Normal Human Bladder Research Articles

Abstract B41: Expression of programmed cell death ligand 1/2 and BCG immunotherapy in bladder cancer

Abstract Background: Immunotherapy based on BCG has been a standard therapy to prevent tumor recurrence and progression in bladder cancer. Activation of immune checkpoint pathways by binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiologic regulation of the immune system. Tumor cells can at times co-opt the PD-1 pathway to escape from immunosurveillance mechanism. We investigated expression of immune checkpoints and their effects on T cells in bladder cancer. Methods: A panel of transitional cell carcinoma cell lines (UC-3, T24, K1919 and 253J) and normal human bladder urothelial cells (SVHUC1) were used to evaluate expression of immune checkpoints including PD-1, PDL1, PD-L2 and CTLA-4. Western blot analysis was used for protein expressions from cell lines and qPCR for RNA expression from human bladder tissues. Human bladder cancer/normal paired tissues were obtained at two time points; surgical resection and then after BCG immunotherapy. Immunohistochemical staining was performed to assess infiltration and distribution of CD4+ and CD8+ T cells in bladder tumor tissues and normal bladder tissues. Immune cells were isolated and T cells were isolated by FACS sorting after immune cell isolation from tumor/normal tissues. Results: PD-L1 and PD-L2 proteins were highly expressed in bladder cancer cell lines compared to normal bladder epithelial cell line (SVHUC-1). In paired human samples (cancer and normal tissues), PD-L1 and 2 were also highly expressed in bladder cancer tissues compared with paired normal tissues. Real time RT-PCR comfirmed the high expression of PD-L1/2 in bladder cancer tissues. Interestingly, normal epithelial tissues regenerated after surgical resection of the tumor and BCG immunotherapy had low PD-L1/2 expression while recurred tissues after surgical resection of the tumor and BCG had high expression of PD-L1/2, which suggest that PD-L1/2 might be involved in tumor relapse after surgical resection and BCG immunotherapy. Immunohistochemical staining with CD4 and CD8 showed that the number of T cells was in bladder cancer tissues compared with paired normal tissues regardless of PD-L1/2 expression. T lymphocytes isolated from human bladder cancer tissues and paired normal tissues showed that infiltrating T cell function is significantly deteriorated in bladder cancer tissue compared with cells from paired normal tissues. Conclusions: PD-L1 and PD-L2 are highly expressed in bladder cancer and though to be involved in relapse after BCG intravesical immunotherapy through suppressing infiltrating T cell function. Our data suggest that immunecheckpoint might be a novel targets in bladder cancer. Citation Format: Jun Hyeok Heo, Hye Young Kim, Ki Chung Park, Sung Joon Hong, Kang Su Cho, Kyung Seok Han. Expression of programmed cell death ligand 1/2 and BCG immunotherapy in bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B41.

Abstract 811: Induction of stem-like cells with malignant properties by chronic exposure of immortalized normal human urothelial cell line to arsenic

