An immunohistochemical study of 88 cases.

Zn-Alpha 2-Glycoprotein (Zn-Alpha 2-GP) and prostatic secretory protein of 94 amino-acids (PSP-94) were recently isolated from the human prostate. Their expression in benign and malignant well-differentiated and poorly differentiated components of 88 prostates with prostatic adenocarcinomas, and in 25 metastases, was evaluated using polyclonal antibodies developed against these antigens. Zn-Alpha 2-GP was present in benign hyperplastic glands in 91.1% of cases, but in only 40.7% (poorly differentiated component) to 48.5% (well-differentiated component) of prostatic adenocarcinomas, and in 8% of metastases. The expression of PSP-94 was present in 89.3% of benign hyperplastic glands, but in only 50% (well-differentiated adenocarcinoma component) to 57.3% (poorly differentiated component) of prostatic adenocarcinomas and 28% of metastases. The expression of these proteins by the tumor was unrelated to the initial stage and the tumor grade. Because of their low frequency in prostatic adenocarcinomas, especially in metastases, Zn-Alpha 2-GP and PSP-94 appear to have a limited diagnostic usefulness. Further studies are needed, however, to explore other clinical applications of these two new prostatic secretory proteins.

Autoradiographic localization of [3H]oestradiol in epididymis, seminal vesicles and prostate of the fetal guinea-pig.

The selective uptake and localization of radioactivity in the fetal male reproductive organs (epididymis, seminal vesicles and prostate) of the guinea-pig (50-60 days of gestation) after in-vivo and in-situ subcutaneous injection of [3H]oestradiol was investigated by autoradiography. In 50-day-old fetuses, the different areas of the epididymis showed selective retention of radioactivity in the nuclei of peritubular and stromal cells surrounding the epididymal duct; no retention was observed in the epididymal epithelium. A similar distribution of silver grains was observed in the 60-day-old fetus. Seminal vesicles and prostate sections from both 50- and 60-day-old fetuses showed concentration and retention of radioactivity only in stromal cells, whereas the epithelium did not exhibit silver grains. In all the tissues studied, the nuclear labelling was abolished after injection of [3H]oestradiol plus a 100-fold excess of non-labelled oestradiol. As the mesenchyme surrounding the epithelia of the epididymis, seminal vesicles and prostate were labelled selectively with [3H]oestradiol, it is suggested that during fetal life of the guinea-pig the mesenchymal stroma of these fetal male reproductive organs may be considered as a target tissue for oestrogen.

Immunohistochemical demonstration of carcinogen-DNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl.

An immunohistochemical procedure was applied which allows accurate localization of DNA lesions within organs and tissues of rats given 3,2'-dimethyl-4-aminobiphenyl (DMAB) using polyclonal antibodies against DMAB-DNA adducts. Dose-related nuclear staining was observed in organs regardless of DMAB-carcinogenic organotropism. In the male accessory sex organs, the lateral lobe of the prostate, a non-target site, demonstrated a similar staining intensity to that found for the ventral prostate and seminal vesicle, target sites. Orchiectomy and pretreatment with ethinyl estradiol resulted in a moderate to slight decrease in binding in the accessory sex organs. No observable decrease in staining intensity was evident in most organs 168 h after the administration of DMAB. These findings suggest that DNA adduct formation itself is not necessarily sufficient for tumor induction.

Stage B prostate adenocarcinoma. Flow cytometric nuclear DNA ploidy analysis.

Over a 16-year period (1966 to 1981), 349 patients underwent radical retropubic prostatectomy for pathologic stage B adenocarcinoma of the prostate. Nuclear DNA content was measured by flow cytometry on available archival material of 283 patients. Two hundred sixty-one patients (92%) had high-quality histograms. The ploidy distribution was as follows: DNA diploid, 177 (68%); DNA tetraploid, 74 (28%); and DNA aneuploid, 10 (4%). The average follow-up was 9.4 years. At the time of follow-up, 53 patients (20%) within the study group had developed tumor progression: 22 local, 23 systemic, and 8 both. The ploidy distribution of the population that developed tumor progression was 27 DNA diploid (51%), 16 DNA tetraploid (30%), and 10 DNA aneuploid (19%). This ploidy distribution is significantly different from that found for the nonprogression group with stage B disease. Overall, 31% of patients with DNA nondiploid tumors had tumors that progressed compared with 15% of patients with DNA diploid tumors. All (100%) DNA aneuploid tumors progressed. The DNA ploidy distribution of all pathologic stage B prostate cancers differs significantly from that found in more advanced stages (C and D1) previously reported for the same time interval. However, the ploidy distribution of stage B tumors that progressed closely resembles that of the stage C and D1 tumors. These results further support the working hypothesis that nuclear DNA content has marked prognostic significance for patients with adenocarcinoma of the prostate. It seems to us that analysis of ploidy by flow or static cytometry will become an essential tool for treating patients with localized prostate cancer.

