UROtsa Cells Research Articles

A time-series analysis of altered histone H3 acetylation and gene expression during the course of MMAIII-induced malignant transformation of urinary bladder cells.

Our previous studies have shown that chronic exposure to low doses of monomethylarsonous acid (MMAIII) causes global histone acetylation dysregulation in urothelial cells (UROtsa cells) during the course of malignant transformation. To reveal the relationship between altered histone acetylation patterns and aberrant gene expression, more specifically, the carcinogenic relevance of these alterations, we performed a time-course analysis of the binding patterns of histone 3 lysine 18 acetylation (H3K18ac) across the genome and generated global gene-expression profiles from this UROtsa cell malignant transformation model. We showed that H3K18ac, one of the most significantly upregulated histone acetylation sites following MMAIII exposure, was enriched at gene promoter-specific regions across the genome and that MMAIII-induced upregulation of H3K18ac led to an altered binding pattern in a large number of genes that was most significant during the critical window for MMAIII-induced UROtsa cells' malignant transformation. Some genes identified as having a differential binding pattern with H3K18ac, acted as upstream regulators of critical gene networks with known functions in tumor development and progression. The altered H3K18ac binding patterns not only led to changes in expression of these directly affected upstream regulators but also resulted in gene-expression changes in their regulated networks. Collectively, our data suggest that MMAIII-induced alteration of histone acetylation patterns in UROtsa cells led to a time- and malignant stage-dependent aberrant gene-expression pattern, and that some gene regulatory networks were altered in accordance with their roles in carcinogenesis, probably contributing to MMAIII-induced urothelial cell malignant transformation and carcinogenesis.

Protective effects of organic and inorganic selenium against the toxicity of 3,5-dimethylaminophenol in UROtsa cells

Protective effects of organic and inorganic selenium against the toxicity of 3,5-dimethylaminophenol in UROtsa cells

Elevated connexin 43 expression in arsenite-and cadmium-transformed human bladder cancer cells, tumor transplants and selected high grade human bladder cancers

Connexin 43 has been shown to play a role in cell migration and invasion; however, its role in bladder cancer is not well defined. Previous studies from our laboratory have shown that the environmental pollutants arsenite and cadmium can cause malignant transformation of the immortalized urothelial cell line UROtsa. These transformed cells can form tumors in immune-compromised mice. The goal of the present study was to determine if connexin 43 is expressed in the normal human bladder, the arsenite and cadmiun-transformed UROtsa cells as well as human urothelial cancer. The results obtained showed that connexin 43 is not expressed in the epithelial cells of the human bladder but is expressed in immortalized cultures of human urothelial cells and the expression is variable in the arsenite and cadmium- transformed urothelial cell lines derived from these immortalized cells. Tumor heterotransplants generated from the transformed cells expressed connexin 43 and the expression was localized to areas of squamous differentiation. Immuno-histochemical analysis of human bladder cancers also showed that the expression of connexin 43 was localized to areas of the tumor that showed early features of squamous differentiation. Treatment of UROtsa cells with various concentrations of arsenite or cadmium did not significantly alter the expression level of connexin 43. In conclusion, our results show that the expression of connexin 43 is localized to the areas of the tumor that show squamous differentiation, which may be an indicator of poor prognosis. This suggests that connexin 43 has the potential to be developed as a biomarker for bladder cancer that may have the ability to invade and metastasize.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

BackgroundEpithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown.MethodsReal-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice.ResultsThis study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin.ConclusionsThe finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells “seeding” a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth.

