Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate.

Prostate cancer is the second most common cause of death from cancer in U.S. men, and advanced, hormone-refractory disease is characterized by painful osteoblastic bone metastases. Endothelin-1, more commonly known as a potent vasoconstrictor, is a normal ejaculate protein that also stimulates osteoblasts. We show here that plasma immunoreactive endothelin concentrations are significantly elevated in men with metastatic prostate cancer and that every human prostate cancer cell line tested produces endothelin-1 messenger RNA and secretes immunoreactive endothelin. Exogenous endothelin-1 is a prostate cancer mitogen in vitro and increases alkaline phosphatase activity in new bone formation, indicating that ectopic endothelin-1 may be a mediator of the osteoblastic response of bone to metastatic prostate cancer.

Herpes simplex virus vector-mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury.

Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrophic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication-defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein, we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before being killed to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared with untreated or control vector-treated groups. The HSV-NTN group had more FG-positive major pelvic ganglion neurons than the control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for the improvement of ED following cavernous nerve injury.

Perceptual learning after test-stimulus exposure in humans.

Exposure to a to-be-tested stimulus produces a reduction in generalization to that stimulus from another similar conditioned stimulus (e.g. Bennett et al., 1994; Symonds and Hall, 1997). Generally, this effect has been interpreted as the result of a loss of effectiveness of the common elements of the stimulus to be conditioned (e.g., latent inhibition). However, Sanjuan et al. (2006) questioned this interpretation after finding that exposing rats to either the test stimulus or to its elements had different effects when the amount of exposure to the common elements was equated. Only exposure to the test stimulus reduced generalization. In the study presented here, this effect was assessed in human participants using a videogame method and colors as stimuli. Generalization after exposure to the test stimulus, or to its elements was assessed. Results show that with people, as in rats, pre-exposure to the test stimulus leads to a greater reduction in generalization than to the elements. Therefore, latent inhibition cannot be the only mechanism responsible for this perceptual learning effect. Results are discussed in terms of current associative theories addressing perceptual-learning phenomenon.

Customizing kernel functions for SVM-based hyperspectral image classification.

Previous research applying kernel methods such as support vector machines (SVMs) to hyperspectral image classification has achieved performance competitive with the best available algorithms. However, few efforts have been made to extend SVMs to cover the specific requirements of hyperspectral image classification, for example, by building tailor-made kernels. Observation of real-life spectral imagery from the AVIRIS hyperspectral sensor shows that the useful information for classification is not equally distributed across bands, which provides potential to enhance the SVM's performance through exploring different kernel functions. Spectrally weighted kernels are, therefore, proposed, and a set of particular weights is chosen by either optimizing an estimate of generalization error or evaluating each band's utility level. To assess the effectiveness of the proposed method, experiments are carried out on the publicly available 92AV3C dataset collected from the 220-dimensional AVIRIS hyperspectral sensor. Results indicate that the method is generally effective in improving performance: spectral weighting based on learning weights by gradient descent is found to be slightly better than an alternative method based on estimating "relevance" between band information and ground truth.

DHX15 promotes prostate cancer progression by stimulating Siah2-mediated ubiquitination of androgen receptor.

Androgen receptor (AR) activation is critical for prostate cancer (PCa) development and progression, including castration resistance. The nuclear export signal of AR (NESAR) has an important role in AR intracellular trafficking and proteasome-dependent degradation. Here, we identified the RNA helicase DHX15 as a novel AR co-activator using a yeast mutagenesis screen and revealed that DHX15 regulates AR activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity. DHX15 and Siah2 form a complex with AR, through NESAR. DHX15 stabilized Siah2 and enhanced its E3 ubiquitin-ligase activity, resulting in AR activation. Importantly, DHX15 was upregulated in PCa specimens and its expression was correlated with Gleason scores and prostate-specific antigen recurrence. Furthermore, DHX15 immunostaining correlated with Siah2. Finally, DHX15 knockdown inhibited the growth of C4-2 prostate tumor xenografts in mice. Collectively, our data argue that DHX15 enhances AR transcriptional activity and contributes to PCa progression through Siah2.

A system for detecting high impact-low frequency mutations in primary tumors and metastases.

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

Adjuvant and salvage treatment options for patients with high-risk prostate cancer treated with radical prostatectomy.

The management of high-risk prostate cancer following radical prostatectomy remains a treatment dilemma. Multimodality approaches incorporating surgery, radiation therapy and systemic agents offer the hope of improved cure rates; however, most randomized studies to date are either immature or negative. The systemic treatment options best studied is androgen deprivation, which has been shown to demonstrate a survival advantage in patients with lymph node-positive disease. Systemic chemotherapy has demonstrated a modest survival advantage in androgen-independent disease. Current studies are exploring its role in the adjuvant and neo-adjuvant setting. Lastly, recent randomized trials have demonstrated a biochemical advantage to adjuvant radiation therapy, but it remains to be seen if this will translate to an improvement is survival end points or if salvage radiation therapy would be just as effective. In this update article, we review the use of external beam radiation therapy and systemic agents in combination with surgery for high-risk prostate cancer patients.

