Normal Prostate Samples Research Articles

Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up-regulates IGF binding protein-3, and suppresses MMP-2 expression.

The type I insulin-like growth factor receptor (IGF-IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF-IR in benign and malignant prostate epithelium. In this study, endogenous IGF-IR gene expression was reduced in stably transfected PC-3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC-3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF-IR gene expression and either up-regulation of IGF binding protein (BP)-3 or down-regulation of matrix metalloproteinase (MMP)-2 expression in androgen-independent PC-3 cells. Moreover, inhibition of IGF-IR gene expression in transfected PC-3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT-PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF-IR gene expression was up-regulated in nine of 12 prostate cancers, whereas IGFBP-3 gene expression was down-regulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF-IR and IGFBP-3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF-IR or IGFBP-3 gene expression in order to improve understanding of the IGF-IR-activated signalling pathways and as a potential treatment for prostate cancer.

TMEFF2 is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cells.

We have identified a gene that is highly expressed in the androgen-dependent prostate cancer cell line, LNCaP. Sequence analysis revealed that it was identical to a recently cloned gene designated TMEFF2, which encodes a transmembrane protein containing an epidermal growth factor (EGF)-like motif and two follistatin domains. This gene was highly expressed only in primary samples of normal prostate and prostate cancer as well as normal brain. Expression of the gene was controlled by androgen as shown by dihydrotestosterone markedly increasing TMEFF2 expression in LNCaP cells. Also, androgen-dependent human prostate cancer xenografts (CWR22) expressed high levels of TMEFF2 and these levels markedly decreased by day 10 after castration of the mice. Furthermore, a large number of androgen-dependent xenografts (CWR22, LuCaP-35, LAPC-4AD, LAPC-9AD) exhibited higher levels of TMEFF2 mRNA than androgen-independent xenografts (CWR22R, LAPC-3AI, LAPC-4AI, LAPC-9AI). Ectopic expression of TMEFF2 in DU145 and PC3 cells resulted in their prominent inhibition of growth. Taken together, the results demonstrate that TMEFF2 is a androgen-regulated gene, which can suppress growth of prostate cancer cells and our xenograft data show that escape of prostate cancer cells from androgen modulation causes them to decrease their expression of this gene, which may result in their more malignant behavior.

The Role of Epigenetic Modifications in Retinoic Acid Receptor β2 Gene Expression in Human Prostate Cancers

The retinoic acid receptor (RAR) β gene is a putative tumor suppressor gene on chromosome 3p24, where a high incidence of loss of heterozygosity is detected in many types of tumors. Retinoic acid suppresses cancer cell growth through binding to RARs, especially RARβ, indicating a critical role in mediating anticancer effects. Selective loss or down-regulation of RARβ mRNA and protein has been reported in prostate cancers (PCas), although the mechanisms remain unclear. We investigated the role of epigenetic modification in RARβ2 gene silencing. Aberrant methylation was detected in 11 of 14 (79%) primary PCas, 9 of 10 (90%) hormone-refractory PCas, and 2 of 4 (50%) PCa cell lines, but not in any normal prostate samples. Chromatin immunoprecipitation assay showed that all RARβ2-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both histones H3 and H4. After exposure to 5-aza-2prime;-deoxycytidine treatment, Trichostatin A and all-trans retinoic acid induced partial demethylation, increased accumulation of acetylated histones, and markedly restored the expression of RARβ2 in RARβ2-negative cells. These data suggest that the RARβ2 gene may be one of the frequently silenced genes by epigenetic modifications in PCa.

Expression of the developmental and oncogenic PAX2 gene in human prostate cancer.

In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma. PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue. Using reverse transcription-polymerase chain reaction combined with Southern hybridization PAX2 expression was detected in 52% of primary cancers and all 3 cell lines. PAX2 expression in these samples was confirmed at a protein level using immunoblotting and immunofluorescence. PAX2 messenger RNA was not detected in any benign or normal prostatic samples. Immunoblotting of protein from benign prostatic hyperplasia samples confirmed the lack of expression of PAX2 protein. The expression of PAX2 in prostate cancer compared to nonmalignant prostates is statistically significant (Fisher's exact test p = 0.0004). These results suggest a possible role for PAX2 in prostate cancer. Although previous studies have suggested a role for PAX2 for supporting proliferation in undifferentiated cells, no correlation of PAX2 expression with Gleason score was found in prostate cancer.