Association of androgen binding sites with the endoplasmic reticulum of rat ventral prostate.

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.

Elevated transferrin receptor content in human prostate cancer cell lines assessed in vitro and in vivo.

Transferrin receptors (TfR) were measured in benign and malignant prostatic cells by performing Scatchard analysis following the administration of 125I-transferrin. Established human prostate cancer cell lines (PC-3 and DU-145) as well as biologically aggressive variants (PC-3 ASC and PC-3 DES) were shown to possess significant levels of high affinity TfR when assessed in vitro. In contrast, TfR content was negligible in cultured stromal cell fractions derived from human benign prostatic hyperplasia (BPH) specimens. Scatchard analysis was also performed on in vivo derived prostatic tissues: tumors resulting from the subcutaneous xenografting of PC-3 ASC cells into athymic, nude mice and fresh BPH surgical specimens. These tissues were dissociated and their stromal and epithelial components separated. TfR were only detected in the epithelial component of both malignant and benign epithelial cells. PC-3 ASC tumor cells exhibited TfR levels comparable to their in vitro expression and these levels were 10-fold greater than in the BPH cells. These findings suggest that elevated TfRs may serve as another useful marker of the transformed phenotype within human prostate tumor systems.

Estrogen and progestin receptors in the reproductive tract of male and female primates.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Evidence of mucin M1 antigens in seminal plasma and normal cells of human prostatic urethra in relation to embryonic development and tumors.

By employing immunoperoxidase methodology, using monoclonal antibodies against the peptide core of gastric mucins (M1 antigens), we demonstrate the presence of M1 mucin-producing cells that are associated with the prostatic urethral epithelium and located mainly in the veru montanum area near the prostatic ductal and utriculus junctions. The significance of these M1 cells is not yet clear. Using an immunoradiometric assay, these M1 mucins were found predominantly in the prostatic fraction obtained from seminal plasma. By chromatography on Sepharose 6B and 2B and cesium chloride gradient centrifugation, we demonstrate that high-molecular-weight components (greater than 10(7) Da) show a density of 1.45 g/ml, similar to mucins, and are immunochemically related to peptidic gastric M1 mucins. The particular location of these M1 antigens in prostatic adult urethra and their fetal expression in cloacal structures suggest that, in males, the prostatic urethral epithelium includes some remnant cells from the enteric cloaca. Finally, the presence of mucin-containing cells in the prostatic urethra could possibly explain the histogenesis of the rare benign villous tumors and primary mucinous adenocarcinomas arising from the prostatic urethral epithelium.

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.

1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.

Immunohistochemical localization of progesterone receptor in benign and malignant human prostate.

A monoclonal antibody to progesterone receptor, KD68, was used to localize this receptor protein in 31 surgical specimens of benign and malignant human prostate. Progesterone receptor was detected almost exclusively in stromal cells. The most striking finding was the periacinar arrangement of stained cells in some specimens of glandular BPH, sometimes associated with close apposition of stained cells to the basement membrane of the acinar epithelium. In both benign and malignant specimens scattered stained cells were observed in some areas of fibromuscular stroma. Except for occasional stained epithelial cells in one benign and one estrogen-treated malignant specimen, both benign and malignant epithelium were negative.

[A pharmacokinetic study of diffusion in adenomatous prostatic tissue of an intravenous bolus of cefamandole]. / Etude pharmacocinétique de la diffusion dans le tissu prostatique adénomateux d'un bolus intra-veineux de céfamandole.

Twenty-three patients undergoing transurethral resection of the prostate for benign prostatic hypertrophy received antibiotic prophylaxis with a second generation cephalosporin, cefamandole, administered by a single IV bolus of 2.5 g. A pharmacokinetic study was performed on blood and resection chips collected at regular intervals. Cefamandole penetrates rapidly into the prostate without any saturation threshold. It diffuses less extensively and persists for a shorter period in elderly subjects, but penetrates to an identical degree regardless of the volume of the adenoma. The prostatic concentration was always higher than the minimal inhibitory concentration for the bacteria generally encountered, except for pseudomonas. The pharmacokinetic study of cefamandole therefore demonstrated that an IV bolus of 2.5 g is perfectly suitable for antibiotic prophylaxis prior to prostatic resection.