EAF2 regulates DNA repair through Ku70/Ku80 in the prostate.

Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells.

In this study, a panel of normal human prostate cells (HPCs) and tumor cells derived from metastases were studied by (1)H NMR spectroscopy to determine whether the malignant transformation of HPCs results in the elevation of choline compounds. Although an elevated choline signal has been observed previously in clinical studies, the contribution of the different Cho compounds to this elevation, as well as their quantification, has not been established until now. Here we have shown that HPCs derived from metastases exhibit significantly higher phosphocholine as well as glycerophosphocholine levels compared with normal prostate epithelial and stromal cells. Thus the elevation of the choline peak observed clinically in prostate cancer is attributable to an alteration of phospholipid metabolism and not simply to increased cell density, doubling time, or other nonspecific effects. Androgen deprivation of the androgen receptor-positive cell lines resulted in a significant increase of choline compounds after chronic androgen deprivation of the LNCaP cell line and in a decrease of choline compounds after a more acute androgen deprivation of the LAPC-4 cell line. These data strongly support the use of proton magnetic resonance spectroscopic imaging to detect the presence of prostate cancer for diagnosis, to detect response subsequent to androgen ablation therapy, and to detect recurrence.

Methylation of the 5' CpG island of the endothelin B receptor gene is common in human prostate cancer.

Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.

Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer.

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.

Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells.

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.

Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue.

In previous studies, we have demonstrated that androgen ablation-induced growth inhibition of androgen-responsive PC-82 and A-2 human prostate cancer xenografts involves not only direct activation of programmed (apoptotic) death of these cells but also indirect activation of this death process via a decrease in tumor angiogenesis secondary to a reduction in tumor vascular endothelial growth factor (VEGF) levels. To determine whether androgens consistently regulate angiogenesis via control of VEGF levels, an additional human (i.e., LnCaP) and two rodent (i.e., Dunning G and H) androgen-sensitive prostate cancer sublines were tested. Androgen ablation causes a decrease in the subsequent growth rate of each of these three additional prostate cancer sublines, and this growth inhibition is consistently associated with a >60% reduction in tumor VEGF levels. To examine whether androgens regulate VEGF levels not only in malignant but also in normal prostatic tissue, male rats were castrated, and the temporal changes in the VEGF content of ventral prostate tissue were determined. One week after castration, VEGF content decreased to <20% within the ventral prostate. Subsequent replacement with exogenous androgen to long-term castrated rats stimulated an 8-fold rise in ventral prostate VEGF content within 1 week. To evaluate whether androgen regulation of VEGF is due to a direct effect of androgen on prostatic cells, the dose-response ability of androgens to increase VEGF levels in media of LnCaP cells grown in vitro was tested. These studies demonstrate that androgens directly stimulate VEGF secretion in these cells. The presence of 4-5-fold higher levels of VEGF in prostatic fluid versus seminal vesicle fluid obtained from benign prostatic hyperplasia and clinically localized prostate cancer patients suggests that elevated levels of VEGF may contribute to the progression of these prostatic conditions by promoting angiogenesis. In summary, one of the mechanisms for androgen sensitivity for the control of the growth of both normal and malignant prostatic tissue is via its stimulation of VEGF levels.

Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate.

Phenylbutyrate (PB), a novel lead compound for prostate cancer therapy, has molecular activities distinct from its metabolite, phenylacetate (PA). Both PB and PA promote differentiation in human prostate cancer cell lines, yet little data exist comparing the cytotoxic effects of each drug. We found that PB is more potent than PA in vitro; PB is 1.5-2.5 times more active at inhibiting growth and inducing programmed cell death than PA at clinically achievable doses against each human prostate cancer line studied. PB is equipotent to sodium butyrate, which induces apoptosis and differentiation through multiple mechanisms. Exposure of prostate cancer cell lines to PB reduces their DNA synthesis, leads to fragmentation of genomic DNA, and causes 50-60% of cells to undergo apoptosis. These PB-induced effects are 2-10 times greater than those of the control or PA. The stereotypical changes of apoptosis can be seen with sodium butyrate at similar concentrations, but not with PA. Prostate cancer cell lines overexpressing P-glycoprotein or possessing heterogeneous molecular alterations, including p53 mutations, are also sensitive to the effects of PB. In vivo, Copenhagen rats treated with oral PB had delayed growth of the androgen refractory Dunning R-3327 MAT-LyLu prostate cancer subline by 30-45% in a dose-dependent manner. These results demonstrate that PB induces cytotoxicity via apoptosis in human prostate cancer, in addition to its differentiating properties.

Methylation of the neutral endopeptidase gene promoter in human prostate cancers.