Osteoprotegerin and rank ligand expression in prostate cancer

Objectives. To investigate the expression of osteoprotegerin (OPG) and RANK ligand (RANKL) in human prostatic tissues. The factors regulating the increased turnover associated with prostate cancer (CaP) bone metastasis are unknown. OPG and RANKL are recently identified regulators of bone resorption and bone remodeling. Methods. Tissues from 28 patients with CaP and from 4 normal organ donors were analyzed by reverse transcriptase-polymerase chain reaction and immunohistochemistry for the expression of OPG and RANKL. Results. OPG and RANKL messages were detected in both normal and cancerous prostate samples. In the normal prostate, OPG protein was detected in luminal epithelial and stromal cells (5% to 65% and 15% to 70%, respectively) and RANKL immunoreactivity was observed in 15% to 50% of basal epithelial cells, 40% to 90% of luminal epithelial cells, and 70% to 100% of stromal cells. OPG was not detected in 8 of 10 primary CaP specimens; RANKL was heterogeneously expressed in 10 of 11 CaP specimens. The percentage of tumor cells expressing OPG and RANKL was significantly increased in all CaP bone metastases compared with nonosseous metastases or primary CaP. Conclusions. CaP bone metastases were consistently immunoreactive for both OPG and RANKL compared with nonosseous metastases or primary CaP. The presence of these crucial bone resorption regulators in CaP bone metastases suggests a mechanism whereby CaP cells may modulate bone turnover and has profound implications for the establishment and development of CaP bone metastases in advanced disease.

Expression of c-kit and kit-ligand in benign and malignant prostatic tissues.

The tyrosine kinase receptor c-kit and its ligand [kit ligand (KL) or stem cell factor (SCF)] exert a broad range of biological activities during organogenesis and normal cell development. Recent studies have revealed that altered c-kit levels occur in a variety of malignancies and cancer cell lines. KL has also been shown to stimulate the growth of malignant cells, as well as to promote chemotaxis. We had previously reported expression of KL in stroma cells of normal human prostate. The present study was undertaken in order to analyze the patterns of expression of c-kit and KL in a well characterized set of prostatic tissues, including normal prostate (n=4), benign prostatic hyperplasia (BPH) (n=53) and adenocarcinoma (n=46) samples. The distribution of c-kit and KL proteins was studied by immunohistochemical analyses, while transcript levels were determined by in situ hybridization with specific RNA probes on a subset of the benign and malignant tissues referred above. In addition, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed to determine levels of c-kit and KL expression in cultures of epithelial and stroma cells, as well as in the prostate cancer cell lines LNCaP, DU145 and PC3. c-kit protein in normal prostate was exclusively detected in mast cells by immunohistochemistry and in situ hybridization. However, c-kit transcripts, but not c-kit protein, were detected in low levels and with an heterogeneous pattern in basal epithelial cells of ducts and acini. c-kit in BPH was detected in epithelial cells in 9 of 53 (17%) specimens. c-kit protein expression in malignant epithelial cells was identified in 1 of 46 (2%) tumors. However, c-kit transcripts were detected in low levels by in situ hybridization in most of the tumors analyzed. KL protein and transcripts in normal prostate were detected in high levels in stroma cells. However, epithelial cells were unreactive for anti-KL antibody, but showed low levels of KL transcripts mainly in cells of the basal layer. Basal epithelial cells in hyperplastic glands showed KL expression in 13 of 53 (24%) specimens. KL protein in tumor cells was noted in 18 of 46 (39%) cases. c-kit transcripts were not found in normal prostate and in the 3 cancer cell lines analyzed by RT-PCR, however, it was present in cultured epithelial cells of BPH, and in cultures of stroma cells from both normal and BPH. The majority of cultured cell lines of epithelial and stromal origin displayed considerable levels of KL. In addition all prostate cell lines studied showed significant levels of KL transcripts. In summary, co-expression of c-kit and KL in a subset of BPH cases may suggest an autocrine mode of signaling. Data from this study reveals that altered patterns of c-kit and KL expression are associated with BPH and adenocarcinoma of prostate. It appears that KL induces mast cells proliferation and maturation and enhances their release of protease. This could explain the accumulation of mast cells at tumor sites, a phenomenon that was not observed in normal prostate or BPH samples.

