Growth factor involvement and oncogene expression in prostatic tumours

The effects of EGF, TGF alpha and 5 alpha-dihydrotestosterone on the growth of a prostatic epithelial cell line have been evaluated in clonal growth assays. Similar bioassay systems have been used to identify tumour-associated growth promoters derived from a human prostatic carcinoma cell line (PC3). Growth factor activity was associated with proteins of Mr 20-30 kDa. In a separate study, EGF receptor concentration and cellular proto-oncogene expression was assessed in prostatic tumour samples. In prostatic carcinoma samples, strong correlation was observed between EGF receptor concentration and c-myc expression. There were no significant correlations between EGF receptor concentration and tumour grade or androgen receptor content in carcinoma samples. EGF receptor concentration was significantly higher in prostatic carcinoma specimens than in BPH.     

Nuclear volume estimates in prostatic intraepithelial neoplasia

OBJECTIVE: Prostatic intraepithelial neoplasia (PIN), the most likely precursor of prostatic adenocarcinoma, is divided into two grades, low and high. Pathologists may encounter difficulties in applying these criteria in daily practice. In view of the clinical significance of high grade PIN as strong predictor of carcinoma, the separation of low and high grade PIN plays an important role in patient management. The aim of the present study was to evaluate three-dimensional nuclear size estimation in normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma as an element in their classification. STUDY DESIGN: We studied 31 formalin-fixed, paraffin-embedded, whole-mounted radical prostastectomy specimens that contained foci of normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma. Hematoxylin-eosin-stained sections were selected for the stereologic estimation of volume-weighted mean nuclear volume by the "point-sampled intercepts" method. On each focus, an average of six fields of vision were systematically chosen. RESULTS: The quantitative results indicate a significant increase in nuclear volume from normal prostatic glands (mean, 209.0 micron 3; SD, 64.6 micron 3) to low grade PIN, high grade PIN and prostatic adenocarcinoma with increments of 49%, 88% and 109%, respectively (F = 29.1, P < .001). Two-group comparisons (Duncan procedure) showed differences between low and high grade PIN and prostatic adenocarcinoma (P < .01). The difference between high grade PIN and prostatic adenocarcinoma was not significant. CONCLUSION: Three-dimensional estimates of nuclear size discriminate low and high grade PIN. Lack of stereologic differences between high grade PIN and prostatic adenocarcinoma further supports high grade PIN as a precursor of prostatic adenocarcinoma.     

Sodium, potassium, calcium, magnesium, zinc, citrate and chloride content of human prostatic and seminal fluid

The ionic composition of human prostatic fluid varied greatly between individuals, reflecting the secretory activity of the gland and the presence or absence of prostatic inflammatory disease. In normal prostatic fluid the major anion was citrate, while chloride concentrations were lower. Their counterions were mainly sodium and potassium, together with calcium, magnesium and zinc. Prostatic secretions from men with prostatitis comprised mainly sodium and chloride. The electrolytes were closely correlated to each other (except for sodium, which was essentially invariant at about 145 nm). The molar changes per mole of citrate were about 0.52, potassium; -0.53, chloride; 0.17, calcium; 0.14, magnesium; and 0.09, zinc. The pH was also associated with citrate, decreasing from 8.0 to 6.2 as the citrate increased. These various ionic changes can be explained as responses to citrate secretion, without the need to propose specific transport mechanisms for the other ions measured. The marked effect of prostatic inflammation on the composition of prostatic fluid can be seen as being due mainly to decreased secretion rather than active modification.     

Absorption,distribution and binding of cadmium and zinc in irrigated rice plants

Summary Rice plants are grown in nutrient solutions (Hoagland-Arnon 1) containing different concentrations of cadmium (0.005; 0.010 and 0.100 ppm) and one concentration of zinc (0.070 ppm) traced respectively with¹⁰⁹Cd and⁶⁵Zn. Distribution and localization of both elements are studied at plant and cellular levels. Contrary to zinc, cadmium remains fixed by radicular sites and its translocation index is very low. Centrifugation of roots homogenates showed that % Cd recovered in the residue increases with Cd concentration and emphasize the high capacity of Cd fixation by walls when Cd external concentration is high. The relative distributions of Cd and Zn present at the same concentration in the growing medium are quite identical. In the 100,000×g supernatant fraction of roots, both elements are associated with compounds of low molecular weight, Cd being bound to higher MW ligands than zinc (1000 to 3000 for zinc). The fact that Cd was recovered in fractions in which most of³⁵S-cysteine was present suggests that in plants, like in animals, peptides rich in — SH residues may play a role in the fixation of heavy metals. Contribution no. B 101488 of the Biology-Radiation Protection and Medical Research Programme DG XII CEC.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

