Ornithine decarboxylase activity during neonatal development of the rat ventral prostate

The relationship between ornithine decarboxylase activity and growth has been examined in ventral prostates from rats aged 2–60 weeks. The incorporation of [¹²⁵I]iododeoxyuridine in vitro was used to assess DNA-synthetic activity. During 2–7 weeks post-partum ornithine decarboxylase activity/prostate and the protein content and wet weight increased exponentially. In contrast, the incorporation of [¹²⁵I]iododeoxyuridine/prostate fell during the first 4 weeks. This decline was reversed at about the time when the systemic concentrations of gonadal androgens start to rise. After 10 weeks post-partum the incorporation of [¹²⁵I]iododeoxyuridine/prostate and ornithine decarboxylase activity/prostate levelled off, whereas the DNA and protein content of the prostate contonued to increae at a slow rate.     

Urokinase plasminogen activator amino-terminal peptides inhibit development of the rat ventral prostate

The plasma membrane urokinase plasminogen activator receptor (uPAR) localizes and enhances activation of pro-uPA. Active uPA, in turn, promotes increased degradation of the extracellular matrix (ECM) by activation of plasminogen. uPAR binds to ECM molecules and integrins, which can affect cellular adhesion, signal transduction, and gene regulation. The current study examines the expression and function of uPAR in developing rat ventral prostates (VPs). We report that newborn VPs express uPAR mRNA and protein. In addition, the function of uPAR-bound uPA during in vitro prostatic development was studied by adding recombinant peptide competitive inhibitors of uPA-uPAR binding. Newborn VP explants were cultured in serum-free media for one week with 10(-8) M testosterone plus chimeric peptides containing a human immunoglobulin G Fc domain and either human uPA amino acids 1-138 (hu-uPA 1-138) as a control or mouse uPA amino acids 1-138 (mo-uPA 1-138) or 1-48 (mo-uPA 1-48). Hu-uPA 1-138-treated VPs underwent normal ductal branching morphogenesis and tissue differentiation. In contrast, VPs treated with mo-uPA 1-138 or mo-uPA 1-48 displayed a dose-dependent perturbation of ductal branching. Differentiation of both epithelial and mesenchymal tissues was also impaired. Mo-uPA 1-48-treated VPs contained significantly more apoptotic cells. These observations suggest that disruption of uPA binding to uPAR results in a retardation of the development of newborn VPs.     

Regional cellular heterogeneity and DNA synthetic activity in rat ventral prostate during postnatal development.

Previous studies have provided evidence that the rat ventral prostate grows primarily, if not exclusively, at its distal tips. However, as yet there have been no analyses in which individual cells in defined regions of the prostatic ductal system have been resolved and quantified. Moreover, the possibility that the prostate might grow differently at different times of postnatal development has received little attention. Our objectives were to identify and quantify the proliferating epithelial and stromal cells in defined regions of the rat ventral prostate during its postnatal development. To this end, 3H-thymidine was administered in vivo to rats of ages 10-60 days. A dissection technique was then used by which the distal, intermediate, and proximal segments of the prostatic ductal system were physically isolated from each other without removing the stromal tissue. Longitudinal sections of these segments were examined for cellular composition and DNA synthetic activity. Regional heterogeneity with respect to cell composition and cell proliferation was seen. In rats of all ages, DNA synthetic activity was seen in epithelial and stromal cells throughout the prostate, rather than only in the distal segment. At Days 10 and 20, significantly higher percentages of epithelial and stromal cells were labeled in the distal than in the proximal segments; but at Days 45 and 60, the percentages of labeled epithelial and stromal cells in the distal, intermediate, and proximal segments were similar. Thus, in all segments, and at all ages, substantial labeling was seen throughout the prostate. These data suggest that the prostate grows in both length and width throughout postnatal development, reminiscent of the growth of a tree.     

Studies of the 3beta-hydroxysteroid oxidoreductase activity in rat ventral prostate

The reduction of ³H-androstanolone into 3β-androstanediol was used to study the 3β-hydroxysteroid oxidoreductase activity of rat ventral prostate. This activity is present only in the cytosol and has a pH optimum of 8.5 with NADH as cofactor. The 3β-hydroxysteroid oxidoreductase activity , as compared to 3α-hydroxysteroid oxidoreductase activity, is much lower in the present study than was observed previously during prostate perfusion $\text{in vivo}$ or prostate organ culture superfusion.     

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.     

Fluorometric microassay of plasminogen activators.