Localization of type 5 17beta-hydroxysteroid dehydrogenase, 3beta-hydroxysteroid dehydrogenase, and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry.

An important source of androgens in the human prostate are those synthesized locally from the inactive adrenal precursor dehydroepiandrosterone (DHEA) and its sulfated derivative DHEA-S. Three beta-HSD (hydroxysteroid dehydrogenase) converts DHEA into androstenedione (4-dione), whereas type 5 17beta-HSD catalyzes the reduction of 4-dione into testosterone in the human prostate and other peripheral intracrine tissues. In the present study, we have used two complementary approaches, namely in situ hybridization and immunocytochemistry, to identify the cells that contain the type 5 17beta-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH). Localization of 3beta-HSD and of the androgen receptor (AR) was also investigated by immunostaining in the same tissue. To find out whether there are any differences between BPH and normal prostate tissue, the localization of type 5 17beta-HSD was reexamined by immunocytochemistry in the normal human prostate samples and also in normal prostate epithelial cell line (PrEC). The in situ hybridization results obtained with a tritiated uridine triphosphate (3H-UTP)-labeled type 5 17beta-HSD riboprobe are in agreement with the immunostaining data obtained with a specific antibody to the enzyme. The immunostaining results obtained from normal prostate tissue and BPH were found to be similar. Thus, in the glandular epithelium, basal cells highly express the messenger RNA and the enzyme, whereas luminal cells show a much lower and variable level of expression. In the stroma and walls of blood vessels, fibroblasts and the endothelial cells lining the blood vessels show positive staining. Similar results are observed when the cellular distribution of 3beta-HSD is investigated. AR immunoreactivity, however, shows a different distribution because, in the epithelium, most of the nuclei of basal cells are negative, whereas the majority of nuclei of the luminal cells show positive staining. A strong reaction for AR is also found in most stromal cell nuclei and in the nuclei of most endothelial cells, as well as in some other cells of the walls of blood vessels. In conclusion, human type 5 17beta-HSD, as well as 3beta-HSD, are highly expressed, not only in the basal epithelial cells and stromal fibroblasts but also in the endothelial cells and fibroblasts of the blood vessels. AR, on the other hand, is highly expressed in the luminal cells. The present data suggest that DHEA is transformed in the basal cells of the glandular epithelium into 4-dione by 3beta-HSD and then into testosterone by type 5 17beta-HSD, whereas dihydrotestosterone is synthesized in the luminal cells after diffusion of testosterone from the underlying layer of basal cells. The potential role of androgen formation and action in blood vessels is unknown and opens new avenues of investigation for a better understanding of the multiple roles of androgens.

LONG TERM ORGAN CULTURE OF HUMAN PROSTATE TISSUE IN A NASA-DESIGNED ROTATING WALL BIOREACTOR

Purpose To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. Materials and Methods Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 × 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. Results We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta 2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. Conclusions The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Androgen regulation of the human pseudoautosomal geneMIC2, a potential marker for prostate cancer

Using the differential display-polymerase chain reaction technique to identify androgen-responsive genes in the human prostatic tumor cell line LNCaP, we cloned an expression tag homologous to the human pseudoautosomal gene MIC2. The role of MIC2 in the prostate had not previously been studied. We used a series of cell lines derived from LNCaP that varied in their degree of differentiation and metastatic potential to assess the relationship between MIC2 expression and androgen responsiveness in prostate cancer. The expression of MIC2 mRNA and its product E2 was upregulated by androgen in a dose- and time-dependent manner in the parental LNCaP line and correlated with the expression of prostate-specific antigen. In the LNCaP sublines and an androgen-repressed invasive human prostate cancer cell line (ARCaP), MIC2 gene expression was not regulated by androgen and was associated with poorer differentiation, decreased androgen sensitivity, and higher metastatic potential. Immunohistochemical analyses indicated that E2 was expressed in tissues from patients with primary prostate cancer (16 of 20), in fetal prostatic tissues (low levels in all 10 fetal tissues assessed), and sporadically in benign prostatic hyperplasia tissues (one of four). The normal prostate tissues did not show positive E2 staining, with the exception of one central-zone section from one of the eight normal prostate samples assessed. These findings suggest that deregulation of expression of the human pseudoautosomal gene MIC2 occurred in the prostate.