Abstract Although there is extensive epidemiologic evidence of increased risk for the development of urothelial carcinoma (UC) associated with arsenic exposure, the mechanisms by which arsenic participates in tumorigenesis are not well understood. To gain mechanistic insights, we recently developed an in vitro arsenic model in an immortalized normal human bladder cell line (HUC1) and in this study we performed extended phenotypic and molecular characterization of the arsenic exposed in vitro model. To assess the impact of arsenic exposure at a time progressive manner, we performed MTT assay using the arsenic exposed and unexposed HUC1 cells. The growth of the HUC1 cells was increased in a time dependent manner after arsenic exposure. In soft agar assay, colonies were observed only in arsenic exposed cells. Similarly, invaded cells in invasion assay were observed only in arsenic exposed cells. To identify whether arsenic exposure induce self-renewal properties, we performed spheres forming assay and our data showed arsenic exposed cells formed more spheres under serum-free medium and non-adherence condition, and the number gradually increased with longer periods of exposure. The sphere forming ability was maintained through two subsequent serial passages, indicating of self-renewal ability. The spheroid cells showed differentiated morphology when cultured in serum containing medium. Finally, to identify epigenetic events associated with the later phenotypic alterations, we performed illumina methylation array and expression array and determined a panel of genes that are epigenetically altered that may be related to arsenic-induced tumorigenesis. Among 29 genes that exhibited reduction of promoter methylation and up-regulation of the expression, 6 genes (EDNRA, GJB2, KRT17, GDF15, S100P, and LCN2) were reported as the oncogenes. Furthermore 2 genes (GJB2 and KRT17) were maintained the alteration even in withdrawal of arsenic for 2.5 months. While, among 13 genes that exhibited induction of promoter methylation and down-regulation of the expression, one gene (TSPYL5) as a tumor suppressor gene was maintained the alteration in withdrawal of arsenic for 2.5 months. In conclusion, chronic arsenic exposure induces malignant phenotype with the acquisition of stem-like activity, and epigenetic alteration might be partial mechanism of tumorigenesis. Validation and detail molecular understanding of later genes are ongoing to determine the arsenic associated pathway alterations in the genesis of UC. Citation Format: Akira Oki, Shahnaz Begum, Mariana Brait, David Sidransky, Mohammad Obaidul Hoque. Induction of stem-like cells with malignant properties by chronic exposure of immortalized normal human urothelial cell line to arsenic. [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 811. doi:10.1158/1538-7445.AM2015-811

Abstract 1322: Expression of programmed cell death ligand 1/2 and BCG immunotherapy in bladder cancer

Abstract Background: Immunotherapy based on BCG has been a standard therapy to prevent tumor recurrence and progression in bladder cancer. Activation of immune checkpoint pathways by binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiologic regulation of the immune system. Tumor cells can at times co-opt the PD-1 pathway to escape from immunosurveillance mechanism. We investigated expression of immune checkpoints and their effects on T cells in bladder cancer. Methods: A panel of transitional cell carcinoma cell lines (UC-3, T24, K1919 and 253J) and normal human bladder urothelial cells (SVHUC1) were used to evaluate expression of immune checkpoints including PD-1, PDL1, PD-L2 and CTLA-4. Western blot analysis was used for protein expressions from cell lines and qPCR for RNA expression from human bladder tissues. Human bladder cancer/normal paired tissues were obtained at two time points; surgical resection and then after BCG immunotherapy. Immunohistochemical staining was performed to assess infiltration and distribution of CD4+ and CD8+ T cells in bladder tumor tissues and normal bladder tissues. Immune cells were isolated and T cells were isolated by FACS sorting after immune cell isolation from tumor/normal tissues. Results: PD-L1 and PD-L2 proteins were highly expressed in bladder cancer cell lines compared to normal bladder epithelial cell line (SVHUC-1). In paired human samples (cancer and normal tissues), PD-L1 and 2 were also highly expressed in bladder cancer tissues compared with paired normal tissues. Real time RT-PCR comfirmed the high expression of PD-L1/2 in bladder cancer tissues. Interestingly, normal epithelial tissues regenerated after surgical resection of the tumor and BCG immunotherapy had low PD-L1/2 expression while recurred tissues after surgical resection of the tumor and BCG had high expression of PD-L1/2, which suggest that PD-L1/2 might be involved in tumor relapse after surgical resection and BCG immunotherapy. Immunohistochemical staining with CD4 and CD8 showed that the number of T cells was in bladder cancer tissues compared with paired normal tissues regardless of PD-L1/2 expression. T lymphocytes isolated from human bladder cancer tissues and paired normal tissues showed that infiltrating T cell function is significantly deteriorated in bladder cancer tissue compared with cells from paired normal tissues. Conclusions: PD-L1 and PD-L2 are highly expressed in bladder cancer and though to be involved in relapse after BCG intravesical immunotherapy through suppressing infiltrating T cell function. Our data suggest that immunecheckpoint might be a novel targets in bladder cancer. Citation Format: Jun Hyeok Heo, Ki Chung Park, Kang Su Cho, Sung Joon Hong, Kyung Seok Han. Expression of programmed cell death ligand 1/2 and BCG immunotherapy in bladder cancer. [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 1322. doi:10.1158/1538-7445.AM2015-1322

Cannabinoid receptor expression in the bladder is altered in detrusor overactivity.