Toxicological characterisation of a thio-arsenosugar-glycerol in human cells

Arsenosugars are water-soluble arsenic species predominant in marine algae and other seafood including mussels and oysters. They typically occur at levels ranging from 2 to 50mg arsenic/kg dry weight. Most of the arsenosugars contain arsenic as a dimethylarsinoyl group (Me2As(O)-), commonly referred to as the oxo forms, but thio analogues have also been identified in marine organisms and as metabolic products of oxo-arsenosugars. So far, no data regarding toxicity and toxicokinetics of thio-arsenosugars are available. This in vitro-based study indicates that thio-dimethylarsenosugar-glycerol exerts neither pronounced cytotoxicity nor genotoxicity even though this arsenical was bioavailable to human hepatic (HepG2) and urothelial (UROtsa) cells. Experiments with the Caco-2 intestinal barrier model mimicking human absorption indicate for the thio-arsenosugar-glycerol higher intestinal bioavailability as compared to the oxo-arsenosugars. Nevertheless, absorption estimates were much lower in comparison to other arsenicals including arsenite and arsenic-containing hydrocarbons. Arsenic speciation in cell lysates revealed that HepG2 cells are able to metabolise the thio-arsenosugar-glycerol to some extent to dimethylarsinic acid (DMA). These first in vitro data cannot fully exclude risks to human health related to the presence of thio-arsenosugars in food.

Para-Phenylenediamine induces apoptosis through activation of reactive oxygen species-mediated mitochondrial pathway, and inhibition of the NF-κB, mTOR, and Wnt pathways in human urothelial cells.

para-Phenylenediamine (PPD) has long been used in two-thirds of permanent oxidative hair dye formulations. Epidemiological studies and in vivo studies have shown that hair dye is a suspected carcinogen of bladder cancer. However, the toxicity effects of PPD to human bladder remains elusive. In this study, the effects of PPD and its involvement in the apoptosis pathways in human urothelial cells (UROtsa) was investigated. It was demonstrated that PPD decreased cell viability and increased the number of sub-G1 hypodiploid cells in UROtsa cells. Cell death due to apoptosis was detected using Annexin V binding assay. Further analysis showed PPD generated reactive oxygen species (ROS), induced mitochondrial dysfunction through the loss of mitochondrial membrane potential and increased caspase-3 level in UROtsa cells. Western blot analysis of PPD-treated UROtsa cells showed down-regulation of phosphorylated proteins from NF-κB, mTOR, and Wnt pathways. In conclusion, PPD induced apoptosis via activation of ROS-mediated mitochondrial pathway, and possibly through inhibition of NF-κB, mTOR, and Wnt pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 265-277, 2017.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

BackgroundThis laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.MethodsReal time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.ResultsIt was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.ConclusionsTumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

Abstract A05: Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53/DNMT3b-mediated activation of MMP2

Abstract Epigenetic-mediated gene activation/silencing plays a crucial role in human tumorigenesis. Eliciting the underlying mechanism behind certain epigenetic changes is essential for understanding tumor biology. An important epigenetic process that is implicated in mammalian development is the methylation of cytosine within the context of a simple dinucleotide site, CpG. Deregulated DNA methylation can be a major driver of pathologic conditions such as neurological and autoimmune diseases, as well as cancers Previous studies in human cancers revealed an unrecognized interplay between Angiogenin (ANG)-MAPK/ERK- matrix metalloproteinase-2 (MMP2) leading to pronounced tumorigenesis. Specifically, we showed that forced ANG expression in a benign human bladder UROtsa cell line induced cellular survival, proliferation, endothelial tube formation and xenograft angiogenesis and growth by mediating the MAPK/ERK-MMP2 axis. Here we provide multiple lines of evidence indicating ANG oncogenic potential and how ANG regulates MMP2 expression. Here we have shown that deletion and site-directed mutagenesis analysis of the MMP2 promoter demonstrated that the p53-binding site is critical for ANG-induced MMP2 promoter activity. To further determine whether ANG facilitates the recruitment of p53 to its targets in cells, we used a ChIP assay to examine p53 binding to the promoter region of the MMP2 gene in UROtsaEmpty, UROtsaANG, RT112 Scr and RT112 KD-ANG. Overexpression of ANG greatly enriched p53 association with the MMP2 promoter and ANG depletion decreased enrichment of p53 with MMP2 promoter. To further explore the mechanism by which ANG regulates MMP2 expression, we investigated whether ANG is also functionally linked to DNA methylation-based transcriptional repression. First, we quantitatively assessed the methylation status of the MMP2 promoter. Overexpression of ANG was associated with a reduction in methylation status of the MMP2 promoter, which led to increased gene expression of MMP2. The alteration of genome-wide DNA methylation in these cells was then analyzed using Illumina whole genome methylation array. ANG expression was noted to be associated with a change in global DNA methylation levels compared to control cells. Mechanistically, ANG negatively regulated DNA methyltransferase 3b (DNMT3b) enzymatic activity by down-regulating its expression and inhibiting its recruitment to the MMP2 promoter. To further define the role of ANG, p53 and MMP2 in tumorigenesis, we collected 78 fresh bladder cancer samples from bladder cancer patients and extracted DNA and RNA for analysis. Muscle invasive bladder cancer specimens had increased levels of ANG, p53 and MMP2 compared to non-muscle invasive bladder cancer. More importantly, disease free survival was adversely and significantly effected if ANG, p53 and MMP2 were overexpressed. In this study we demonstrate that ANG is overexpressed in approximately 45% of bladder tumors resulting in overexpression of p53 and MMP2, a reduction in DNMT3b and deep changes in the methylation status genome wide. Such molecular alterations are associated with higher rates of muscle invasive bladder cancers as well as a reduction in disease specific survival, thus attesting to ANG, p53 and MMP2 ability to further risk stratify patients that may require a more aggressive, even personalized, management plan. Therefore, the study provides novel evidence that ANG plays an important role in the promoter activation of the tumor oncogenic gene MMP2, which likely contributes to ANG oncogenic activity. Citation Format: Rafael Peres, Hideki Furuya, Ian Pagano, Yoshiko Shimizu, Kanani Hokutan, Joanna Gawecka, Charles Joel Rosser. Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53/DNMT3b-mediated activation of MMP2. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A05.