Quantification of matrix metalloproteinases and tissue inhibitors of metalloproteinase in prostatic tissue: analytical aspects.

The balance between matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) has been seen as important during tumor invasion and progression. The determination of these components needs a special strategy of tissue preparation. This analytical problem has not been considered for prostatic tissue. We adapted an extraction method consisting of two extraction steps with 0.25% Triton X-100/CaCl2 solution and two heat extraction steps at 60 degrees C for 4 min. This combination allowed a complete extraction of MMP (measured as enzyme activity) and TIMP-1 (measured with an ELISA test) from cancerous and normal prostatic tissue samples. The median values for cancerous vs. normal MMPs (50.8 mU/g wet tissue and 1,580 mU/g protein vs. 88.8 and 2,497) and TIMP-1 (4.49 micrograms/g wet tissue and 96.7 micrograms/g protein vs. 12.4 and 237.8) were significantly lower, whereas the respective ratios for MMP/ TIMP-1 (11.1 vs. 4.0 on wet weight and 15.5 vs. 5.3 on protein basis) were significantly higher. An optimized extraction procedure was elaborated for determining MMPs and TIMP-1 in prostatic tissue samples. The increased ratio of MMP/TIMP-1 can be interpreted as an indicator of the imbalance between MMP and TIMP, characteristic of prostate carcinoma tissue.

Chromosome 16 in primary prostate cancer: a microsatellite analysis.

Cytogenetic and molecular genetic analyses of prostate cancer specimens have revealed nonrandom chromosomal deletions, affecting chromosomes 7q, 8p, 10q and 16q. Based on these data, we designed this study to further characterize the altered region(s) on chromosome 16 by evaluating 16 microsatellite markers on a population composed of 32 paired normal and primary prostatic tumor samples. The 16 microsatellites selected mapped to 11 distinct loci on 16q and 5 loci on 16p. No alterations were identified affecting 16p. However, 16 of 31 (51%) informative cases showed molecular alterations in at least one of the loci analyzed on 16q, consisting of 18 deletions and 11 bandshifts. Moreover, most of the deletions clustered at 6 microsatellite loci, mapping to the 16q22.1-23.1 region. Our results suggest that microsatellite alterations on the long arm of chromosome 16 are frequent events in prostate cancer, and that the 16q22.1-23.1 region might harbor a tumor suppressor gene involved in prostate cancer.

DNA flow cytometric study of the hyperplastic and neoplastic canine prostate

Flow cytometric DNA measurements were carried out on 45 formalin-fixed, paraffin-embedded canine prostate tissues. The tissues were categorized as normal, hyperplastic, or neoplastic on the basis of light microscopic examination, and DNA ploidy was compared with histologic classification. Ten normal prostate samples showed diploid DNA histograms, but with proliferation greater than that found in the normal human prostate. Thirteen specimens of benign prostate hyperplasia showed diploid or near-diploid DNA histograms. Of 22 prostatic carcinomas, 12 were diploid and 10 were aneuploid, with the majority of the aneuploidy being near-triploid. The frequency of DNA aneuploidy recognized in canine prostatic carcinoma is similar to findings in human prostatic carcinoma if all their grades of malignancy are included.

Receptors for luteinizing hormone‐releasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostate cancers and in benign prostate hyperplasia