High-grade prostatic intraepithelial neoplasia

High-grade prostatic intraepithelial neoplasia is considered the most likely precursor of prostatic carcinoma. The only method of detection is biopsy; prostatic intraepithelial neoplasia (PIN) does not significantly elevate serum prostate-specific antigen concentration and cannot be detected by ultra-sonography. The incidence of PIN in prostate biopsies averages 9% (range, 4%-16%), representing 115,000 new cases of PIN diagnosed each year in United States. PIN has a high predictive value as a marker for adenocarcinoma, and its identification warrants repeated biopsy for concurrent or subsequent invasive carcinoma. Carcinoma will develop in most patients with PIN within 10 years. PIN is associated with progressive abnormalities of phenotype and genotype that are intermediate between normal prostatic epithelium and cancer, indicating impairment of cell differentiation and regulatory control with advancing stages of prostatic carcinogenesis. Androgen deprivation therapy decreases the prevalence and extent of PIN, suggesting that this form of treatment may play a role in chemoprevention.     

Identification of three antigens in human brain associated with similar antigens on human leukaemic cells.

Monoclonal antibodies prepared against a non-T and non-B acute-lymphocytic-leukaemia cell line were tested for reactivity against human brain tissue. Several of the monoclonal antibodies were found to react specifically with brain fractions. Three antigens, 44H4, 44D7 and 44D10, were identified in white matter. Although 44D10 was absent from grey matter, the levels of 44H4 and 44D7 antigens present in grey matter were 2- and 4-fold higher respectively than in white matter. Fractionation of white matter indicated that all three antigens were absent from the multilamellar compact myelin, but associated with a membrane fraction of higher density. All three antigens, which required detergent for solubilization from the membranes, were purified by affinity to monoclonal antibodies and/or were analysed by immunoblotting. The 44H4 and 44D10 antigens were single polypeptide chains with Mr 94000 and 80000 respectively when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Monoclonal antibody 44D7 reacted with a complex of a Mr greater than 120000 under non-reducing conditions in the presence of sodium dodecyl sulphate. This complex dissociated on reduction into four bands with Mr values of 80000, 57000, 47000 and 41000. The brain antigens are present on proteins similar to, or identical with, those isolated from acute-lymphocytic-leukaemia cells.     

Creatine kinase and ATPase in human seminal fluid and prostatic fluid

A creatine kinase assay based on estimation of creatine liberated from creatine phosphate was accurate and reproducible for use with seminal or prostatic fluid, after allowance was made for acid phosphatase interference. Comparison of this method with one which relies on enzymic coupling of ATP formation to NADP+ oxidation shows that the latter under-estimates creatine kinase activity by a factor of about 3. This discrepancy could be due to the high ATPase activity found in prostatic and seminal fluid. Uncritical use of the NADP+ assay might account for different seminal creatine kinase values reported in the literature. Interrelationships between ATPase, creatine kinase and zinc suggest that seminal ATPase is a prostatic secretory product while creatine kinase may be multiglandular in origin.     

Detection of prostatic tissue and seminal plasma peptides reacting with human seminal fluid acid phosphatase antibodies

IgG1 monoclonal antibody to purified seminal fluid phosphatase was raised by fusion of spleen cells from immunized mice with cell line Sp2/O-Ag 14 using simple method of screening for antiphosphatase antibody secreting clones. All molecular forms of catalytically active seminal fluid phosphatase and prostatic tissue phosphatase, resolved by chromatofocusing in pH gradient, react with this monoclonal antibody and with rabbit antiserum to purified seminal fluid phosphatase. Peptides of Mr 25,000 to 76,000 and of Mr 13,000 to 76,000 were adsorbed from the prostatic tissue extract and from seminal plasma on the monoclonal antibody-Sepharose column.     

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.     

The localization of sulfated glycoconjugates synthesized by the corneal epithelium of chick embryos

The localization of sulfated glycoconjugates in the corneal epithelium of 19-d-old chick embryo was investigated biochemically using epithelia labeled in vitro with [35S]sulfate, which exhibited autoradiographically a similar distribution of silver grains to that labeled in ovo. The radiolabeled tissues were dissociated into single cells by incubation in 0.25% trypsin containing 0.02% EDTA at 37 degrees C for 40 min. The proteoglycans and sulfated glycoproteins which were associated with the cells and those released into the dissociation medium were separated by DEAE-Sepharose CL-6B and analyzed on Sepharose CL-6B and SDS-PAGE. About 86% of the proteoglycans was released into the dissociation medium and more than 50% of the cell-associated ones was affected by trypsin. This indicates that the proteoglycans are mostly localized in an extracellular compartment. On the other hand, the extent of release of sulfated glycoproteins into the medium on dissociation of tissues was distinctly different depending upon their molecular weight (Mr): almost all of the sulfated glycoproteins of the family with Mr 48,000-70,000 (32% of the total sulfated glycoproteins) were recovered as intact molecules with the cells, whereas approximately 50% of those with Mr 70,000-150,000 (36%) and about 70% of those with Mr over 150,000 (28%) were released into the dissociation medium. These results indicate that the family with Mr 48,000-70,000 is localized intracellularly and that with Mr 70,000-150,000 in a compartment poorly affected by trypsin; in contrast to those, that with Mr more than 150,000 is localized in an extracellular compartment like the proteoglycans.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-specific antigen), at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high Mr basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K(m) values were in the 10(2)-10(3) m(-1) x s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex vivo. PAP cleavage sites differed from those of hK3 and were mainly at P1 = Gln residues or between residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP. Purified human semenogelin was cleaved by purified and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identified ex vivo by analysis of seminal plasma.     

Short-term effects of prolactin on prostatic function in rats with lisuride-induced hypoprolactinaemia

The effects of a single injection of ovine prolactin on prostatic function were monitored in intact, intact androgenized and castrated-androgenized rats rendered hypoprolactinaemic after 7 days of treatment with a potent dopamine agonist, lisuride. Hypoprolactinaemia was associated with reductions in ventral prostate weight, polyamine levels, lateral lobe zinc and the concentration of the ventral prostate protein prostatein, but an elevation in the level of cytosolic oestradiol binding. Whether these differences attained statistical significance depended on whether the animals were intact, intact-androgenized or castrated-androgenized. With the exception of ventral prostate weight and lateral lobe zinc concentrations, a single injection of prolactin restored or reversed these changes towards control levels within 12 h, which could not be explained by an indirect effect of the hormone on adrenal or testicular function. No effects of lisuride or prolactin were observed with regard to the content of fructose in the coagulating gland or in the degree of prolactin binding to prostatic membranes.     

Conversion factors of high- to low-molecular-weight forms of atrial natriuretic factor

The atrial natriuretic factor (ANF) is comprised of a 126-amino-acid precursor (pro-ANF) and its biologically active fragments. Partially purified pro-ANF and its larger fragments (greater than 10,000 daltons) have been referred to as high-molecular-weight (Mr) ANF, the partially purified smaller fragments (less than 10,000 daltons) as low Mr ANF. In vitro, mild proteolysis of high Mr ANF yielded low Mr ANF and enhanced biological activity. In the rat, pro-ANF was the predominant atrial form; however, low Mr ANF was largely released from isolated perfused hearts, which suggests that conversion of pro-ANF to low Mr ANF occurred immediately before or during secretion. High Mr ANF was also found in the perfusate of isolated rat hearts and in the plasma of rats, which suggests that some pro-ANF was secreted with low Mr ANF. Evidence for extraatrial conversion and activation of pro-ANF comes from two studies. 1) Intra-renal-arterial injection of high Mr ANF had little renal vascular action, whereas its i.v. injection caused renal vascular dilation, which suggests that the renal vasodilatory action of high Mr ANF became activated during circulation. 2) When high Mr ANF was incubated with rat blood or rat platelets in vitro, its natriuretic activity was converted to low Mr ANF within minutes; the platelet-induced conversion was associated with enhanced activity in relaxing aortic smooth muscle.     

The contribution of prostatic acid phosphatase and prostatic specific antigen in the diagnosis of prostatic cancer

Prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) were measured by immunochemical methods using test preparations from two different companies. In 66 patients with benign hyperplasia of the prostate a good correlation was found only between PSA levels (orthogonal regression analysis: y = 1.77 x -0.68; r = 0.995). Discrimination analysis between benign hyperplasia and new prostatic cancer (28 patients), using ROC curves, revealed a sensitivity for prostatic cancer of about 30 percent using both PAP methods and of about 58 percent using both PSA methods at the 95-percentile of benign hyperplasia. The PSA methods were both more sensitive in detecting prostatic cancer than the PAP methods.     

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.     

Polyamines and prostatic cancer

The importance of polyamines in prostatic growth and differentiation has prompted studies to evaluate the clinical relevance of the ornithine decarboxylase/polyamine system in prostatic cancer. These studies show that differences in biological behaviour of prostatic (cancer) cells are associated with changes in polyamine levels and/or the activity of their metabolic enzymes. Faulty antizyme regulation of polyamine homoeostasis may play an important role in the growth and progression of prostatic carcinoma. Treatment of human prostate carcinoma cells with inhibitors of polyamine metabolic enzymes or polyamine analogues induces cell growth arrest or (apoptotic) cell death. Our recent in vitro studies using conformationally restricted polyamine analogues show that these compounds inhibit cell growth, probably by inducing antizyme-mediated degradation of ornithine decarboxylase. Sensitivity of human prostate cancer cells for these compounds was increased in the absence of androgens. These results suggest that these analogues might have chemotherapeutic potential in case prostatic cancer has become androgen-independent. Pilot data in an in vivo model show that these analogues have effects on tumour cell proliferation, vascularity, blood perfusion and tissue hypoxia. Overall, these studies show that polyamines may serve as important biomarkers of prostatic malignancy and provide a promising target for chemotherapy of prostatic cancer.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.