Immunohistochemical (IHC) evidence shows that cannabinoid receptors (CB) are expressed in human bladders and cannabinoid agonists are known to inhibit detrusor contractility. However, the mechanism for this inhibition remains unknown. In addition, the role of CB in detrusor overactivity (DO) is under-investigated. The aim of this study was to compare CB expression in normal and DO human bladders and to further characterise these receptors. Polymer chain reaction (PCR) was used to detect differences in CB transcripts in bladder samples. Differences in CB protein expression was assessed by IHC. Immunofluorescence (IF) was used to evaluate co-localisation of CB with nerve fibres. Receptor density and binding affinity were measured using the cannabinoid radioligand [(3)H]-CP-55,940. There were higher levels of CB1 transcripts in the urothelium of patients with DO and lower levels in the detrusor, compared with normal bladders. Radioligand binding revealed CB density of 421 ± 104 fmol/mg protein in normal human bladders. IHC confirmed these findings at the protein level. IF staining demonstrated co-localisation of CB1 with choline acetyltransferase-(ChAT)-positive nerves in the detrusor and co-localisation with PGP9.5 in both urothelium and detrusor. CB2 was co-localised with both ChAT and PGP9.5 in the urothelium and the detrusor. Cannabinoid receptor expression is reduced in the detrusor of patients with DO, which may play a role in the pathophysiology of the disease. Co-localisation of CB receptors with cholinergic nerves may suggest that CB1, being localised on pre- and postsynaptic terminals, could influence neurotransmitter release. Our findings suggest the potential role of cannabinoid agonists in overactive bladder pharmacotherapy.

The role of muscarinic receptor subtypes on carbachol-induced contraction of normal human detrusor and overactive detrusor associated with benign prostatic hyperplasia

The aim of this study was to compare the effect of antimuscarinic antagonists on carbachol-induced contraction of normal human bladder and detrusor overactivity associated with benign prostatic hyperplasia (DO/BPH). Samples of human bladder muscle were obtained from patients undergoing total cystectomy for bladder cancer (normal bladder), and those undergoing retropubic prostatectomy for BPH. All of the patients with DO/BPH had detrusor overactivity according to urodynamic studies. Detrusor muscle strips were mounted in 10-ml organ baths containing Krebs solution, and concentration–response curves for carbachol were obtained in the presence of antimuscarinic antagonists (4-DAMP, methoctramine, pirenzepine, tolterodine, solifenacin, trospium, propiverine, oxybutynin, and imidafenacin) or vehicle. All antagonists competitively antagonized concentration–response curves to carbachol with high affinities in normal bladder. The rank order of mean pA2 values was as follows: trospium (10.1) > 4-DAMP (9.87), imidafenacin (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). The effects of these antimuscarinic antagonists did not change when tested with DO/BPH bladder, suggesting that each antimuscarinic antagonist has a similar effect in this condition. Schild plots showed a slope corresponding to unity, except for propiverine with DO/BPH detrusor. In conclusion, M3-receptors mainly mediate contractions in human bladder strips with normal state and DO/BPH.

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 ...

Loss of STAG2 causes aneuploidy in normal human bladder cells.

The aim of this study was to determine how the function of human stromal antigen 2 (STAG2) plays an important role in proper chromosome separation. STAG2 mRNA in normal bladder cells and bladder tumor cells was evaluated by RT-PCR. The protein levels of STAG2 in normal bladder cells and bladder tumor cells were determined by western blot. A cell proliferation assay was used to measure the growth of tumor cells and STAG2-inhibited normal cells, and STAG2- inhibited normal cells were subjected to karyotype analysis. Both STAG-2 mRNA and protein expression levels were lower in bladder cancer cells compared to the controls. Knockdown of STAG2 caused aneuploidy in normal bladder cells, leading to a decreased expression of the cohesin complex components SMC1, SMC3 and RAD21, but there was no obvious effect of STAG2 knockdown on cell proliferation. Our study indicated that abnormal expression of STAG2 could cause aneuploidy in normal bladder cells.