Using sensitive multipoint micromethods, we estimated membrane receptors for [D-Trp6]-luteinizing hormone-releasing hormone ([ D-Trp6]-LH-RH), somatostatin (SS-14), human prolactin (hPRL), and epidermal growth factor (EGF) in experimental Dunning rat prostate cancers and in samples of normal human prostate, benign prostatic hyperplasia (BPH), and human prostate cancer (PC) obtained from biopsy, after prostatectomy, or at autopsy. In the Dunning R-3327 rat prostate adenocarcinoma specimens, the receptors were characterized in untreated animals and following in vivo treatment with microcapsules of the agonist [D-Trp6]-LH-RH and the somatostatin analog RC-160. Two populations of binding sites were found for [D-Trp6]-LH-RH, one with high affinity and low capacity and another with low affinity and high capacity. Treatment with [D-Trp6]-LH-RH and RC-160 alone or with the combination of these analogs increased the binding capacity (Bmax) of the low-affinity binding sites for [D-Trp6]-LH-RH and decreased Bmax for hPRL and EGF. Therapy with [D-Trp6]-LH-RH also reduced Bmax of SS-14 binding and dissociation binding constant of high-affinity binding sites for [D-Trp6]-LH-RH, whereas administration of RC-160 or the combination treatment with both analogs increased Bmax of SS-14 binding. These findings are compatible with the view that analogs of LH-RH and SS-14 might exert some direct inhibitory effects on the Dunning prostate cancer. Among 13 human BPH samples examined, only one had receptors for [D-Trp6]-LH-RH, and seven specimens exhibited binding for prolactin. [D-Trp6]-LH-RH receptors were found in all seven samples of human PC but not in any of the eight specimens of normal human prostate. All samples of normal human prostate, BPH, and human PC exhibited binding sites for EGF but not for SS-14. Our findings on the membrane receptors in the human and rat prostate cancers raise the intriguing possibility that LH-RH, acting as a growth factor, along with EGF and prolactin, might be involved in complex interactions that contribute to the promotion of prostate cancer in man.

Glutathione S-transferases in human prostate

A number of human prostatic tissue biopsies have bben analyzed glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61–90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 ± 44 nmol/min per mg with a range of 97–237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19–0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in M r from approximately 224 000 to 28 500 and in p I from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity wiht five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an M r of approx. 24 000 and a near-neutral p I and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Y f, subunit 7 or π). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes α, μ and π. These are characterized as Y a, Y b, Y′ b and Y f (analagous alternative nomenclature subunits 1, 3, 4 and 7).

Sex Steroid Receptors in Normal and Hyperplastic Human Prostate

The concentrations of sex steroid receptors (per unit DNA) were measured in normal periurethral and peripheral prostatic tissue samples from seven men (mean age 64 years; range 54-71 years) undergoing cystectomy for bladder cancer, and in hyperplastic nodules from 15 men with BPH (mean age 69 years; range 60-89). Occupied androgen (AR) and estrogen (ER) receptors were measured with an improved exchange procedure, where receptor-binding sites were stabilized by a combinatorial procedure involving careful washout of extracellular secretory products (including proteases) prior to homogenization, inclusion of 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 20 mM molybdate in the exchange medium, and long-term incubation at 0-4 degrees C. Bound radioligands were separated by a hydroxylapatite (HAP) batch adsorption procedure. Maximal specific exchange binding of 3H-R 1881 or 3H-estradiol in total homogenates of human prostate samples was achieved after incubation periods of about 72 h at 0-4 degrees C. In contrast, progestin receptors (PR) were readily available for binding 3H-R 5020; thus overnight binding at 0-4 degrees C was routinely used to measure PR. Binding specificities and equilibrium binding constants (calculated from 8-point Scatchard plots, correcting for nonsaturable binding) were found to be characteristic for AR, PR, and ER, respectively. The receptor results obtained in this study demonstrate that no significant differences existed in total AR per unit DNA between hyperplastic and either central or peripheral prostatic tissue samples; PR was present in both zones of normal prostatic tissue as often as in BPH samples, with PR concentrations significantly lower in hyperplastic samples; and ER was irregularly detected in both normal and hyperplastic tissue in low concentration relative to AR and PR; the frequency of ER detection was much lower in BPH than in normal prostate tissue. Studies of steroid receptor content relative to enzyme markers specific for epithelial and stromal cells in BPH samples showed a positive correlation between acid phosphatase activity (a specific marker for epithelial cells) and both AR and PR. No correlation was observed between AR or PR with either prolyl hydroxylase or myosin ATPase (specific markers for stromal cells). These observations suggest that PR, as well as AR, is primarily associated with the epithelial elements of prostate. Because of the relative infrequency of ER, similar correlation of ER with enzyme markers was not possible.