Effect of fibronectin-gelatin coating on 3D assembly of urothelial tissue constructs in vitro

Event Abstract Back to Event Effect of fibronectin-gelatin coating on 3D assembly of urothelial tissue constructs in vitro James P. Turner1, Julianne T. Jett2 and Jiro Nagatomi1 1 Clemson University, Bioengineering, United States 2 Clemson University, Biological Sciences, United States Introduction: Currently, limited solutions are available to regenerate the damaged urothelial layer caused by congenital bladder disorders, cancer of the bladder, and trauma. The goal of the present study is to develop a 3D urothelial tissue construct in vitro using the layer-by-layer cell assembly technique. Specifically, urothelial cells differentiated from human adipose-derived stem cells (hADSCs) were subjected to coating with alternating layers of fibronectin and gelatin prior to seeding and culturing. Cell morphology, phenotypic marker expression, and barrier function of the constructs were examined. Materials and Methods: For urothelial differentiation, hADSCs were cultured in defined keratinocyte serum-free media (KSFM) with supplementation. UROtsa cells were cultured in DMEM supplemented with 10% FBS and 1% Glutamax. Cells were coated via suspension in 9 alternating layers of fibronectin and gelatin at 0.04 mg/mL concentration. Coated cells were placed in either fibronectin-coated or fibronectin-gelatin multilayer-coated Transwell® inserts. To achieve an 8-layered tissue, hADSCs and UROtsa cells were seeded at 8.0x105 and 2.0x106 cells per well, respectively and cultured for at least 24 hours and up to 10 days. At the end of the prescribed time period, cells on insert membranes were fixed and sectioned for H&E staining, and immunostaining for CK-17, CK-20, UPIb, and ZO-1. Permeability across hADSC and UROtsa multilayers was measured using fluorescently labeled dextran (4 kDa, FITC; 70 kDa rhodamine B) and a microplate reader. Results and Discussion: Fibronectin-gelatin-coated, hADSC-derived urothelial cells on fibronectin-coated membrane showed aggregation after 24 hours indicating incomplete formation of a multilayer. In contrast, fibronectin-gelatin-coated UROtsa cells cultured on a fibronectin-gelatin-coated membrane displayed uniform layering and expression of tight junction protein ZO-1. Uncoated cells or cells seeded on an uncoated membrane exhibited a scattered appearance and less expression of ZO-1. Together, these results suggest that the fibronectin-gelatin coating facilitates cell-cell and membrane adhesion in UROtsa cells, which is critical for formation of 3D tissue constructs. In addition, permeability studies showed that there was significant difference in transcellular and paracellular transports between cell-seeded and empty culture inserts. However, results were similar between coated and uncoated hADSCs and UROtsa cells indicating that further maturation time may be necessary to create barrier function in the 3D multilayered cell constructs. Conclusion: The results of the present study provided evidence that the cellular microenvironment modified by ECM proteins would make a significant impact on the formation of 3D multilayered cell structure. Further experimentation is, however, needed to determine the effect of other parameters such as cell maturation and numbers of hADSC-derived urothelial cells on the barrier function of the multilayer construct, which is not only useful for regenerative medicine, but also as test platforms for drug screening and other experimentations in vitro. Keywords: Regenerative Medicine, Tissue Engineering, stem cell, 3D scaffold Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials in constructing tissue substitutes Citation: Turner JP, Jett JT and Nagatomi J (2016). Effect of fibronectin-gelatin coating on 3D assembly of urothelial tissue constructs in vitro. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00319 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers James P Turner Julianne T Jett Jiro Nagatomi Google James P Turner Julianne T Jett Jiro Nagatomi Google Scholar James P Turner Julianne T Jett Jiro Nagatomi PubMed James P Turner Julianne T Jett Jiro Nagatomi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase.

BackgroundThe glycosylphosphatidylinositol-anchored extracellular membrane serine protease prostasin is expressed in normal bladder urothelial cells. Bladder inflammation reduces prostasin expression and a loss of prostasin expression is associated with epithelial-mesenchymal transition (EMT) in human bladder transitional cell carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of various cancers including bladder cancer, but the molecular mechanisms underlying the anticancer effect of NSAIDs are not fully understood.MethodsThe normal human bladder urothelial cell line UROtsa, the normal human trophoblast cell line B6Tert-1, human bladder transitional cell carcinoma cell lines UM-UC-5 and UM-UC-9, and the human breast cancer cell line JIMT-1 were used for the study. Expression changes of the serine proteases prostasin and matriptase, and cyclooxygenases (COX-1 and COX-2) in these cells following ibuprofen treatments were analyzed by means of reverse-transcription/quantitative polymerase chain reaction (RT-qPCR) and immunoblotting. The functional role of the ibuprofen-regulated prostasin in epithelial tight junction formation and maintenance was assessed by measuring the transepithelial electrical resistance (TEER) and epithelial permeability in the B6Tert-1 cells. Prostasin’s effects on tight junctions were also evaluated in B6Tert-1 cells over-expressing a recombinant human prostasin, silenced for prostasin expression, or treated with a functionally-blocking prostasin antibody. Matriptase zymogen activation was examined in cells over-expressing prostasin.ResultsIbuprofen increased prostasin expression in the UROtsa and the B6Tert-1 cells. Cyclooxygenase-2 (COX-2) expression was up-regulated at both the mRNA and the protein levels in the UROtsa cells by ibuprofen in a dose-dependent manner, but was not a requisite for up-regulating prostasin expression. The ibuprofen-induced prostasin contributed to the formation and maintenance of the epithelial tight junctions in the B6Tert-1 cells. The matriptase zymogen was down-regulated in the UROtsa cells by ibuprofen possibly as a result of the increased prostasin expression because over-expressing prostasin leads to matriptase activation and zymogen down-regulation in the UROtsa, JIMT-1, and B6Tert-1 cells. The expression of prostasin and matriptase was differentially regulated by ibuprofen in the bladder cancer cells.ConclusionsIbuprofen has been suggested for use in treating bladder cancer. Our results bring the epithelial extracellular membrane serine proteases prostasin and matriptase into the potential molecular mechanisms of the anticancer effect of NSAIDs.

Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications.

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffold's 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 μm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications.

Abstract 1171: Increased expression of PDK4 in bladder cancer cells

Abstract Introduction and Objectives. Cancer cells have a high rate of glucose metabolism due to an increased requirement for biomolecules and energy. In some solid tumors, cancer cells divert pyruvate to lactate even in the presence of oxygen (Warburg effect) providing a survival advantage in an acidic and anaerobic environment. Pyruvate dehydrogenase complex (PDC) catalyzes oxidative decarboxylation of pyruvate to acetyl-CoA and links glycolysis to energetic and anabolic functions of the tricarboxylic acid cycle. PDC activity is regulated by pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). In a laser capture, microarray pilot study we found PDK4 expression to be increased 33-fold in high-grade invasive vs low-grade bladder cancers. The aim of this study was to examine the expression of PDK and PDP genes in bladder cancer cells. Methods. Bladder cancer (HTB2, HTB4, HTB5, HTB9, HT1376) and benign urothelial (UROtsa) cell lines were cultured in defined media. Total RNA was extracted and quantitative PCR performed. Cell numbers were counted using a Coulter counter. Cell cycle and apoptosis analyses were performed using fluorescence-activated cell sorting. Transient transfections with PDK4 gene specific siRNAs were performed. Results. In the present study, PDK4 expression was increased (20-100 fold; p<0.01) in bladder tumor vs. benign cell lines. Expression of the other three PDK isoforms was increased 3-5 fold. Investigation of transcription factors known to regulate the expression of PDK4 revealed overexpression of PPARα (8-22 fold; p<0.05-0.001) and HIF1α (3.7 - 13.4 fold; p<0.01 only in HT1376). Expression of PDP1 and PDP2 was also assessed (0.4 - 2.2 fold, not significant). The ratio of PDK/PDP expression was found to be 101 (PDK4/PDP1) and 138 (PDK4/PDP2) for HTB9; 196 and 42 for HT1376; 94 and 50 for HTB5 and 57 and 26.3 for HTB4. To examine the impact of PDK4 inhibition, cells were treated with a known inhibitor, dichloroacetate (DCA), for 0-72 h. A significant decrease in cell number was detected in a dose dependent manner (10-50 mM). HTB2, a low grade cell line, showed a significant decline in cell number (70%) when incubated with 50 mM DCA for 72 h (p< 0.01). HTB5, a high-grade cell line with a higher PDK4 expression, had a greater decline (92%) with same treatment (p< 0.01). Further, we observed growth inhibition by cell count with siRNA knockdown of PDK4. Our preliminary data on cell cycle analysis showed an enhanced number of tumor cells in G1 phase and a decrease in S-phase. Conclusions. PDK4 gene expression was found to be higher in bladder cancer cell lines. Both inhibition and knock down of PDK4 resulted in impaired cell growth with cell cycle arrest at the G1 phase. Taken together these data suggest that PDK4 may prove to be a novel therapeutic target in the management of bladder cancer. Source of Funding. The Leo & Anne Albert Charitable Trust Citation Format: Dharamainder Choudhary, Andrew Mikhalyuk, Carol Pilbeam, John Taylor. Increased expression of PDK4 in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2015-1171

Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation.

In humans, arsenic is primarily metabolized by arsenic (+3 oxidation state) methyltransferase (As3MT) to yield both trivalent and pentavalent methylated metabolites. We recently reported that the putative N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) can biotransform monomethylarsonous acid (MMA(III)) to dimethylarsinic acid, conferring resistance of human cells to arsenic exposure. To further decipher the role of N6AMT1 and its interaction with As3MT in arsenic biomethylation, we examined the relative contribution of N6AMT1 and As3MT in metabolizing arsenic using several newly modified UROtsa human urothelial cells, ie, UROtsa cells with either a constant level of N6AMT1 or As3MT in combination with an inducible level of As3MT or N6AMT1, respectively. Our analysis confirmed the involvement of N6AMT1 in MMA(III) biomethylation but not for inorganic arsenic. In a comparable level of N6AMT1 and As3MT, the effect of N6AMT1 mediated MMA(III) biomethylation was obscured by the action of As3MT. Furthermore, we showed that the levels of N6AMT1 and As3MT proteins varied among and within human normal and cancerous tissues. Overall, the data showed that N6AMT1 has a role in MMA(III) biomethylation, but its effect is relatively minor and limited compared with As3MT. In addition, the varied levels and distributions of N6AMT1 and As3MT among human tissues may potentially contribute to the tissue specificity and susceptibility to arsenic toxicity and carcinogenicity.

MP36-20 DEFINING THE MOLECULAR DRIVERS OF HIGH-GRADE NON-INVASIVE UROTHELIAL CARCINOMA OF THE BLADDER

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-20 DEFINING THE MOLECULAR DRIVERS OF HIGH-GRADE NON-INVASIVE UROTHELIAL CARCINOMA OF THE BLADDER Haiping Zhou, Yan Liu, Herbert Lepor, Moon-shong Tang, Chuanshu Huang, and Xue-Ru Wu Haiping ZhouHaiping Zhou More articles by this author , Yan LiuYan Liu More articles by this author , Herbert LeporHerbert Lepor More articles by this author , Moon-shong TangMoon-shong Tang More articles by this author , Chuanshu HuangChuanshu Huang More articles by this author , and Xue-Ru WuXue-Ru Wu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.748AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High-grade non-invasive urothelial carcinoma of the bladder (HG-NIUCB) is a highly enigmatic entity that carries a high risk of progression to the invasive stage. The molecular and genetic underpinning of HG-NIUCB remains unclear, thus presenting a significant challenge in clinical management. This study employs second-generation genetically engineered mouse modeling to create a HG-NIUCB model, helping define its molecular drivers and provide an in vivo platform for investigating its mechanism(s) of progression. METHODS A human-relevant mutant of fibroblast growth factor receptor 3 (FGFR3-S249C) was engineered in transgenic mice such that its expression in bladder epithelium is triggered upon doxycycline treatment. Additionally, a compound mouse line was created that expressed not only the FGFR3-S249C mutant but also a Simian virus large T antigen. Parallel studies were performed by transfecting UroTsa, an SV40T antigen-immortalized human urothelial cell line with the FGFR3-S248C mutant. The phenotypic and molecular alterations were examined using histopathology, Real-time PCR, Western blotting, cell cycle analysis, immunofluorescence and immunohistochemistry. RESULTS Urothelium-specific and inducible expression of the FGFR3-S249C mutant in adult mice resulted in an increased expression of mitogenic signals such as phosphorylated AKT and MAPK but did not elicit urothelial proliferation or tumorigenesis. A compensatory tumor barrier was observed in FGRF3-S249C-expressing urothelial cells in the form of p53 and RB1 pathway activation, as the possible cause of the lack of tumorigenicity for FGFR3-S249C. Interestingly, functional inactivation of p53 and RB1 by urothelial expression of SV40T in FGFR3-S249C-expressing cells led to the formation of exclusively HG-NIUCB that bears strong resemblance to the human counterpart. An identical collaborative effect in tumor promotion was observed in UroTsa cell line transfected with the FGFR3-S249C mutant. CONCLUSIONS Despite the prevalence of FGFR3 mutations in human bladder cancer, our results suggest that cooperative genetic events are needed to render FGFR3 mutations tumorigenic. A combination of FGFR3 mutations and p53/RB pathway deficiency may be a new biomarker set for HG-NIUCB and inhibition of FGFR3 mutation or its downstream signaling pathway may be of therapeutic value in controlling this important but elusive urothelial carcinoma variant. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e437 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Haiping Zhou More articles by this author Yan Liu More articles by this author Herbert Lepor More articles by this author Moon-shong Tang More articles by this author Chuanshu Huang More articles by this author Xue-Ru Wu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP45-20 PDK4 – A POSSIBLE THERAPEUTIC TARGET FOR HIGH GRADE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2015MP45-20 PDK4 – A POSSIBLE THERAPEUTIC TARGET FOR HIGH GRADE BLADDER CANCER Andrew Mikhalyuk, Dharamainder Choudhary, Carol Pilbeam, and John Taylor Andrew MikhalyukAndrew Mikhalyuk More articles by this author , Dharamainder ChoudharyDharamainder Choudhary More articles by this author , Carol PilbeamCarol Pilbeam More articles by this author , and John TaylorJohn Taylor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1518AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The activity of pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation and dephophorylation via pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP), respectively. Inactivation of PDC by PDK promotes the flux of pyruvate into lactate formation. Some cancer cells overexpress PDK and this shift in metabolism may help sustain cell growth in the acidic and hypoxic tumor microenvironment. In a laser capture microdissection-coupled microarray gene expression analysis of low grade bladder cancer (LGBC) vs high grade muscle invasive (MIBC) samples, we found a 33-fold (p<0.05) higher PDK4 expression in MIBCs. The goal of this study was to explore the role of PDK4 in bladder cancer cell lines to ascertain its potential as a novel therapeutic target. METHODS Bladder cancer cell lines (HTB4, HTB5, HTB9, HT1376) were cultured in defined media as specified by ATCC. Total RNA was extracted and quantitative PCR was performed. Cell numbers were counted using an automated cell counter. Cell cycle and apoptosis analyses were performed by fluorescence-activated cell sorting. Transient transfections with PDK4 gene specific siRNAs were performed. RESULTS PDK4 expression was elevated in all bladder cancer cell lines (20-100 fold; p<0.02-0.003) as compared to benign UROtsa cells. On the other hand, lower overexpression of both PDP1 and PDP2 was observed in all the cancer cell lines (0.3- 2.2 fold; not significant). To determine the functional significance of PDK4 expression, we treated cells with dichloroacetate (DCA), a PDK inhibitor, for 0-72 h. A significant decrease in cell number was detected in a dose dependent manner (10-50 mM). HTB2, a low grade cell line, showed a significant decrease in cell number (70%) when incubated with 50 mM DCA for 72 h (p< 0.001). The high grade cell line, HTB5, showed a higher decrease (92%) with the same treatment (p< 0.001). These results suggest that bladder cancer cells are utilizing an aerobic glycolytic pathway, also known as The Warburg effect. Our preliminary data on cell cycle analysis with 50 mM DCA treatment at 72 h showed an increased number of cells arrested at the G1 phase (from 53% to 72%) and a decrease in number in S-phase (from 39% to 20%). In addition, preliminary data suggested that inhibiting PDK4 expression with siRNA resulted in growth inhibition. CONCLUSIONS These findings suggest a novel role of PDK4 in bladder cancer and provide the first evidence that inhibiting PDK activity may be a viable antitumor therapy in MIBC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e543 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew Mikhalyuk More articles by this author Dharamainder Choudhary More articles by this author Carol Pilbeam More articles by this author John Taylor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Bladder uptake of liposomes after intravesical administration occurs by endocytosis.

Liposomes have been used therapeutically and as a local drug delivery system in the bladder. However, the exact mechanism for the uptake of liposomes by bladder cells is unclear. In the present study, we investigated the role of endocytosis in the uptake of liposomes by cultured human UROtsa cells of urothelium and rat bladder. UROtsa cells were incubated in serum-free media with liposomes containing colloidal gold particles for 2 h either at 37°C or at 4°C. Transmission Electron Microscopy (TEM) images of cells incubated at 37°C found endocytic vesicles containing gold inside the cells. In contrast, only extracellular binding was noticed in cells incubated with liposomes at 4°C. Absence of liposome internalization at 4°C indicates the need of energy dependent endocytosis as the primary mechanism of entry of liposomes into the urothelium. Flow cytometry analysis revealed that the uptake of liposomes at 37°C occurs via clathrin mediated endocytosis. Based on these observations, we propose that clathrin mediated endocytosis is the main route of entry for liposomes into the urothelial layer of the bladder and the findings here support the usefulness of liposomes in intravesical drug delivery.

E2F1-mediated FOS induction in arsenic trioxide-induced cellular transformation: effects of global H3K9 hypoacetylation and promoter-specific hyperacetylation in vitro.

Background: Aberrant histone acetylation has been observed in carcinogenesis and cellular transformation associated with arsenic exposure; however, the molecular mechanisms and cellular outcomes of such changes are poorly understood.Objective: We investigated the impact of tolerated and toxic arsenic trioxide (As2O3) exposure in human embryonic kidney (HEK293T) and urothelial (UROtsa) cells to characterize the alterations in histone acetylation and gene expression as well as the implications for cellular transformation.Methods: Tolerated and toxic exposures of As2O3 were identified by measurement of cell death, mitochondrial function, cellular proliferation, and anchorage-independent growth. Histone extraction, the MNase sensitivity assay, and immunoblotting were used to assess global histone acetylation levels, and gene promoter-specific interactions were measured by chromatin immunoprecipitation followed by reverse-transcriptase polymerase chain reaction.Results: Tolerated and toxic dosages, respectively, were defined as 0.5 μM and 2.5 μM As2O3 in HEK293T cells and 1 μM and 5 μM As2O3 in UROtsa cells. Global hypoacetylation of H3K9 at 72 hr was observed in UROtsa cells following tolerated and toxic exposure. In both cell lines, tolerated exposure alone led to H3K9 hyperacetylation and E2F1 binding at the FOS promoter, which remained elevated after 72 hr, contrary to global H3K9 hypoacetylation. Thus, promoter-specific H3K9 acetylation is a better predictor of cellular transformation than are global histone acetylation patterns. Tolerated exposure resulted in an increased expression of the proto-oncogenes FOS and JUN in both cell lines at 72 hr.Conclusion: Global H3K9 hypoacetylation and promoter-specific hyperacetylation facilitate E2F1-mediated FOS induction in As2O3-induced cellular transformation.Citation: Rahman S, Housein Z, Dabrowska A, Mayán MD, Boobis AR, Hajji N. 2015. E2F1-mediated FOS induction in arsenic trioxide–induced cellular transformation: effects of global H3K9 hypoacetylation and promoter-specific hyperacetylation in vitro. Environ Health Perspect 123:484–492; http://dx.doi.org/10.1289/ehp.1408302

Differential effects of selective frankincense (Ru Xiang) essential oil versus non-selective sandalwood (Tan Xiang) essential oil on cultured bladder cancer cells: a microarray and bioinformatics study.

BackgroundFrankincense (Boswellia carterii, known as Ru Xiang in Chinese) and sandalwood (Santalum album, known as Tan Xiang in Chinese) are cancer preventive and therapeutic agents in Chinese medicine. Their biologically active ingredients are usually extracted from frankincense by hydrodistillation and sandalwood by distillation. This study aims to investigate the anti-proliferative and pro-apoptotic activities of frankincense and sandalwood essential oils in cultured human bladder cancer cells.MethodsThe effects of frankincense (1,400–600 dilutions) (v/v) and sandalwood (16,000–7,000 dilutions) (v/v) essential oils on cell viability were studied in established human bladder cancer J82 cells and immortalized normal human bladder urothelial UROtsa cells using a colorimetric XTT cell viability assay. Genes that responded to essential oil treatments in human bladder cancer J82 cells were identified using the Illumina Expression BeadChip platform and analyzed for enriched functions and pathways. The chemical compositions of the essential oils were determined by gas chromatography–mass spectrometry.ResultsHuman bladder cancer J82 cells were more sensitive to the pro-apoptotic effects of frankincense essential oil than the immortalized normal bladder UROtsa cells. In contrast, sandalwood essential oil exhibited a similar potency in suppressing the viability of both J82 and UROtsa cells. Although frankincense and sandalwood essential oils activated common pathways such as inflammatory interleukins (IL-6 signaling), each essential oil had a unique molecular action on the bladder cancer cells. Heat shock proteins and histone core proteins were activated by frankincense essential oil, whereas negative regulation of protein kinase activity and G protein-coupled receptors were activated by sandalwood essential oil treatment.ConclusionThe effects of frankincense and sandalwood essential oils on J82 cells and UROtsa cells involved different mechanisms leading to cancer cell death. While frankincense essential oil elicited selective cancer cell death via NRF-2-mediated oxidative stress, sandalwood essential oil induced non-selective cell death via DNA damage and cell cycle arrest.

Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder

To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

P2Y receptor modulation of ATP release in the urothelium.

The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8 µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM). Selective P2 receptor agonists, αβ-methylene-ATP, ATP-γ-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100 µM, P = 0.002). RT4 urothelial cells break down nucleotides (100 µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.