Normal Human Urothelium Research Articles

Knockdown of Keratin 6 Within Arsenite-Transformed Human Urothelial Cells Decreases Basal/Squamous Expression, Inhibits Growth, and Increases Cisplatin Sensitivity.

Urothelial carcinoma (UC) is prevalent, especially in elderly males. The high rate of recurrence, treatment regime, and follow-up monitoring make UC a global health and economic burden. Arsenic is a ubiquitous toxicant that can be found in drinking water, and it is known that exposure to arsenic is associated with UC development. Around 25% of diagnosed UC cases are muscle-invasive (MIUC) which have poor prognosis and develop chemoresistance, especially if tumors are associated with squamous differentiation (SD). The immortalized UROtsa cell line is derived from normal human urothelium and our lab has malignantly transformed these cells using arsenite (As3+). These cells represent a basal subtype model of MIUC and the tumors derived from the As3+-transformed cells histologically and molecularly resemble clinical cases of the basal subtype of MIUC that have focal areas SD and expression of the basal keratins (KRT1, 5, 6, 14, and 16). Our previous data demonstrate that KRT6 protein expression correlates to areas of SD within the tumors. For this study, we performed a lentiviral knockdown of KRT6 in As3+-transformed UROtsa cells to evaluate the effects on morphology, gene/protein expression, growth, colony formation, and cisplatin sensitivity. The knockdown of KRT6 resulted in decreased expression of the basal keratins, decreased growth, decreased colony formation, and increased sensitivity to cisplatin, the standard treatment for MIUC. The results of this study suggest that KRT6 plays a role in UC cell growth and is an exploitable target to increase cisplatin sensitivity for MIUC tumors that may have developed resistance to cisplatin treatment.

The Urothelial Transcriptomic Response to Interferon Gamma: Implications for Bladder Cancer Prognosis and Immunotherapy.

Interferon gamma (IFNγ) is central to the inflammatory immune response, such as that entrained by BCG immunotherapy for bladder cancer. However, immune-mediated tumour cell killing is subject to modulation by immunoinhibitory "checkpoint" receptors such as PD-L1. We investigated the effects of IFNγ on barrier-forming in vitro-differentiated normal human urothelium using mRNA-sequencing, and showed canonical upregulation of MHC class I/II and de novo expression of the T cell tropic CXCL9-11 chemokines. Normal urothelium constitutively expressed immunoinhibitory B7 family member VSIR (VISTA), while CD274 (PD-L1) expression was induced/upregulated by IFNγ. We generated a urothelial IFNγ response gene signature. When applied to the unsupervised clustering of non-muscle-invasive bladder cancers, the IFNγ-signature predicted longer recurrence-free survival. In muscle-invasive cancers, the IFNγ-signature split the basal/squamous consensus subtype, with significantly worse overall survival when weak or absent. This study offers novel insights into strategies to enhance immunotherapy via the IFNγ and VISTA/PD-L1 nexus.

Integrated proteogenomic characterization of urothelial carcinoma of the bladder

BackgroundUrothelial carcinoma (UC) is the most common pathological type of bladder cancer, a malignant tumor. However, an integrated multi-omics analysis of the Chinese UC patient cohort is lacking.MethodsWe performed an integrated multi-omics analysis, including whole-exome sequencing, RNA-seq, proteomic, and phosphoproteomic analysis of 116 Chinese UC patients, comprising 45 non-muscle-invasive bladder cancer patients (NMIBCs) and 71 muscle-invasive bladder cancer patients (MIBCs).ResultProteogenomic integration analysis indicated that SND1 and CDK5 amplifications on chromosome 7q were associated with the activation of STAT3, which was relevant to tumor proliferation. Chromosome 5p gain in NMIBC patients was a high-risk factor, through modulating actin cytoskeleton implicating in tumor cells invasion. Phosphoproteomic analysis of tumors and morphologically normal human urothelium produced UC-associated activated kinases, including CDK1 and PRKDC. Proteomic analysis identified three groups, U-I, U-II, and U-III, reflecting distinct clinical prognosis and molecular signatures. Immune subtypes of UC tumors revealed a complex immune landscape and suggested the amplification of TRAF2 related to the increased expression of PD-L1. Additionally, increased GARS, related to subtype U-II, was validated to promote pentose phosphate pathway by inhibiting activities of PGK1 and PKM2.ConclusionsThis study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in urothelial carcinoma of the bladder.