Monitoring of permeability of different analytes in human normal and cancerous bladder tissues in vitro using optical coherence tomography

We report our preliminary results on quantification of glucose and dimethyl sulfoxide (DMSO) diffusion in normal and cancerous human bladder tissues in vitro by using a spectral domain optical coherence tomography (SD-OCT). The permeability coefficients (PCs) of a 30 % aqueous solution of glucose are found to be and in normal and cancerous bladder tissues, respectively. The PCs of 50 % DMSO are calculated to be and in normal and cancerous bladder tissues, respectively. The obtained results show a statistically significant difference in permeability of normal and cancerous tissue and indicate that the PC of 50 % DMSO is about 1.13-and 1.21-fold higher than that of 30 % glucose in normal bladder and cancerous bladder tissues, respectively. Thus, the quantitative measurements with the help of PCs from OCT images can be a potentially powerful method for bladder cancer detection.

Abstract 3127: The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model

Abstract Bladder cancer is the second most common malignancy of the genitourinary tumors in the world and tenth most common cancer in Taiwan. The most common type of bladder cancer is urothelial carcinoma (UC, formerly known as transitional cell carcinoma, TCC). The incidence rate is particularly high in southwestern coast of Taiwan. Treatment for superficial non-invasive bladder carcinoma is currently using intravesical chemotherapy with mitomycin C and BCG after transurethral resection of bladder tumor (TUR-BT). However, this therapy usually causes adverse effects including urinary frequency, urinary urgency, hematuria and tuberculosis infection. Therefore, we intend to find a complementary and alternative medicine for bladder cancer treatment which can bring a good result with less side effects. In our previous study, we found that a traditional Chinese Medicine (TCM), Guizhi Fuling Wan (GFW) had an effect on growth inhibition of human bladder cancer cell lines as same as mitomycin-C, doxorubicin and cisplatin, but had mild toxicicity on normal human bladder epithelial cells. In this study we investigated whether GFW could be used as an intravesical chemotherapy agent in our orthotopic bladder cancer mouse model and compared its adverse effect with mitomycin-C and BCG. A mouse bladder cancer cell line, MB49, was selected and evaluated its response to GFW treatment. Cell Counting Kit-8 (CCK-8) assay was employed to analyze cell viability and flow cytometry was used to determine cell cycle and apoptosis. MB49 cells were implanted into female C57BL/6 mice to generate our orthotopic bladder tumor model which was confirmed under a microscope in histology by hematoxylin and eosin staining. Our data showed that GFW was a potent inhibitor of the proliferation of mouse bladder cancer cells in vitro as well as inhibited the tumor growth on MB49 bladder cancer orthotopic model. In addition, bladder hemorrhage which was observed in bladder cancer mice had completely disappeared 17 days after the intravesical GFW-treatment. These results support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer. Citation Format: Chi-Chen Lu, Ling-Huei Tseng, Syue-Yi Chen, Jiann-Der Wu, Shu-Fen Wu, Michael W.Y. Chan, Yu-Wei Leu, Cheng-Da Hsu. The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2014-3127

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.

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.

Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

PD8-01 HUMAN URINE-DERIVED STEM CELLS ORIGINATE FROM PARIETAL STEM CELLS

INTRODUCTION AND OBJECTIVES: The aim of this study is to identify the origin of urine-derived cells (USCs) to better understand their development and optimize their use. METHODS: Urine specimens were collected from 6 healthy male donors, and from 3 women who received male donor kidneys. After isolation, cultured USCs were assessed for expression of mesenchymal stem cells (MSC)/pericytes (CD73, CD 90, CD 105, CD29, CD44 and CD146) and hematopoietic stem cell surface markers (CD 31 and CD 34), using flow cytometry and immunofluorescent staining. Both X and Y chromosomes in USCs from gender-mismatched kidney transplant patients were assessed with FISH and PCR. In addition, USCs were examined in renal cell transcripts (PAX8, NR3C2, and L1CAM) with real-time PCR, and assessed in podocyte-specific protein markers (podocin and synaptopodin) using Western blotting and immunofluorescemt staining. Normal human kidney, ureter, and bladder tissues from donors were used as controls. RESULTS: USC clones from both healthy individuals and gender-mismatched kidney transplant patients were small and had a rice grain-shaped appearance. USCs expressed MSC markers (CD73, CD 90, CD 105, CD44, CD29, and CD146), but did not express hematopoietic stem cell surface markers (CD34 and CD31). Nine of 12 USC clones from the gender-mismatched kidney transplant patients showed X/Y chromosome character, indicating that the USCs were from the upper urinary system. Real-time PCR showed that USCs expressed genes for kidney-related markers such as PAX8, NR3C2, and L1CAM. Immunofluorescent staining showed that cultured USCs and parietal cells/podocytes in nephron glomeruli within kidney all expressed CD31-/ CD146+ and podocyte-specific protein markers (podocin and synaptopodin), but no cells were expressed in ureter or bladder tissues. Western blotting showed that cultured USC at late passages (p>8) expressed podocin and synaptopodin as well. Taken together, USCs appeared to originate from parietal cells or podocytes in the renal glomerulus. CONCLUSIONS: Stem cells isolated from human urine are CD31-/CD146+ cells with self-renewal and multipotent differentiation characteristics. USCs could be an alternative source of stem cells for regenerative medicine therapy USCs are most likely of nephron glomerular podocyte origin, probably from parietal stem cells.

PD8-02 ENHANCE MYOGENSIS AND INNERVATION AFTER IMPLANTATION OF URINE-DERIVED STEM CELLS EXPRESSING VEGF IN COLLAGEN-I HYDROGEL

INTRODUCTION AND OBJECTIVES: The aim of this study is to identify the origin of urine-derived cells (USCs) to better understand their development and optimize their use. METHODS: Urine specimens were collected from 6 healthy male donors, and from 3 women who received male donor kidneys. After isolation, cultured USCs were assessed for expression of mesenchymal stem cells (MSC)/pericytes (CD73, CD 90, CD 105, CD29, CD44 and CD146) and hematopoietic stem cell surface markers (CD 31 and CD 34), using flow cytometry and immunofluorescent staining. Both X and Y chromosomes in USCs from gender-mismatched kidney transplant patients were assessed with FISH and PCR. In addition, USCs were examined in renal cell transcripts (PAX8, NR3C2, and L1CAM) with real-time PCR, and assessed in podocyte-specific protein markers (podocin and synaptopodin) using Western blotting and immunofluorescemt staining. Normal human kidney, ureter, and bladder tissues from donors were used as controls. RESULTS: USC clones from both healthy individuals and gender-mismatched kidney transplant patients were small and had a rice grain-shaped appearance. USCs expressed MSC markers (CD73, CD 90, CD 105, CD44, CD29, and CD146), but did not express hematopoietic stem cell surface markers (CD34 and CD31). Nine of 12 USC clones from the gender-mismatched kidney transplant patients showed X/Y chromosome character, indicating that the USCs were from the upper urinary system. Real-time PCR showed that USCs expressed genes for kidney-related markers such as PAX8, NR3C2, and L1CAM. Immunofluorescent staining showed that cultured USCs and parietal cells/podocytes in nephron glomeruli within kidney all expressed CD31-/ CD146+ and podocyte-specific protein markers (podocin and synaptopodin), but no cells were expressed in ureter or bladder tissues. Western blotting showed that cultured USC at late passages (p>8) expressed podocin and synaptopodin as well. Taken together, USCs appeared to originate from parietal cells or podocytes in the renal glomerulus. CONCLUSIONS: Stem cells isolated from human urine are CD31-/CD146+ cells with self-renewal and multipotent differentiation characteristics. USCs could be an alternative source of stem cells for regenerative medicine therapy USCs are most likely of nephron glomerular podocyte origin, probably from parietal stem cells.

Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: Combined application of atomic force microscopy and modulated Raman spectroscopy

Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM–Raman combined methodologies for cancer profiling and screening with translational significance.

An Update of the Interstitial Cell Compartment in the Normal Human Bladder

Aims. Interstitial cells, also called myofibroblasts, most probably play a major role in the pathogenesis of the overactive bladder. However, no specific phenotypic marker has been identified. We investigated whether N-cadherin could play a role as a discriminatory marker for interstitial cells in the human bladder. Methods. Bladder biopsies (n = 16) were collected from macroscopically nonpathological locations during cystectomy which was performed because of bladder cancer. Tissue was analyzed for expression of N-cadherin. N-cadherin+ cells were phenotyped using antibodies against PGP9.5, smoothelin, vimentin, and C-kit. Findings were related to bladder tissue histology and ultrastructure of myofibroblastic cells. Results. N-cadherin+/vimentin+ cells with branched cell bodies were found in the lamina propria and detrusor layer. They were closely associated with neurons and showed no colocalization of PGP9.5 or smoothelin. A second type of N-cadherin+ cells was found at the boundary of detrusor bundles and in the lamina propria. These cells colocalization C-kit. We assumed that N-cadherin+/vimentin+ cells are similar to the ultrastructurally defined myofibroblasts. Conclusions. N-cadherin can play a role as a discriminatory marker for interstitial cells in the human bladder, as the interstitial compartment of the human bladder houses a population of cells from mesenchymal origin, immunopositive for N-cadherin, vimentin, and C-kit.

Use of donor bladder tissues for in vitro research

To evaluate deceased non-heart beating (DNHB) donors and deceased heart beating (DHB) brain-stem dead donors, as sources of viable urological tissue for use in biomedical research. To identify sources of viable human bladder tissue as an essential resource for cell biological research aimed at understanding human diseases of the bladder and for developing new tissue engineering and regenerative medicine strategies for bladder reconstruction. Typically, normal human urinary tract tissue is obtained from adult or paediatric surgical patients with benign urological conditions, but few surgical procedures yield useful quantities of healthy bladder tissue for research. Research ethics committee approval was obtained for collection of donor bladder tissue. Consent for DHB donors was undertaken by the Donor Transplant Coordinators. Tissue Donor Coordinators were responsible for consent for DNHB donors and the retrieval of bladders was coordinated through the National Blood Service Tissue Banking Service. All retrievals were performed by practicing urologists and care was taken to maintain sterility and to minimise bacterial contamination. Two bladders were retrieved from DNHB donors and four were retrieved from DHB donors. By histology, DNHB donor bladder tissue exhibited marked urothelial tissue damage and necrosis, with major loss or absence of urothelium. No cell cultures could be established from these specimens, as the urothelial cells were not viable in primary culture. Bladder urothelium from DHB donors was intact, but showed some damage, including loss of superficial cells and variable separation from the basement membrane. All four DHB bladder specimens yielded viable urothelial cells that attached in primary culture, but cell growth was slow to establish and cultures showed a limited capacity to form a functional barrier epithelium and a propensity to senesce early. We have shown that normal human bladder urothelial cell cultures can be established and serially propagated from DHB donor bladders. However, our study suggests that rapid post-mortem changes to the bladder affect the quality and viability of the urothelium, rendering tissue from DNHB donors an inadequate source for urothelial cell culture. Our experience is that whereas patients are willing to donate surgical tissue for research, there is a barrier to obtaining consent from next of kin for retrieved tissues to be used for research purposes.

Lack of Decorin Expression by Human Bladder Cancer Cells Offers New Tools in the Therapy of Urothelial Malignancies

Decorin, a multifunctional small leucine-rich extracellular matrix proteoglycan, has been shown to possess potent antitumour activity. However, there is some uncertainty whether different cancer cells express decorin in addition to non-malignant stromal cells. In this study we clarified decorin expression by human bladder cancer cells both in vivo and in vitro. In addition, the effect of adenovirus-mediated decorin expression on human bladder cancer cells in vitro was examined. We first demonstrated using the publicly available GeneSapiens databank that decorin gene expression is present in both normal and malignant human bladder tissues. However, when we applied in situ hybridization with digoxigenin-labeled RNA probes for decorin on human bladder carcinoma tissue samples derived from a large radical cystectomy patient cohort (n = 199), we unambiguously demonstrated that invasive and non-invasive bladder carcinoma cells completely lack decorin mRNA. The cancer cells were also negative for decorin immunoreactivity. Instead, decorin expression was localized solely to original non-malignant stromal areas of bladder tissue. In accordance with the aforementioned results, human bladder cancer cells in vitro were also negative for decorin expression as shown by RT-qPCR analyses. The lack of decorin expression by bladder cancer cells was shown not to be due to the methylation of the proximal promoter region of the decorin gene. When bladder cancer cells were transfected with a decorin adenoviral vector, their proliferation was significantly decreased. In conclusion, we have shown that human bladder cancer cells are totally devoid of decorin expression. We have also shown that adenovirus-mediated decorin gene transduction of human bladder cancer cell lines markedly inhibits their proliferation. Thus, decorin gene delivery offers new potential therapeutic tools in urothelial malignancies.

Pseudomonas aeruginosa-mannose–sensitive hemagglutinin inhibits epidermal growth factor receptor signaling pathway activation and induces apoptosis in bladder cancer cells in vitro and in vivo

ObjectivesPseudomonas aeruginosa-mannose–sensitive hemagglutinin (PA-MSHA), a peritrichous P. aeruginosa strain with MSHA fimbriae, has been shown to be a valuable anticancer drug in many kinds of cancers. However, the effect of PA-MSHA on bladder cancer has not been elucidated. In this study, we focused on the antitumor activities and related mechanisms of PA-MSHA on bladder cancer in vitro and in vivo. Materials and methodsSV-40-immortalized normal uroepithelial cells (SV-HUC-1) and human bladder cancer cell lines (T24, 5637, and HT-1376) were treated with PA-MSHA or PA (heat-killed P. aeruginosa). At first, the effect of PA-MSHA on cancer cell proliferation was measured using Cell Counting Assay Kit-8 (CCK-8), whereas the changes of cell morphology were observed by transmission electron microscopy. The early apoptosis induced by PA-MSHA was evaluated by flow cytometry, and the expression level of apoptosis-related molecules was detected using Western blot assay. We then investigated the activation of the epidermal growth factor receptor signaling pathway stimulated by PA-MSHA; the expression and phosphorylation of several key regulators involved in the EGFR signaling pathway were detected. At last, xenograft tumor in nude mice was used to further investigate the antitumor effect of PA-MSHA in vivo. ResultsOur results showed that PA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cell lines. Furthermore, cells stimulated with PA-MSHA exhibited an inactivation of EGFR signaling. In vivo, PA-MSHA treatment significantly suppressed tumor growth and induced apoptosis in xenografts tumor in nude mice. ConclusionsPA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cells in vitro and in vivo, which is associated with the inactivation of EGFR signaling pathway, and it might be used as a potential therapeutic agent for bladder cancer.

Optimisation of wavelength modulated Raman spectroscopy: towards high throughput cell screening.

In the field of biomedicine, Raman spectroscopy is a powerful technique to discriminate between normal and cancerous cells. However the strong background signal from the sample and the instrumentation affects the efficiency of this discrimination technique. Wavelength Modulated Raman spectroscopy (WMRS) may suppress the background from the Raman spectra. In this study we demonstrate a systematic approach for optimizing the various parameters of WMRS to achieve a reduction in the acquisition time for potential applications such as higher throughput cell screening. The Signal to Noise Ratio (SNR) of the Raman bands depends on the modulation amplitude, time constant and total acquisition time. It was observed that the sampling rate does not influence the signal to noise ratio of the Raman bands if three or more wavelengths are sampled. With these optimised WMRS parameters, we increased the throughput in the binary classification of normal human urothelial cells and bladder cancer cells by reducing the total acquisition time to 6 s which is significantly lower in comparison to previous acquisition times required for the discrimination between similar cell types.