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Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Whether prostate cancer recurrence can be predicted by microvessel density (MVD) measurements is controversial. One reason for the lack of agreement may be the differing antibodies used to determine MVD. We evaluated MVD using 2 different antibodies against endothelial cells, CD31 and CD34, on 102 patients who underwent radical prostatectomy without adjuvant hormonal therapy. The tumors from these cases were identified, and areas with the highest Gleason pattern were immunostained. Average MVD determined by CD31 (MVD/CD31) staining was significantly lower than that obtained by MVD/CD34 staining (60.1 vs 80.3). By using Kaplan-Meier analysis, prostate-specific antigen (PSA) recurrence was correlated with MVD/CD31 and MVD/CD34. MVD/CD34 and MVD/CD31 were associated strongly with PSA recurrence on a univariate level. However, only MVD/CD34 was an independent predictor of PSA failure. Therefore, some of the confusion about MVD value as a prognostic indicator may be due to the antibodies used.

The bladder is a dynamic organ that responds to the stress of outlet obstruction by rapidly increasing in mass and cellular content to compensate for increased urethral resistance. If the outlet obstruction is released prior to decompensation, the hypertrophied bladder will shrink, returning to normal size and cellularity. However, with chronic obstruction the bladder will continue to increase in mass, developing drastic alterations in the amount and composition of the extracellular matrix and, ultimately, losing the ability to function. The extensive tissue remodeling associated with each of these changes requires different bladder components to undergo cellular proliferation, cellular hypertrophy, and even programmed cell death (apoptosis). Each of these cellular processes is known to be regulated by various peptides that are referred to as growth factors. Herein we provide an overview of the growth factors that are known to influence the bladder in addition to a variety of experimental animal studies that putatively identify a role of four particular growth factors [basic fibroblast growth factor (bFGF), transforming growth factor-ß (TGF-ß), nerve growth factor (NGF), and epidermal growth factor (EGF)] in the obstructive bladder response.

The urinary bladder responds to distension induced by a number of different stresses with rapid and substantial increases in bladder mass and concomitant alterations in the contractile responses to neuronal stimulation, pharmacological simulation by autonomic agonists, and membrane depolarization. Furosemide, sucrose, or diabetes-induced diuresis, as well as outlet obstruction and overdistension all produce similar effects on the bladder. Accompanying the increases in bladder mass and contractile changes are increases in DNA synthesis and [3H]-thymidine uptake. Autoradiographic studies have localized the increased DNA synthesis following bladder distension initially to the urothelium, followed by slower increases in labelling of the lamina propria and extramural connective tissue. The net result of these compartmental differences in DNA synthesis is a reorganization of the structural relationships between smooth-muscle cells, the connective-tissue matrix, and the extrinsic connective-tissue lamina. This may contribute to the functional changes which occur after severe overdistension. Increases in the expression of heat-shock protein-70, basic fibroblast growth factor, N-ras, and c-myc, and decreases in transforming growth factor-beta occurred acutely after obstruction, suggesting that these changes may play a role in obstruction-induced bladder hypertrophy. Removal of the obstruction induces apoptosis of urothelial and connective tissue elements in the bladder, accompanied by increases in transforming growth factor-beta and decreases in basic fibroblast growth factor genes, and a reversal of the bladder dysfunction. Therefore the bladder hyperplasia after outlet obstruction and the regression following removal of the obstruction seem to be directly opposing processes governed by gene expression.

Previously, increased expression of mRNA encoding the p53 tumor suppresor protein was described during castration-induced regression of the rat ventral prostate gland with Northern blot techniques. This activity was confirmed with a ribonuclease protection assay that demonstrated a 16-fold induction of p53 transcripts in ventral prostate RNA within 72 hrs after castration. The induced expression of p53 mRNA correlated with increased detection of p53 protein in nuclei of regressing prostate epithelial cells. Immunohistochemical staining with anti-p53 antibody was strongly reactive for epithelial nuclei in castrated glands but unreactive for nuclei of control adult glands. In contrast to the upregulation of p53 in regressing prostate glands with a large proportion of apoptotic cells, expression of p53 mRNA was decreased in rat prostate glands that were stimulated to regrow by testosterone replacement.Copyright 1994, 1999 Academic Press

Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as acute or chronic toxicities. Alternately, suppression of apoptosis through, for example, interference in cell signaling can result in pathological accumulation of aberrant cells and diseases such as tumors. Investigations into the mechanisms underlying apoptosis have extended into many areas, driven by increasingly sophisticated instrumental and molecular biology techniques. This symposium summary explores related aspects of apoptosis, including control of cell population size and function, specific gene activity and regulation, chromatin condensation and scaffold detachment, oxidative stress-induced cell proliferation versus death by apoptosis or necrosis, and hepatotoxicant-induced apoptosis versus necrosis. Insights into the mechanisms governing apoptosis and increasing appreciation of the relevance of apoptotic cell death are redirecting research in toxicology and carcinogenesis and are yielding novel therapeutic approaches for the control of toxicity, disease, and ultimately perhaps senescence.

