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In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4',6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell.

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4′ ,6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell. © 2017 by the authors.

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4′ ,6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell. © 2017 by the authors.

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4',6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell.

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4′ ,6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell. © 2017 by the authors.

We investigated the mechanisms by which flavonoids (FLAV) alter neurotensin receptor (NTR) function in PC3 cells in order to shed light on the role of NT in the negative effects of fat intake on PC incidence and the positive effects of Asian diets. We found that NTR is subject to heterologous regulation by PKC. Conventional PKCs appear to exert negative effects, whereas novel PKCs exert positive effects. Our results suggest that FLAV modulate NTR by a number of mechanisms, one of which could be to alter the balance between conventional and novel PKCs. Other potential mechanisms supported by experiments reported here involve receptor tyrosine kinases, lipoxygenases and cellular metabolism. We also show here that NTR is localized to membrane caveolae, and that the agonist NT caused a decrease in the level of NTR in these signaling modules. Beta cyclodextrin, which disrupts caveolae by removing cholesterol from membranes, displaced NTR from caveolae and produced FLAV-like effects on NTR. Although FLAV did not displace NTR from caveolae, this suggested that FLAV might interact with membranes to induce similar conformational changes in NTR that alter its function. These findings have implications regarding mechanisms that regulate NTR function and the design of agents to block NT-induced growth signaling in PC., The original document contains color images.

We propose that AR phosphorylation at serines 213 and 650 regulate differential target gene expression and recruitment to gene promoters via altered interaction with other cellular transcription factors. To test this hypothesis we have conducted yeast two-hybrid analysis with the N-terminus of wild type AR as well as AR S213A and AR S213E variants. Our preliminary analysis indicates that the screen is preferentially isolating proteins with a known role in gene transcription and we are currently assessing the phosphorylation-dependence of the putative AR interacting proteins. Additionally, we have generated PC3 cells stably transfected with wild type, S650A and S650E AR. We have shown that the cells activate endogenous target genes in response to androgens and are currently investigating classes of genes affected by differential AR phosphorylation. Further, we have optimized conditions to isolate pools of hyper- and hypo-phosphorylated AR in order to isolate proteins that interact with AR in a phosphorylation-dependent manner. These proteins were identified via mass spectrometry., Grant No. PC050571. The original document contains color images.

Arginine deprivation by arginine deiminase (ADI) is a novel therapy that is effective against prostate cancers that lack argininosuccinate synthetase (ASS), the rate-limiting enzyme for de novo arginine synthesis. We show that in ASS negative CWR22Rv1 prostate cancer cells, ADI rapidly induced autophagy through AMPK/mTOR/S6K as well as ERK1/2 pathways in order to delay the onset of caspase-independent apoptosis. Inhibiting autophagy with chloroquine or Beclin1 siRNA accelerated and enhanced ADI-induced apoptosis. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer., The original document contains color images.

We have been successful in creating the necessary constructs for generating prostate cancer cells inducible for FGFR4. Despite our best efforts, however, we have not been able to successfully incorporate the pVgRXR regulatory plasmid into prostate cancer cells. We proceeded by transiently transfecting PC3 prostate cancer cells to look at effects on downstream signaling components and found no significant differences in MAPK activity, NCAM expression or STAT1 and STAT5 localization when comparing the various FGFR4 constructs, including FL-FGFR4 and PTD-FGFR4. We have also made a very exciting discovery recently that may be important for understanding the role of FGFR4 in prostate cancer progression. Using a yeast-two-hybrid assay, we found that FGFR4 interacts with IKKbeta and leads to its tyrosine phosphorylation. We are currently investigating if there are any differences in binding tyrosine phosphorylation with the G388R polymorphism. Though not specifically relating to cell adhesion, understanding the role of FGFR4 in altering IKKbeta activity may be important for our understanding of prostate cancer progression., The original document contains color images.

We have been successful in creating the necessary constructs for generating prostate cancer cells inducible for FGFR4. Despite our best efforts, however, we have not been able to successfully incorporate the pVgRXR regulatory plasmid into prostate cancer cells. We proceeded by transiently transfecting PC3 prostate cancer cells to look at effects on downstream signaling components and found no significant differences in MAPK activity, NCAM expression or STAT1 and STAT5 localization when comparing the various FGFR4 constructs, including FL-FGFR4 and PTD-FGFR4. We have also made a very exciting discovery recently that may be important for understanding the role of FGFR4 in prostate cancer progression. Using a yeast-two-hybrid assay, we found that FGFR4 interacts with IKKbeta and leads to its tyrosine phosphorylation. We are currently investigating if there are any differences in binding tyrosine phosphorylation with the G388R polymorphism. Though not specifically relating to cell adhesion, understanding the role of FGFR4 in altering IKKbeta activity may be important for our understanding of prostate cancer progression.