Lectins as Biomarkers of IC/BPS Disease: A Comparative Study of Glycosylation Patterns in Human Pathologic Urothelium and IC/BPS Experimental Models.

Pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) remains poorly understood, as well as its effective diagnosis and therapy. Studying changes in tissue glycosylation patterns under pathological conditions is a promising way of discovering novel biomarkers and therapeutic targets. The glycobiology of IC/BPS is largely understudied, therefore we compared glycosylation patterns of normal human urothelium with the urothelium of IC/BPS patients using a selection of 10 plant-based lectins with different monosaccharide preferences. We also compared lectin binding to human urothelium with the two most cited experimental models of IC/BPS, specifically, TNFα-treated human urothelial cell line RT4 and cyclophosphamide-induced chronic cystitis in C57BL6/J mice. Furthermore, binding of four of the selected lectins (ConA, DSL, Jacalin and WGA) was evaluated qualitatively by means of fluorescence microscopy, and quantitatively by fluorescence intensity (F.I.) measurements. Our results reveal a significant reduction in F.I. of Jacalin, as well as a prominent change in the WGA labeling pattern in the urothelium of IC/BPS patients, suggesting their potential use as promising additional biomarkers for histopathological diagnosis of IC/BPS. We have also shown that urothelial glycosylation patterns between selected experimental models and patients with IC/BPS are similar enough to offer an adequate platform for preclinical study of IC/BPS glycobiology.

Induction of APOBEC3-mediated genomic damage in urothelium implicates BK polyomavirus (BKPyV) as a hit-and-run driver for bladder cancer

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells.

Invasive urothelial carcinomas of the bladder (UCB) characteristically show a loss of differentiation markers. The transcription factor Grainyhead-like 3 (GRHL3) plays an important role in the development and differentiation of normal urothelium. The contribution to UCB progression is still elusive. Differential expression of GRHL3 was assessed in normal human urothelium and in non-invasive and invasive bladder cancer cell lines. The contribution of GRHL3 to cell proliferation, viability and invasion in UCB cell lines was determined by gain- and loss-of-function assays in vitro and in an organ culture model using de-epithelialized porcine bladders. GRHL3 expression was detectable in normal human urothelial cells and showed significantly higher mRNA and protein levels in well-differentiated, non-invasive RT4 urothelial carcinoma cells compared to moderately differentiated RT112 cells. GRHL3 expression was absent in anaplastic and invasive T24 cells. Ectopic de novo expression of GRHL3 in T24 cells significantly impaired their migration and invasion properties in vitro and in organ culture. Its downregulation improved the invasive capacity of RT4 cells. The results indicate that GRHL3 may play a role in progression and metastasis in UCB. In addition, this work demonstrates that de-epithelialized porcine bladder organ culture can be a useful, standardized tool to assess the invasive capacity of cancer cells.

Mutational signature modelling in vitro recapitulates bladder cancer initiation

Smoking is the best established risk factor for bladder cancer and direct mutagenesis by smoke carcinogens on the cellular genome can be described in clonal populations as mutational signatures. A mutational signature summarises a lifetime of DNA-damage (caused by interaction between mutagenic processes, tissue-specific gene transcription, and the DNA-repair machinery) as mutational classes in the context of their local genomic chemistry. Our aim was to establish a novel experimental model for understanding carcinogenic initiation in normal human urothelium. Finite normal human urothelial cell cultures were established in vitro and differentiated to form functional barrier epithelia capable of activating the smoke-derived procarcinogen benzo[a]pyrene (BaP), via CYP1A1, into mutagenic metabolites. New in vitro approaches were developed allowing chronic BaP-exposure, subsequent clonal expansion of NHU cells for whole genome DNAseq and mutational signature derivation. This approach retains tissue heterogeneity during the carcinogenic exposure, modelling the early selection pressures in urothelium that lead to clonal expansion and cancer. The single-base (SBS) and double-base signatures (DBS) derived from BaP-exposed urothelial tissues were dominated by C>A and CC>AA transversions, respectively. The InDel (ID) signature described cytosine deletions in homopolymer regions. Comparison to the Catalogue of Somatic Mutations in Cancer (COSMIC) using the Signal pipeline showed homology with SBS4, DBS2 and ID3, respectively; which have been detected by others in bladder cancer patients. Mutation enrichment in specific genes (eg KMT2D and CDKN1A) mirrored in situ observations by others; effectively modelling the competitive advantage mutations convey in the carcinogenic tissue environment. BaP is detected in urine, metabolically activated by urothelial CYP1A1, and therefore may be a bladder mutagen. However, C>A transversions contribute a minor fraction of all mutations recorded in bladder tumours. Widespread DNA damage caused by chronic BaP exposure did not trigger any of the APOBEC-like mutational processes (SBS2/13) that dominate bladder tumour genomes, indicating a missing factor that the new model system is well positioned to identify.