Neutral endopeptidase 24.11 (NEP) is a cell surface peptidase expressed by prostatic epithelial cells that cleaves and inactivates neuropeptide growth factors implicated in the growth of androgen-independent prostate cancer (PC). Decreased NEP expression in hormone-refractory metastatic PCs can result from hormonal therapies because NEP transcription is induced by androgens and down-regulated by androgen withdrawal. NEP is encoded by a gene that contains a 5' CpG island spanning a transcriptional regulatory region. In this study, we investigate whether DNA hypermethylation of the NEP promoter accompanies decreased NEP expression in PC cell lines and whether it occurs in human PC tissues in vivo. DNA isolated from PC cell lines and from normal and neoplastic human prostate tissues was restriction-digested with a methylation-sensitive restriction endonuclease and analyzed by Southern blot using a 5' sequence-specific NEP probe. Methylation-specific PCR was performed using PCR primers designed to discriminate between methylated and unmethylated alleles, and reverse transcription-PCR using NEP-specific primers was performed on cDNA extracted from PC cells treated with 5-aza-2'-deoxycytidine. Methylation of the NEP promoter was present in androgen-independent PC cell lines but not in androgen-dependent or small-cell derived PC cell lines and in 3 of 21 (14%) primary PCs from patients with androgen-dependent disease. Exposure of PC cells to the demethylating agent 5-aza-2'-deoxycytidine led to an increase in NEP transcripts in DU-145 and PC-3 cells. These data show that hypermethylation of the 5' CpG NEP island is associated with a loss of NEP expression in PC. Loss of NEP expression via hypermethylation of the NEP promoter may contribute to the development of neuropeptide-stimulated PCs.

Stimulus generalization, context change, and forgetting.

Forgetting is often attributed to retrieval failure caused by background contextual cues changing over time. However, generalization between stimuli may increase over time and make them increasingly interchangeable. If this effect occurs with contextual cues, it might cancel any effect of a changing context. The authors review the evidence and suggest a resolution of this paradox. Although generalization gradients can change over time, the effect is not always strong. Increased responding to nontarget stimuli is not often shown, and few studies have demonstrated such changes with contextual cues in a way that rules out other interpretations. Even this example of forgetting may be caused by retrieval failure. The physical contexts manipulated in learning and memory experiments themselves occur within a superordinate temporal context and can thus be forgotten with no inherent challenge to a context-change account of forgetting.

The role of an intensive care unit in a community hospital. A ten-year review with observations on utilization past, present, and future.

A retrospective ten-year review of the surgical-intensive-care-unit utilization practices at Riverside Community Hospital revealed that, according to the author's criteria, an average of 32% of over 800 yearly admissions could have been safely managed in a less intensive and expensive environment. The admission practices, monitoring parameters, length of stay, and intensity of nursing interaction were evaluated and compared with those of published reports. Prospective payment by diagnosis-related groups will likely force a change in the existing use of surgical intensive care units. Surgeons are urged to examine the utilization of their hospitals' intensive care units and actively work with their hospital administration to establish intermediate care units so that patients will not be jeopardized by the impending fiscal constraints of diagnosis-related groups.

Extra-hippocampal projections of CCK neurons of the hippocampus and subiculum.

A population of neurons in the hippocampus and subiculum contains cholecystokinin (CCK). Following transection of the dorsal fornix, a major afferent pathway of the hippocampus and associated structures. CCK levels were reduced in the septum and hypothalamus. A microdissection analysis indicated that the loss of CCK occurred in nuclei receiving direct projections from the hippocampus and subiculum, suggesting that CCK-containing neurons in the hippocampus and subiculum project to extrahippocampal regions.

Evaluation of the effect of spinal cord injury on serum PSA levels.

OBJECTIVES: Prostatic structure and secretory activity are thought to be influenced by autonomic innervation of the prostate. Prostatic denervation is especially likely in patients with spinal cord injury (SCI) at the level of the cauda equina or the conus medullaris, where the peripheral nerve supply to the prostate may be specifically damaged. This may result in changes in serum prostate-specific antigen (PSA) levels, either directly or indirectly. Therefore, we measured serum PSA levels and also studied the influence of factors such as age, catheterization, duration of SCI, urinary tract infection, and history of cystitis on serum PSA values in men with SCI. METHODS: Serum PSA levels were determined in 79 men with SCI (age older than 40 years) using banked sera by the Abbott MEIA PSA assay. Variables such as age, catheterization, duration of SCI, urine culture results, and history of cystitis were obtained from a review of patient records. Comparisons were made with a randomly selected, non-SCI control population of 501 men, 40 to 89 years old, who underwent serum PSA determination at our institution. Statistical comparisons were performed using the Mann-Whitney U test (nonparametric), since the populations were not normally distributed. Multivariate logistic regression analysis was used to assess the correlation between the various factors and the serum PSA levels in men with SCI. RESULTS: No statistically significant differences were found in the median serum PSA values between the SCI group and the non-SCI control population. The age-specific PSA values obtained in the SCI group were also comparable to those reported for the general population at large. Age (P <0.03) and the presence of a catheter (P <0.0002) were the only two factors that were correlated with higher serum PSA values in the SCI group by regression analysis. CONCLUSIONS: Men with SCI tended to have serum PSA value distributions that were similar to those of the general population. However, those in the SCI group who had indwelling catheters were more likely to have higher PSA values at baseline, as were older men with SCI.