RNA and protein products encoded by the testosterone-repressed prostate message-2 gene (TRPM-2) are induced to high levels, coordinate with the onset of cell death, in numerous rodent models of inducible tissue damage. These models include cell death initiated by hormonal stimuli (prostate regression), pressure insult (renal atrophy after ureteral obstruction), developmental stimuli (necrosis of interdigital tissue), and cytotoxic injury (chemotherapeutic regression of a tumor). Sequence analysis of cDNA encoding TRPM-2 revealed its close homology with a product referred to as SGP-2 or clusterin expressed constitutively by Sertoli cells; however, the immunologically related polypeptides expressed in regressing tissues differ in molecular mass from the forms secreted by the testis. Although the function(s) of the products encoded by the TRPM-2 gene remains unclear, their presence provides a remarkable and early indicator of programmed cell death in many types of mammalian cells.

In this paper we show that the expression of the herpes simplex virus type 1 (HSV-1) gene for thymidine kinase (tk) in HSV-transformed cells is subject to regulation by two viral products synthesized during productive infection of these cells with a tk- mutant of HSV-1. The cell line used in this study is a derivative of tk-deficient mouse L cells that, after exposure to UV-inactivated HSV-1, had acquired the HSV-1 gene for tk (which we term a resident viral gene) and consequently expressed the tk+ phenotype (LVtk+ cells). Productive infection of these cells with HSV-1(tk-) at appropriate multiplicities caused significant enhancement of the viral tk activity. The results of several experiments allow us to conclude that this enhancement was due to increased synthesis of tk specified by the HSV-1 gene resident in the LVtk+ cells and that a specific protein made early after infection with HSV-1(tk-) mediated the enhancement, probably by increasing the production of mRNA from the viral tk gene resident in the LVtk+ cells. Our data also indicate that another HSV-1(tk-) product acted to turn off tk synthesis. The finding that tk activity continued to increase for a longer time after infection of the LVtk+ cells at 2 PFU/cell than after infection at higher multiplicities suggested the synthesis of a product which inhibited tk synthesis and whose concentration reached critical levels earlier at higher multiplicities of infection. Inhibition of DNA synthesis after infection, a treatment that depresses the synthesis of late viral proteins, prolonged the synthesis of tk in LVtk+ cells infected at either 2 or 5 PFU/cell. Infection of the LVtk+ cells with HSV-2(tk-) resulted in only small increases in tk activity, indicating some type specificity in recognition of viral products that can modify the expression of the HSV-1 tk gene resident in these cells.

The interaction of sex steroid hormone receptors with the nuclear matrix (NM) of target and non-target tissue was investigated using a simple in vitro binding assay. Steroid receptors can recognize acceptor sites on the NM of target cells; androgen receptor binds with the highest apparent affinity to rat prostate NM; similarly estrogen receptor binds with the highest apparent affinity to uterine NM. Furthermore, the steroid receptor-NM interaction depends upon the hormonal status of the animal. The binding of androgen receptor to rat prostate NM was drastically reduced upon hormone withdrawal (castration) and fully recovered upon hormonal stimulation. When NM were prepared by an alternate method (DNase I digestion prior to high salt extraction) known to digest “active” chromatin, no preferential receptor binding to target tissue NM was observed. Although the NM fraction contains less than 1% of the total nuclear DNA, the matrix-associated DNA sequences seem to be, at least in part, responsible for specific receptor recognition. DNA extracted from the prostate NM was shown to be a potent competitor for androgen receptor binding as measured by DNA-cellulose competition experiments. Moreover, this DNA recognition also depends upon the hormonal status of the animal. These studies are consistent with the notion that hormonal manipulation induces changes in the NM-associated DNA sequences of steroid hormone target tissue.

Purpose: Previous studies have demonstrated that partial outlet obstruction and acute overdistension of the rabbit bladder rapidly stimulate the activation of similar patterns of gene expression. Since bladder overdistension has been shown to reduce the blood flow of the bladder and since unilateral ischemia of rabbit bladder has been found to induce similar physiologic and histologic changes to the bladder as found in association with partial outlet obstruction, we tested whether experimental ischemia of the rabbit bladder could induce the same molecular response. Materials and Methods: Unilateral ischemia was created by surgically ligating the major arteries entering the rabbit bladder to one side. Bladders were recovered from control rabbits and from rabbits at 1 hour, 8 hours, 1, 3, or 7 days after unilateral ischemia and were divided into ischemic and nonischemic portions. These tissues were used for in vitro ^3^5S-methionine and^3 H-thymidine incorporation assays. Ribonucleic acids extracted from these tissues were examined by Northern blot assay for the expression of a number of different mRNA transcripts. Results: 1) Bladder DNA synthesis was elevated 4-fold at 24 hours after ischemia; 2) protein synthesis was increased 2-fold at 8 and 24 hours after ischemia; 3) early response genes (c-fos and c-jun) mRNAs were induced by 1 hour of ischemia; hsp-70 mRNA was highly induced by 8 hours of ischemia, and bFGF mRNA was elevated 3- to 5-fold by 8 hours of ischemia. Conclusion: The early molecular response of rabbit bladder to ischemia is similar to responses observed in bladder overdistension or partial outlet obstruction.