Macroscopic somatic clonal expansion in morphologically normal human urothelium.

Knowledge of somatic mutation accumulation in normal cells, which is essential for understanding cancer development and evolution, remains largely lacking. In this study, we investigated somatic clonal events in morphologically normal human urothelium (MNU; epithelium lining the bladder and ureter) and identified macroscopic clonal expansions. Aristolochic acid (AA), a natural herb-derived compound, was a major mutagenic driving factor in MNU. AA drastically accelerates mutation accumulation and enhances clonal expansion. Mutations in MNU were widely observed in chromatin remodeling genes such as KMT2D and KDM6A but rarely in TP53, PIK3CA, and FGFR3 KMT2D mutations were found to be common in urothelial cells, regardless of whether the cells experience exogenous mutagen exposure. Copy number alterations were rare and largely confined to small-scale regions, along with copy-neutral loss of heterozygosity. Single AA-associated clones in MNU expanded to a scale of several square centimeters in size.

Procarcinogen Activation and Mutational Signatures Model the Initiation of Carcinogenesis in Human Urothelial Tissues In Vitro

Disparity between genome-wide mutations in bladder and other cancers where smoking is a risk factor raises questions about carcinogenesis in different epithelia. To develop an experimental model of bladder carcinogenesis, we clonally expanded in vitro differentiated normal human urothelial (NHU) cells following exposure to an exemplar procarcinogen and used whole-genome DNA sequencing to derive mutational signatures. Benzo[a]pyrene (BaP) was activated by endogenous cytochrome P450 (cytochrome P450 family 1 subfamily A member 1 [CYP1A1]) to create genomically modified NHU cells. Comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) showed that mutations induced by BaP in NHU cells were similar to smoking-associated signatures in bladder and other cancers, including single- and doublet-base substitution signatures characterised by C > A transversions (COSMIC_SBS4 and COSMIC_DBS2, respectively), and an insertion/deletion signature of C deletions in homopolymer regions (COSMIC ID3). Our study provides the first direct evidence that BaP is activated locally in the urothelium, initiating the well-described smoking-associated mutational signatures. An absence of other common bladder cancer (BLCA)-associated genomic signatures points strongly to other primary causes of BLCA, which the new experimental approach described here is well placed to investigate. Mutational signatures ignore whether genes are affected, but tissue-specific drivers (KMT2D, KMT2C, and CDKN1A) were significantly overmutated in this model, providing insight on the emergent selection pressures. Patient summaryIn a carefully controlled laboratory setting, we exposed normal human urothelial tissues to a procarcinogen (benzo[a]pyrene) found in cigarette smoke. We show that the urothelial tissues activated the carcinogen and led to mutations forming across the genome in a characteristic pattern. This particular “mutational signature” is found in bladder tumours and other smoking-induced cancers (eg, lung); however, our study highlights that there are other unknown mutational processes in bladder cancer that is not the direct result of smoke carcinogens, and this will require further investigation.

Abstract 1932: Cationic amphiphilic drugs as a potential new treatment option for patients with high risk non-muscle invasive bladder cancer