A tripartate prodrug of S-trityl-L-cysteine was synthesized and investigated for antiprostatic potency versus the free anticancer agent (S-trityl- L-cysteine) in PSA-secreting cells (LNCap) and non-PSA-secreting cells (PC3). The prodrug did not inhibit cell growth as much as S-trityl-Lcysteine itself was not selective for PSA-secreting cells, and did not release S-trityl-L-cysteine when treated with cell-free PSA. Interestingly, S-trityl-L-cysteine itself selectively inhibited the growth of LNCap versus PC3 cells., The original document contains color images.

The purpose is to define the relationship between neurotensin (NT) and protein kinase C (PKC) isotypes and to investigate the mechanism by which flavonoids (FLAV) inhibit NT growth signaling in PC3 cells. The long-range scope is to determine the significance of NT in the negative effects of high fat intake on PC incidence and the positive effects of diets containing FLAV. Our results show that NT-induced growth signaling involves and requires activation of several PKC isotypes (most notably PKC epsilon and delta) that arachidonic acid metabolism and EGF receptor activation participate in the NT signaling process that cell metabolism and ATP levels can influence NT receptor function and that activated PKC (most notably PKC alpha and beta) can feed back to regulate the ability of NT receptor to bind NT and to initiate signaling. FLAV was found to exert differential effects on PKC isotype activation and downregulation. Thus FLAV could alter the balance between conventional and novel PKC acbvity which could influence growth responses to NT. These findings have implications regarding mechanisms that regulate NT receptor function and the design of agents to block NT-induced growth signaling in PC., The original document contains color images.

The purpose is to define the relationships between neurotensin (NT) and protein kinase C (PKC) isotypes and to investigate the mechanism by which flavonoids (FLAV) inhibit NT-induced signaling in PC3 cells. The long-range scope is to determine the significance of NT as a participant in the negative effects of high fat intake on PC incidence and growth, and the positive effects of diets containing large amounts of FLAV. Our results show that NTinduced growth signaling in PC3 cells involves and requires the activation of several PKC isotypes, that arachidonic acid release and lipoxygenase activity participate in the signaling process, that cellular metabolism and ATP levels are important inputs to NT receptor function, and that activated PKC feeds back to regulate the ability of NT receptor to bind and to initiate signaling. These findings have implications regarding general mechanisms of G protein-linked receptor function and the design of new agents to block NT-induced growth signaling in PC., The original document contains color images.

We have made excellent progress in the preliminary stages of our project to examine the role of FGFR4 G388R in altering cell adhesion in prostate cancer. This includes acquiring expertise in the passage and transfection for gene expression studies using prostate cancer cell lines. A key accomplishment is the demonstration of feasibility of ponasterone; A inducibility of FGFR4 expression in PC3 cells. We hope to successfully create these inducible cells to use in our studies of FGFR4 G388R and its role in prostate cancer progression., The original document contains color images.

The main purpose of the proposed study was to evaluate the toxicity and efficacy of multiple targeting vectors for the treatment of prostate cancer in mouse models. It included Avastin, an anti-vascular endothelial growth factor (VEGF) antibody and PAI2, a protein that targets uroplasminogen activation (uPA) receptors in the PA system; both to be used in combination therapy together with labeling with alpha-emitting radionuclide, Bismuth-213. New methods for the labeling of Avastin were developed with labeling efficiency in excess of 90%. The resulting conjugate was tested for in vitro stability and was found to be stable with less than 20% leaching over a period of 5 half-lives of the isotope. Enhancement of plasminogen activation expression was achieved with VEGF with PC3 cell line showing the maximum enhancement, DU145 showing some enhancement whereas LN3-LNCaP showing no change in its other negative uPA expression. VEGF secretion was estimated for the three cell lines and based on that PC3 was chosen for the tumour model development. Subcutaneous and orthotopic tumour models were successfully developed in mice and are subject of the ongoing in vivo studies. Successful labeling and stability testing of the Avastin conjugate is world first and remains the most significant finding of the study to-date., The original document contains color images.