Histological localization of estrogen receptors in normal and diseased human prostates by immunocytochemistry.

The role of estrogens and estrogen receptors (ER) in the human prostate remains unresolved. In this study we have used the monoclonal ER antibody H222 to investigate the histological localization of ER in normal and diseased human prostates by immunocytochemistry. Prostate tissue was obtained from 3 young organ donors (Group I-normal prostate), from 14 prostates removed by radical prostatectomy or radical cystoprostatectomy, which had caused no or only mild obstructive symptoms (Group II-non-obstructive prostate), and from 11 prostates removed by suprapubic prostatectomy, which had caused severe obstructive symptoms due to a large benign prostatic hyperplasia (BPH) (Group III-obstructive prostate). In prostates of all groups ER were found to be in nuclei of the prostatic urethra and of the periurethral prostatic duct. In striking contrast, ER in the interglandular prostatic stroma was not as homogeneous among the different groups. We observed a low concentration of ER in the stroma of normal prostates, the highest concentration in non-malignant stroma of non-obstructive prostates, and no ER at all in stroma of obstructive prostates. Based on the immunocytochemical localization of ER in normal and diseased human prostate, our results indicate that stromal growth in obstructive BPH may not be mediated via ER. However, we cannot exclude that an increase of stromal ER concentration (as observed in non-obstructive prostates) is directly involved in induction of BPH, leading further prostate growth thereafter into an estrogen independent state.

Morphological and immunohistochemical investigations of the utriculus prostaticus from the fetal period up to adulthood.

We investigated the utriculus prostaticus from the fetal period up to adulthood in 148 prostates. During the second half of gestation the utriculus had a simple tubular or a cystic form and was lined with metaplastic squamous epithelium which showed immunohistochemical positivity for different keratins, carcinoembryonic antigen, and peanut agglutinin binding sites. After birth, alveolar outgrowths of the utriculus developed. After puberty, the utriculus had become a complicated and variable structure. The epithelium no longer differed from that of the prostate glands either morphologically or immunohistochemically. Within the epithelium numerous endocrine cells were found containing neuron-specific enolase, chromogranin, and serotonin. The utriculus and ejaculatory ducts were embedded in a fibrous stroma with, after birth, numerous plexus-like blood vessels. This fibrous zone was peripherally bordered by a layer of smooth muscle. There was no evidence for a function of the utriculus differing from that of the prostate glands. Since the epithelium of both structures is identical immunohistochemically, the epithelium of the sinus urogenitalis most likely particpates in the lining of the utriculus during embryogenesis.

Species- and organ-specificity of secretory proteins derived from human prostate and seminal vesicles.

Polyclonal antibodies against semenogelin (SG) isolated from human seminal vesicle secretion and acid phosphatase (PAP), beta-microseminoprotein (beta-MSP), and Prostate-Specific Antigen (PSA) derived from human prostatic fluid, as well as a monoclonal antibody against beta-MSP were used for immunocytochemical detection of the respective antigens in different organs from different species. SG immunoreactivity was detected in the epithelium of the pubertal and adult human and in monkey seminal vesicle, ampulla of the vas deferens, and ejaculatory duct. PAP, beta-MSP, and PSA immunoreactivities were detected in the pubertal and adult human prostate and the cranial and caudal monkey prostate. With the exception of a weak PSA immunoreactivity in the proximal portions of the ejaculatory duct, none of the latter antisera reacted with seminal vesicle, ampullary, and ejaculatory duct epithelium. Among the non-primate species studied (dog, bull, rat, guinea pig) only the canine prostatic epithelium displayed a definite immunoreactivity with the PAP antibody and a moderate reaction with the PSA antibody. No immunoreaction was seen in bull and rat seminal vesicle and canine ampulla of the vas deferens with the SG antibody. The same was true for the (ventral) prostate of rat, bull, and dog for beta-MSP. The epithelium of the rat dorsal prostate showed a slight cross-reactivity with the monoclonal antibody against beta-MSP and one polyclonal antibody against PSA. The findings indicate a rather strict species-dependent expression of human seminal proteins which show some similarities in primates, but only marginal relationship to species with different physiology of seminal fluid.

Primary explant culture of human prostate tissue: a model for the study of prostate physiology and pathology.

An explant of human prostate tissue containing viable acini will give rise in vitro to an outgrowth of epithelial cells of presumed basal cell origin. These cells can be passaged by trypsinization, undergo numerous population doublings (up to 50-60), and attempt a normal pattern of differentiation, which may succeed, to varying degrees, depending on conditions of culture. The system can serve as a useful model for analysis of the role of hormones, growth factors, cytoskeletal elements, cell-cell interactions, and of the basement membrane both in normal physiology and in pathology of the prostate. The system also should prove useful in the evaluation of the possible roles of chemical carcinogens, radiation, and viral or cellular oncogenes in carcinogenesis. Moreover, the model system should be useful in the evaluation of chemical, physical, or biological agents for treatment or prevention of prostate cancers.

Serum prostate-specific antigen and prostate pathology in men having simple prostatectomy.

Prostate-specific antigen (PSA) is a sensitive and specific serum marker for monitoring disease activity in men with prostatic carcinoma. Despite reports of elevation of levels of this analyte in men with benign prostatic hyperplasia, no information is available correlating the serum levels with the actual prostatic abnormalities in men having prostatectomy for presumed benign disease. In the present investigation, the authors compared preoperative serum PSA levels with prostate disease in 81 men with bladder outlet obstruction. Five pathologic groups were found: incidental high-grade carcinoma (n = 3), low-grade carcinoma (n = 11), acute inflammation (n = 16) with or without chronic inflammation, Prostatic intraepithelial neoplasia (PIN) (n = 25), and benign hyperplasia (n = 26). Serum PSA levels were significantly elevated in both low- and high-grade carcinoma, acute inflammation, and PIN when compared with the patients with benign hyperplasia with and without chronic inflammation. Within the four groups with elevated levels, use of PSA levels could separate only the high-grade cancer patients who were subsequently shown to have metastatic disease. Only one patient with simple hyperplasia had PSA levels in the abnormal range.

Study of estramustine binding protein (EMBP): purification of rat EMBP and establishment of RIA.

Estramustine binding protein (EMBP) was purified from the ventral prostate of the rat using DEAE-cellulose chromatography, concanavalin-A affinity chromatography and DEAE-sepharose chromatography. At the final step of the purification, two different peaks (Peaks A and B) of A280 nm were obtained. Peak A had a high binding activity to [3H] estramustine. On the other hand, Peak B had a low binding activity. On the analysis of polyacrylamide gel electrophoresis, Peak A gave two protein bands, whereas Peak B gave a single band. The molecular weight of the markedly stained band of Peak A was approximately 27,000, whereas that of Peak B was 18,000, as estimated by analysis of Fargusson's plot. The antibody against Peak B was used for establishing a radioimmunoassay (RIA) of EMBP. The sensitivity of this assay system was sufficient to measure of 1 ng of EMBP. The dilution curve of rat prostatic cytosol was paralleled with the standard curve. As a result obtained from this RIA, the mean concentration of immunoreactive EMBP was 8.01 ng/mg cytosol protein in human benign hyperplastic prostate (BPH) and 4.28 ng/mg protein in human prostatic carcinoma (PC), respectively. These results here obtained indicate that human prostate has an immunoreactive protein to the purified EMBP obtained from the ventral lobe of rat prostate.

Studies on the mechanism of action of Win 49596: a steroidal androgen receptor antagonist.

Win 49596 is an orally active antiandrogen in the rat. This report describes a series of in vitro and in vivo studies which were performed to characterize the mechanism of action of this compound. In vitro competition and Lineweaver-Burk analyses indicate that Win 49596 binds competitively to the rat ventral prostate androgen receptor with a Ki of 2.2 +/- 0.4 microM. Similar to other androgen antagonists, the relative binding affinity (RBA) of Win 49596 was greater after 1 h of incubation with androgen receptor than after an 18 h incubation (RBA of 2.2 versus 0.05, respectively). Win 49596 did not bind to rat cytosolic uterine estrogen or progesterone receptors or thymus glucocorticoid receptors. Furthermore, Win 49596 did not inhibit rat ventral prostate 5 alpha-reductase or 3 alpha-oxidoreductase, rat adrenal 3 beta-hydroxysteroid dehydrogenase or human placental aromatase activity in vitro at concentrations as high as 10 microM. A series of in vivo studies demonstrated that Win 49596 inhibited the uptake of [3H]testosterone as well as testosterone-induced nuclear accumulation of androgen receptor in the rat ventral prostate. Collectively, these results support direct androgen receptor antagonism as the mechanism for the antiandrogenic effects of Win 49596.