Abstract Urothelial carcinoma of the bladder (UCB) is the fifth most common cancer in the western world. UCB present either as non-muscle-invasive BC (NMIBC) or as muscle-invasive carcinomas (MIBC). NMIBC is characterized by a high risk of recurrence and even progression to MIBC based on clinicopathological risk factors. Despite radical surgery and extensive chemotherapy, the outcome of MIBC patients is poor. High-risk NMIBC is treated with adjuvant intravesical instillations with Bacillus Calmette-Guérin (BCG). However, a substantial number of patients do not tolerate BCG or do not respond. Novel therapy for patients who do not tolerate BCG or in who are BCG unresponsive is a clear unmet clinical need. Drug repositioning of existing FDA-approved drugs could be a promising opportunity. Recently, cationic amphiphilic drugs (CADs), including commonly used antidepressants, antihistamines and antipsychotics were found to preferentially induce lysosomal cell death in transformed cells. In this study, we evaluated the potential anti-tumor efficacy and toxicity of these drugs in in vitro assays, in vivo preclinical models, and in and ex-vivo cultured patient-derived bladder cancer tissue slices. Treatment with CADs resulted in a significant dose-dependent decrease in proliferation and viability of multiple human UCB cell lines (a.o. RT4, TCC-SUP, and UM-UC-3) in vitro. Consistent with previous data regarding the ability of CADs to induce lysosomal membrane permeabilization in other solid cancer cells, we observed lysosomal puncta formation, which is a hallmark of lysosomal leakage. Strikingly, instillation of CADs in preclinical in vivo models of orthotopically growing human UM-UC-3 UCB resulted in a significant reduction in orthotopic as well as metastatic tumor burden. In line with these data, treatment of 39 patient-derived ex vivo cultured UCB slices with penfluridol resulted in significant anti-tumor responses ranging from complete regression to a significant reduction of the tumor.In vivo experiments revealed that normal mouse urothelium is not affected upon bladder instillation of CADs (weekly instillation of penfluridol for 5 weeks) in non-tumor bearing nude mice. In addition, penfluridol treatment of ex vivo cultured normal human urothelium only marginally affected the tissue. In conclusion, we demonstrated that CADs can target UCB cells and might be a promising novel approach for treatment of UCB. Citation Format: Geertje van der Horst, Arjanneke F. van de Merbel, Eline Ruigrok, Maaike H. van der Mark, Emily Ploeg, Marja Jäätelä, Janneke van Uhm, Rob C. Pelger, Chris H. Bangma, Joost Boormans, Gabri van der Pluijm, Ellen C. Zwarthoff. Cationic amphiphilic drugs as a potential new treatment option for patients with high risk non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1932.

Specificity of the Metallothionein-1 Response by Cadmium-Exposed Normal Human Urothelial Cells.

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next-generation sequencing and RT-PCR; protein expression was assessed by Western blotting using isoform-specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H, and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post-exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium, providing candidate transcript and long-lived protein biomarkers of cadmium exposure.

Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers.

Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

Aquaporin 3 Expression Loss in Urothelial Carcinoma: Association with Tumor Invasion Depth, but not with Grading?

We have previously provided molecular evidence of expression of aquaporin 3 (AQP3) in normal human urothelium and in UBC of various stages. Whereas former studies demonstrated that loss of AQP3 was associated with invasive and high-grade disease and worse progression-free and cancer-specific survival, this report investigates the expression of AQP3 in associated CIS. Contrary to what we had expected, all CIS specimens were shown to exhibit strong AQP3 expression, suggesting loss of AQP3 in UBC is primarily associated with the ability of tumor cells for invasion but not with grading as sign of dedifferentiation.

MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL Bernadette Zwaans, Sarah Bartolone, Peter Levanovich, Monica Liebert, Michael Chancellor, and Laura Lamb Bernadette ZwaansBernadette Zwaans More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , Peter LevanovichPeter Levanovich More articles by this author , Monica LiebertMonica Liebert More articles by this author , Michael ChancellorMichael Chancellor More articles by this author , and Laura LambLaura Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.770AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radiation cystitis (RC) is a chronic inflammatory condition of the bladder that results from extensive exposure to radiation therapy for the treatment of pelvic malignancies. RC is a severely debilitating condition due to its symptoms (e.g. hematuria) and the lack of reliable treatment options. The underlying mechanism that contributes to RC development is not well understood. In this study, we aim to investigate the contribution of radiation-induced urothelial damage to the development of RC. We hypothesize that radiation exposure weakens the urothelial layer by reducing cellular proliferation, survival and differentiation ability of basal urothelial cells. Consequently, the damaged urothelium becomes more permeable to urine, which can cause irritation to the underlying tissue. METHODS Normal human basal urothelial (UR15) cells, developed by Jim Bassuk at the University of Washington, were used to study the effect of radiation therapy on the urothelium. UR15 cells, immortalized using HPV E6/E7, were cultured in serum-free keratinocyte media and differentiated in 10% DMEM. Cells were irradiated and the effect of radiation on cellular proliferation, survival, and differentiation was determined. RESULTS Exposure of undifferentiated UR15 cells to radiation treatment resulted in a dose-dependent decline in cellular proliferation that lasted beyond 2 weeks in culture. This decline in cellular proliferation was both due to an increase in apoptosis (Annexin V) as well as a decrease in the number of cell divisions. Additionally, irradiated UR15 cells had reduced differentiation capacity as measured by the formation of tight junctions (E-cadherin), a marker of urothelial differentiation. Ongoing studies are determining cell survival of differentiated urothelial cells as well as identifying changes to urothelial permeability in response to radiation. CONCLUSIONS Using a novel urothelial radiation model our studies concluded that radiation exposure to the bladder damages the urothelium, at least in part by reducing proliferation and differentiation of basal urothelial cells. Furthermore, a decrease in E-cadherin localized to the cell membrane suggests an increase in bladder permeability. This model may be advantages to investigate therapeutic options that may treat or prevent radiation cystitis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e275 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bernadette Zwaans More articles by this author Sarah Bartolone More articles by this author Peter Levanovich More articles by this author Monica Liebert More articles by this author Michael Chancellor More articles by this author Laura Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor–Independent Pathway Characterized by Mitochondrial Stress

Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca2+ concentration that was unresolved. The sustained elevation in cytosolic Ca2+ concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway.

MP20-03 NLRP3 INFLAMMASOME PLAYS A KEY ROLE IN UROTHELIAL DEFENSE AGAINST UROPATHOGENIC E. COLI

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder II1 Apr 2015MP20-03 NLRP3 INFLAMMASOME PLAYS A KEY ROLE IN UROTHELIAL DEFENSE AGAINST UROPATHOGENIC E. COLI Feng He, Yan Liu, Ellen Shapiro, Herbert Lepor, and Xue-Ru Wu Feng HeFeng He More articles by this author , Yan LiuYan Liu More articles by this author , Ellen ShapiroEllen Shapiro More articles by this author , Herbert LeporHerbert Lepor 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.2015.02.975AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inflammasomes are relatively recently discovered multi-protein complexes that function to activate proinflammatory cytokines particularly interleukin (IL) 1β and 18 with which to attack pathogens and cause pyroptosis. Initially found in myeloid cells, inflammasomes are also present and operative in certain epithelial cells. Whether inflammasomes are involved in innate immunity of urothelium against uropathgenic E. coli (UPEC) is presently unknown and a subject of this study. METHODS Primary cultured normal human urothelium and bladder cancer cell lines (UroTsa, RT4 and T24) were surveyed by RT-PCR and Western blotting for the expression of the receptors of major inflammasomes (NLRP1, NLRC4, NLRP3 and AIM2). NLRP3 was chosen for additional studies because of its reproducible, high-level expression in urothelial cells. The cultured cells were exposed to NLRP3 ligands (lipopolysaccharide, flagellin and ATP) and to UPEC strains and controls (UTI89, CFT073, NU14 and NU14-1). Activation status of NLRP3 was assessed by ELISA and Western blotting of secreted IL-1β and IL-18, cleavage of pro-caspase-1 and lactate dehydrogenase (LDH) release (marker for pyroptosis). Cultured cells also underwent NLRP3 knockdown or were treated with an NLRP3 inhibitor (glybenclamide) and a caspase-1 inhibitor and their responses to UPEC assessed using ELISA of IL-1β and IL-18. RESULTS Urothelial cells responded robustly to LPS, flagellin and ATP in a dose-dependent manner by secreting IL-1β and IL-18 and converting pro-caspase 1 to active caspase 1, forming specks of adaptor molecule ASC and releasing LDH, all of which are hallmarks of NLRP3-mediated inflammasome activation. Type 1-piliated UPEC strains (UTI89, CFT073 and NU14) but not the non-type 1-piliated control (NU14-1), also induced rapid activation of NLRP3, peaking at 4 h post-infection. Specific knockdown of NLRP3 by shRNA or addition of NLRP3 inhibitor glybenclamide or caspase-1 inhibitor-I significantly blunted UPEC-induced inflammasome activation. Finally, co-immunoprecipitation (co-IP) and reverse co-IP showed direct binding between NLRP3 and its adaptor molecule ASC in LPS- and UTI89-treated cells but not in untreated controls, further establishing NLRP3 inflammasome activation by UPEC. CONCLUSIONS Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that this activation may play an important role in innate urothelial defenses during urinary tract infection. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e224 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Feng He More articles by this author Yan Liu More articles by this author Ellen Shapiro More articles by this author Herbert Lepor More articles by this author Xue-Ru Wu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors.