Autoradiographic Localization of Estrogen and Androgen Target Cells in Human and Rat Prostate Carcinoma

The distribution of estrogen target cells within the Dunning R3327-H rat prostate tumor following intravenous injection of tritiated estradiol into rat hosts was compared to the distribution obtained following incubation of a 2mm.3 sample of the tumor with tritiated estradiol in organ culture. No difference was observed, indicating that the in vitro method was an effective approach for autoradiographic analysis of tumor biopsy samples. Subsequently, tumor samples were excised from solid tumors of R3327-H and R3327-MAT LyLu tumors growing in Copenhagen rats. These tumor models were chosen as representatives of hormone sensitive (R3327-H) and hormone insensitive (R3327-MAT LyLu) tumors. Normal rat dorsal prostate and human tumor biopsy samples were also studied. Autoradiographic studies were performed in vitro utilizing tritiated estradiol and tritiated dihydrotestosterone to compare the distribution of estrogen and androgen target cells.The present research demonstrated that 1) similar patterns of nuclear uptake of steroids are obtained with in vivo and in vitro autoradiographic techniques, 2) estradiol receptors occur primarily in extra-acinar epithelioid cells in both rat and human prostate carcinomas, 3) these epithelioid cells are not characteristic of the normal rat dorsal prostate, 4) androgen receptors occur in both acinar and stromal epithelioid cells in rat and primarily in acinar epithelial cells in human tumors and 5) in vitro autoradiographic methods can provide insight into differences in sensitivity to steroids which may be of diagnostic importance in the treatment of cancer.

Sex steroid receptors in normal and hyperplastic human prostate.

The concentrations of sex steroid receptors (per unit DNA) were measured in normal periurethral and peripheral prostatic tissue samples from seven men (mean age 64 years; range 54-71 years) undergoing cystectomy for bladder cancer, and in hyperplastic nodules from 15 men with BPH (mean age 69 years; range 60-89). Occupied androgen (AR) and estrogen (ER) receptors were measured with an improved exchange procedure, where receptor-binding sites were stabilized by a combinatorial procedure involving careful washout of extracellular secretory products (including proteases) prior to homogenization, inclusion of 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 20 mM molybdate in the exchange medium, and long-term incubation at 0-4 degrees C. Bound radioligands were separated by a hydroxylapatite (HAP) batch adsorption procedure. Maximal specific exchange binding of 3H-R 1881 or 3H-estradiol in total homogenates of human prostate samples was achieved after incubation periods of about 72 h at 0-4 degrees C. In contrast, progestin receptors (PR) were readily available for binding 3H-R 5020; thus overnight binding at 0-4 degrees C was routinely used to measure PR. Binding specificities and equilibrium binding constants (calculated from 8-point Scatchard plots, correcting for nonsaturable binding) were found to be characteristic for AR, PR, and ER, respectively. The receptor results obtained in this study demonstrate that no significant differences existed in total AR per unit DNA between hyperplastic and either central or peripheral prostatic tissue samples; PR was present in both zones of normal prostatic tissue as often as in BPH samples, with PR concentrations significantly lower in hyperplastic samples; and ER was irregularly detected in both normal and hyperplastic tissue in low concentration relative to AR and PR; the frequency of ER detection was much lower in BPH than in normal prostate tissue. Studies of steroid receptor content relative to enzyme markers specific for epithelial and stromal cells in BPH samples showed a positive correlation between acid phosphatase activity (a specific marker for epithelial cells) and both AR and PR. No correlation was observed between AR or PR with either prolyl hydroxylase or myosin ATPase (specific markers for stromal cells). These observations suggest that PR, as well as AR, is primarily associated with the epithelial elements of prostate. Because of the relative infrequency of ER, similar correlation of ER with enzyme markers was not possible.

Mitogenic Factors in Prostatic Tissue and Expressed Prostatic Secretion

Expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue contain a polypeptide growth factor(s) that stimulates the uptake of tritium-labeled thymidine by cultured 3T3 fibroblasts. Mitogenic activity was present in expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue. The apparent molecular weights of the mitogenic fractions were estimated to be 300,000, 150,000 and 60,000 daltons for prostatic tissue extracts, and 30,000 daltons for expressed prostatic secretions. Bioassays yielded a mean of 27 units of mitogenic activity per mg. protein in expressed prostatic secretions obtained from men with normal and enlarged prostate glands. There was no difference in bioassayable mitogenic activity in the expressed prostatic secretions from normal and benign prostatic hyperplasia samples but gel filtration studies revealed a high molecular weight component present only in samples from men with prostatic enlargement. A dialyzable low molecular weight inhibitor of fibroblast growth was found in the prostatic tissues and expressed prostatic secretions. We report the characterization studies and discuss the possible roles of growth factors in the pathogenesis of benign prostatic hyperplasia.