A new method for determining plasminogen activator levels has been developed. Data are presented which demonstrate measurements of trypsin, plasmin, urokinase, and plasminogen activation. The assay is based on the digestion of N-terminal-blocked protamine and subsequent measurement of the exposed amino groups using the fluorogenic amine reagent, Fluram. The soluble substrate provides an assay which is linear with respect to both time and concentration and which is sensitive enough to allow measurements on a microscale. As little as 1 ng of trypsin, 0.002 CTA units (established by the Committee on Thrombolytic Agents of the NIH) of plasmin, and 0.01 CTA units of urokinase can be detected under the conditions described.Interference with the amine determination due to Fluram-positive material found in biological samples is minimized with the high dilutions attainable with the system. Plasminogen activator in the urine of the female mouse can be detected using 1 μl of urine in a 200-μl test system.     

Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

Increased gene expression of plasminogen activators and inhibitors in left ventricular hypertrophy

In the early stages of left ventricular hypertrophy (LVH) acute adaptive changes occur in the coronary vasculature as it remodels. Plasminogen activators (PAs) and inhibitors (PAIs) have the potential effects of proteolytic degradation that is relevant to tissue remodeling and angiogenesis. Our study focused on the possible roles of PAI-1, PAI-2, uPA and tPA in myocyte hypertrophy and angiogenesis in the early and late stages of pressure overload induced left ventricular hypertrophy (LVH). We divided seventeen adult swine, weighing 24.2 ± 6.5 kg, into four groups: control, sham-operated, early LVH and late heart failure LVH group. At surgery we placed a fixed constrictor on the ascending aorta immediately above the aortic valve. This increased LV systolic pressure from 133 ± 15 to 193 ± 24 mm Hg after the surgery. We subdivided the early group into groups of 3 animals each that we euthanized at 8, 24 and 72 h after operation and obtained heart samples for analysis. In the late heart failure group individual animals were euthanized at 55, 59, 62 and 72 days after the detection of congestive heart failure. We also obtained tissue samples from the control and sham-operated swine. Sections for histologic analysis were fixed in 10% buffered formalin. We isolated RNA, size fractionated it using 1% formaldehyde-agarose gel electrophoresis and then did Northern blots. The mRNAs from both PAI-1 and PAI-2 showed a remarkable increase at 8 and 24 h after acute aortic constriction and returned to control by 72 h. Regional differences showed that most of the increases were in the endocardium. Three animals in the late heart failure LVH group were determined to be in congestive heart failure at about 2 months after the onset of aortic constriction. In these animals PAI-1 and PAI-2 were increased in both the left and right ventricles but remained low in an animal of the same elevation in aortic pressure seen by the LV who did not have congestive failure. These data suggest that PA and PAI gene expressions change before morphologic changes occur in the early stages of developing LVH. Also at the time of onset of congestive heart failure this increased expression reappears. PAs and PA inhibitors mRNA levels vary in the different regions of the heart reflecting changing wall stresses. Thus, the PAs and PA inhibitors may play an important role in angiogenesis that occurs during the early stages of LVH. The increased expression in the late stage of LVH may reflect further changes in wall stresses since these animals also showed overt clinical signs of heart failure.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

Osteopontin modulates prostate carcinoma invasive capacity through RGD-dependent upregulation of plasminogen activators

Osteopontin, a non-collageneous bone matrix protein, is produced in several human tumors but its role in cancer progression has been only partially elucidated. In this study we investigated the potential role of osteopontin in the malignancy of prostate cancer cells. Chemotaxis and chemoinvasion analyses revealed a dose-dependent increase in PC3 cell movement induced by osteopontin and a strict dependence of cell invasion on alphavbeta3 integrin function. The pattern of protease expression was modified by osteopontin and was characterized by an upregulation of plasminogen activators. Our findings suggest that osteopontin may confer selective malignant potential to prostate cancer cells through the enhancement of their invasive and proteolytic capability.     

Isolation and characterization of plasminogen activators from hyperlastic and malignant prostate tissue

Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen framents in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates $\text{pyro-Glu-gly-Arg}\text{-p}\text{NA and H-}\text{D}\text{-Ile-Pro-Arg}\text{-p}\text{NA}$ were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase.     

Isolation and characterization of plasminogen activators from hyperplastic and malignant prostate tissue

Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen fragments in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates pyro-Glu-Gly-Arg-pNA and H-D-Ile-Pro-Arg-pNA were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase.