In vivo expression of prostate-specific adenoviral vectors in a canine model.
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The activity and expression of transgene beta-galactosidase (lacZ) by replication-deficient adenoviral vectors (Ad-lacZ) containing prostate-specific promoters were compared using an in vivo canine model. The prostate tissue-specific promoters were prostate-specific antigen, probasin, and mouse mammary tumor virus long-terminal repeat, which were fused separately to an Escherichia coli lacZ gene. Dogs underwent laparotomy, and adenoviral vectors were delivered by direct intraprostatic injection. At 72 hours postinjection, the prostate and various other organs were harvested to evaluate the degree of prostate expression and dissemination of adenoviral vectors. Expression of lacZ in tissues was determined by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining, beta-galactosidase assay, and E. coli lacZ reverse transcriptase-polymerase chain reaction (PCR). The presence of adenoviral DNA sequences in canine tissues was determined by PCR using primers specific for the type 5 adenoviral genome. All three of the prostate-specific adenoviruses tested effectively expressed the lacZ gene in the canine prostate, but expression levels were lower than that of the control viral vector AdRSVlacZ following intraprostatic injection. By PCR, adenoviral vector DNA was detected in other organs and tissues, including the bladder and vas deferens. However, reverse transcriptase-PCR analysis revealed that prostate-specific Ad-lacZ vectors only transcribed lacZ mRNA in the prostate and not in nonprostatic tissues. Thus, these novel prostate-specific adenoviral vectors each have equal in vivo expression exclusively in the prostate and may potentially be used for prostate cancer gene therapy. (+info)
Potassium channels in rat prostate epithelial cells.
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Voltage-dependent K(+) channels were identified and characterized in primary culture of rat prostate epithelial cells. A voltage-dependent, inactivating K(+) channel was the most commonly observed ion channel in both lateral and dorsal cells. The K(+) current exhibited a voltage threshold at -40 mV. Averaged half-inactivation potential (V(1/2)) and the slope factor (k) values were -26 mV and 6, respectively. It showed a monoexponential decay with an inactivation time constant of about 600 ms at +60 mV. The deactivation time constant at -60 mV was 30 ms and the reversal potential was estimated at -80 mV, suggesting that current was carried by potassium ions. The scorpion venom peptides charybdotoxin (5 nM) and margatoxin (1 nM), inhibited K(+) current at all membrane potentials with a rapid and a slow reversibility respectively. Both tetraethylammonium (10 mM) and 4-aminopyridine (50 microM) reduced K(+) current by approximately 40%. We conclude that plasma membranes of lateral and dorsal rat prostate epithelial cells contain Kv K(+) channels that have biophysical and pharmacological properties consistent with those of the Kv1.3 family. (+info)
Growth regulation of prostatic stromal cells by prostate-specific antigen.
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BACKGROUND: Prostate-specific antigen (PSA) is a serine protease that can cleave insulin-like growth factor-binding protein-3 (IGFBP3), thereby decreasing its affinity for insulin-like growth factor-I (IGF-I). Dissociation of the IGF-I-IGFBP3 complex renders IGF-I available to bind to its receptor and stimulates cellular proliferation. We evaluated the potential for PSA to modulate the effects of IGF-I and IGFBP3 on the proliferation of human benign prostatic hyperplasia (BPH)-derived fibromuscular stromal cells in primary cultures. METHODS: We cultured BPH-derived stromal cells for 48 hours in serum-free RPMI-1640 medium supplemented with 0.2% bovine serum albumin and studied the effects of IGF-I, IGFBP3, PSA, and ZnCl(2) at varying concentrations. Differences in cell growth between control and treated cultures were evaluated by use of Dunnett's test. Concentration-related trends were evaluated by linear regression of log-transformed concentrations of test reagents on BPH-derived stromal cell number responses. Statistical tests were two-sided. RESULTS: We observed a concentration-dependent proliferative response of BPH-derived stromal cells to IGF-I. IGFBP3 inhibited this response in a concentration-dependent fashion. IGFBP3 alone had no effect on stromal cell proliferation. When stromal cells were incubated with PSA alone or with PSA, IGF-I, and IGFBP3, an increase in stromal cell numbers that was dependent on PSA concentration was evident in both instances. Zinc, an endogenous inhibitor of PSA enzymatic activity, was able to attenuate the stimulatory effect of PSA at intraprostatic physiologic concentrations. CONCLUSIONS: These results are consistent with the idea that PSA can modulate in vitro interactions between IGF-I and IGFBP3 and suggest that PSA may play a role in the regulation of human prostatic fibromuscular cell growth. (+info)
Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor.
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An important biological feature of prostate cancer (PCa) is its marked preference for bone marrow as a metastatic site. To identify factors that may support the growth of PCa in bone marrow, expression of receptor and nonreceptor tyrosine kinases by androgen-independent PCa bone marrow metastases was assessed. Bone marrow biopsies largely replaced by PCa were analyzed using reverse transcriptase-polymerase chain reaction amplification with degenerate primers that amplified the conserved kinase domain. Sequence analyses of the cloned products demonstrated expression of multiple kinases. Expression of the receptor and nonreceptor tyrosine kinases, alpha platelet-derived growth factor receptor and Jak 1, respectively, was confirmed by immunohistochemistry. In contrast, the type 1 insulin-like growth factor receptor, thought to play a role in PCa development, was lost in metastatic PCa. These results implicate several specific growth factors and signaling pathways in metastatic androgen-independent PCa and indicate that loss of the type 1 insulin-like growth factor receptor contributes to PCa progression. (+info)
Combined treatment with the 5 alpha-reductase inhibitor PNU 157706 and the antiandrogen flutamide on the Dunning R3327 prostatic carcinoma in rats.
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The steroid 5 alpha-reductase enzyme catalyzes the conversion of testosterone to the potent androgen 5 alpha-dihydrotestosterone (DHT). PNU 157706, a novel, potent and selective dual 5 alpha-reductase inhibitor, was reported to be effective in inhibiting the growth of established tumors in the Dunning R3327 rat prostatic carcinoma model. We have studied the efficacy of combined treatment with PNU 157706 and the antiandrogen flutamide in this prostatic tumor in rats. Rats with tumor diameters of about 1 cm were treated orally 6 days a week for 9 weeks with PNU 157706 (10 mg/kg per day) alone or in combination with flutamide (1 and 5 mg/kg per day). Animals were killed 24 h after the last treatment and ventral prostates were removed for testosterone and DHT determination. PNU 157706 reduced the growth of established tumors by 36%; flutamide showed a slight effect at 1 mg/kg per day (24% inhibition), while at the dose of 5 mg/kg per day it reduced tumor growth by 48%. The combination of PNU 157706 with the lower dose of flutamide caused an additive tumor growth inhibition (60%) and the combination with the higher dose of flutamide resulted in a better inhibition of tumor growth (68%) than did either treatment alone. Castration resulted in marked tumor growth inhibition (76%). Ventral prostate weight was more markedly reduced by PNU 157706 treatment than by flutamide; combined treatment was as effective as castration. Prostatic DHT content was markedly reduced by PNU 157706 (93%), whereas prostatic testosterone increased (137%). Concomitant treatment with flutamide partially antagonized the testosterone increase induced by PNU 157706 and did not modify the already considerable suppression of DHT. These data show that the inhibitory effects of PNU 157706 and flutamide on Dunning prostatic tumor growth are additive, thus supporting the rationale of this combination therapy in advanced prostate cancer, in order to achieve adequate androgen blockade with minimal side-effects. (+info)
Loss of responsiveness to transforming growth factor beta induces malignant transformation of nontumorigenic rat prostate epithelial cells.
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Transforming growth factor (TGF)-betas are multifunctional growth factors, the properties of which include the potent inhibition of epithelial cell growth. Expression patterns of TGF-betas and TGF-beta receptors in the normal prostate indicate that these growth regulators play key roles in prostatic development and proliferative homeostasis. Importantly, TGF-beta receptor levels are frequently diminished in malignant human prostate tissue. To test the hypothesis that loss of TGF-beta responsiveness is causally involved in the tumorigenic process, we have used retroviral transduction to introduce a dominant-negative mutant type II TGF-beta receptor (DNR) into the premalignant rat prostatic epithelial cell line, NRP-152. High-level expression of the DNR abolished the ability of TGF-beta to inhibit cell growth, to promote cell differentiation, and to induce apoptosis, and it partially blocked the induction of extracellular matrix gene expression. When injected into nude mice, NRP-152-DNR cells formed carcinomas at 13 of 34 sites, compared with 0 of 30 sites for parental and control cells (P = 0.0001). We conclude that the type II TGF-beta receptor is an important tumor suppressor in the prostate, and furthermore, that loss of TGF-beta responsiveness can contribute early in the tumorigenic process by causing the malignant transformation of preneoplastic cells. (+info)
Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium.
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The present study demonstrates that fibroblasts associated with carcinomas stimulate tumor progression of initiated nontumorigenic epithelial cells both in an in vivo tissue recombination system and in an in vitro coculture system. Human prostatic carcinoma-associated fibroblasts grown with initiated human prostatic epithelial cells dramatically stimulated growth and altered histology of the epithelial population. This effect was not detected when normal prostatic fibroblasts were grown with the initiated epithelial cells under the same experimental conditions. In contrast, carcinoma-associated fibroblasts did not affect growth of normal human prostatic epithelial cells under identical conditions. From these data, we conclude that in this human prostate cancer model, carcinoma-associated fibroblasts stimulate progression of tumorigenesis. Thus, carcinoma-associated fibroblasts can direct tumor progression of an initiated prostate epithelial cell. (+info)
A very rapid effect of androgen on initiation of protein synthesis in prostate.
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The initiation of protein synthesis by ribosomal particles of rat ventral prostate was studied by measuring ribosomal binding of an initiator (35-S)methionyl-tRNAf. The binding activity is dependent on ribosomes, GTP, and a prostate cytosol protein fraction. The 40S but not the 60S ribosomal subunit particles are active. The cytosol activity decreases rapidly within one hour after the rat is castrated. This loss is prevented by an intraperitoneal injection of 17beta-hydroxy-5-alpha-androstan-3-one (5-alpha-dihydrotestosterone). The cytosol activity can be stimulated almost immediately (within 10 min) after an intravenous injection of low dose (15 mug per rat) of 5-alpha-dihydrotestosterone into the castrated rat. (+info)