While DNA damage response pathways are well characterized in cancer cells, much less is known about their status in normal cells. These pathways protect tumour cells from DNA damage and replication stress and consequently present potential therapeutic targets. Here we characterize the response of human telomerase reverse transcriptase (hTERT)-immortalized normal human urothelial (NHU) and bladder cancer cell lines to agents that disrupt the DNA damage response. Effects of replication and DNA damage response inhibitors on cell cycle progression, checkpoint induction and apoptosis were analysed in hTERT-NHU and bladder cancer cell lines. The primary signalling cascade responding to replication stress in malignant cells (ataxia telangiectasia-mutated (ATM) and Rad3-related-checkpoint kinase 1 (ATR-CHK1)) is not activated in hTERT-NHU cells after treatment with a replication inhibitor and these cells do not depend upon CHK1 for protection from apoptosis during replication stress. Instead, ATM signalling is rapidly activated under these conditions. Intriguingly, an ATM inhibitor suppressed S-phase checkpoint activation after exposure to replication inhibitors and stopped entry of cells into S-phase indicating G1 checkpoint activation. Consistent with this, hTERT-NHU cells treated with the ATM inhibitor showed increased levels of cyclin-dependent kinase inhibitor p19(INK4D), reduced levels of cyclin D1 and CDK4, and reduced phosphorylation of the retinoblastoma protein. In contrast, a bladder cancer cell line cotreated with ATM and replication inhibitors progressed more slowly through S phase and showed a marked increase in apoptosis. Taken together, our findings suggest that ATM and CHK1 signalling cascades have different roles in tumour and normal epithelial cells, confirming these as promising therapeutic targets.

Biomimetic Urothelial Tissue Models for the in Vitro Evaluation of Barrier Physiology and Bladder Drug Efficacy

The bladder is an important tissue in which to evaluate xenobiotic drug interactions and toxicities due to the concentration of parent drug and hepatic/enteric-derived metabolites in the urine as a result of renal excretion. Breaching of the barrier provided by the bladder epithelial lining (the urothelium) can expose the underlying tissues to urine and cause harmful effects (e.g., cystitis or cancer). Human urothelium is most commonly represented in vitro as immortalized or established cancer-derived cell lines, but the compromised ability of such cells to undergo differentiation and barrier formation means that nonimmortalized, normal human urothelial (NHU) cells provide a more relevant cell culture system. The impressive capacity for urothelial self-renewal in vivo can be harnessed in vitro to generate experimentally-useful quantities of NHU cells, which can subsequently be differentiated to form a functional or "biomimetic" urothelium. When seeded onto permeable membranes, these barrier-forming human urothelial tissue models enable the modeling of serum and luminal (intravesical) exposure to drugs and metabolites, thus supporting efficacy/toxicity assessments. Biomimetic human urothelial constructs provide a potential step along the preclinical trail and may support the extrapolation from rodent in vivo data to determine human relevance. Early evidence is beginning to demonstrate that human urothelium in vitro can provide information that supersedes conventional rodent studies, but further validation is needed to support widespread adoption.

The significance of the expression of interferon regulatory factor 3 and its alternative splicing isoforms in bladder cancer

Objective To evaluate the role of interferon regulatory factor 3 (IRF-3) and its alternative splicing isoforms in bladder cancer.Methods RNA was isolated from 3 cell lines (normal human urothelium cells,769-P,EJ) and 48 cases of bladder cancer tissues and adjacent normal tissues from Peking university first hospital between Jun.2012 to Jan.2013 by Trlzol method.RT-PCR was used to examine IRF-3 and its four alternative splicing isoforms (IRF-3a,-3c,-3d,-3e).Sequencing technology was used to find the point mutations in open reading frame of IRF-3 cDNA.Results The expression of IRF-3d is negative in normal human urothelium cells and the level of IRF-3d is lower than that in 769-P and EJ cell lines.In the 48 cases of pathological specimens,IRF-3 and IRF-3a expressed widely and no significant difference was observed between tumor and normal tissues.IRF-3d and-3e showed tumor-specific expression (P＜0.05).Although IRF-3c expressed more widely in tumor than in normal tissues,no statistical significance was observed.There was a correlation between IRF-3a and bladder tumor staging.Conclusions IRF-3 ubiquitously expressed in tumor and normal tissue (P =0.01).Its alternative splicing isoforms IRF-3d and-3e had significant correlations with bladder tumor occurrence.IRF-3a expression was also associated with bladder tumor staging. Key words: Interferon regulatory factor 3; Alternative splicing isoforms; Point mutation; Bladder tumor

Correlation between urothelial differentiation and sensory proteins P2X3, P2X5, TRPV1, and TRPV4 in normal urothelium and papillary carcinoma of human bladder.

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns.