Human Bladder Epithelial Cells Research Articles

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) β-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

Role of uropathogenic Escherichia coli outer membrane protein T in pathogenesis of urinary tract infection.

OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines.

Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

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

Luteolin decreases the attachment, invasion and cytotoxicity of UPEC in bladder epithelial cells and inhibits UPEC biofilm formation

Urinary tract infection (UTI), primarily caused by uropathogenic Escherichia coli (UPEC), is one of the most common infectious diseases worldwide. Emerging antibiotic resistance requires novel treatment strategies. Luteolin, a dietary polyphenolic flavonoid, has been confirmed as a potential antimicrobial agent. Here, we evaluated the sub-MICs of luteolin for potential properties to modulate the UPEC infection. We found that luteolin significantly decreased the attachment and invasion of UPEC J96 or CFT073 in human bladder epithelial cell lines T24. Meanwhile, obvious decreased expression of type 1 fimbriae adhesin fimH gene, lower bacterial surface hydrophobicity and swimming motility, were observed in luteolin-pretreated UPEC. Furthermore, luteolin could attenuate UPEC-induced cytotoxicity in T24 cells, which manifested as decreased activity of lactate dehydrogenase (LDH). Simultaneously, the inhibition of luteolin on UPEC-induced cytotoxicity was confirmed by ethidium bromide/acridine orange staining. Finally, the luteolin-pretreated UPEC showed a lower ability of biofilm formation. Collectively, these results indicated that luteolin decreased the attachment and invasion of UPEC in bladder epithelial cells, attenuated UPEC-induced cytotoxicity and biofilm formation via down-regulating the expression of adhesin fimH gene, reducing the bacterial surface hydrophobicity and motility.

Contribution of pharmaceuticals, fecal bacteria and endotoxin to the inflammatory responses to inland waters

The increasing contamination of freshwater with pharmaceuticals, surfactants, pesticides and other organic compounds are of major concern. As these contaminants are detected at trace levels in the environment it is important to determine if they elicit biological responses at the observed levels. In addition to chemical pollutants, there is also a concern for increasing levels of bacteria and other microorganisms in freshwater systems. In an earlier study, we observed the activation of inflammatory systems downstream of a wastewater treatment plant (WWTP) in southern Sweden. We also observed that the water contained unidentified components that were pro-inflammatory and potentiated the immune response in human urinary bladder epithelial cells. In order to determine if these effects were unique for the studied site or represent a common response in Swedish water, we have now performed a study on three WWTPs and their recipient waters in central Sweden. Analysis of immune responses in urinary bladder epithelial cells, monocyte-like cells and blood mononuclear cells confirm that these waters activate the immune system as well as induce pro-inflammatory responses. The results indicate that the cytokine profiles correlate to the endotoxin load of the waters rather than to the levels of pharmaceuticals or culturable bacteria load, suggesting that measurements of endotoxin levels and immune responses would be a valuable addition to the analysis of inland waters.

In vitro studies of epithelium-associated crystallization caused by uropathogens during urinary calculi development

Infectious urinary stones account for about 10% of all urinary stones. In 50% of cases urolithiasis is a recurrent illness, which can lead to the loss of a kidney if not properly treated. One of the reasons for recurrence of the disease may be the ability of bacteria to invade urothelial cells, persist in the host cells and serve as potential reservoirs for infection. Various uropathogens are associated with the formation of bacteria-induced urinary stones but Proteus mirabilis is the most commonly isolated (70%). An in vitro model was used in this study to analyze intracellular growth and crystallization in the presence of P. mirabilis, Klebsiella pneumoniae and Escherichia coli. Human ureter (Hu 609) and bladder (HCV 29) epithelial cell lines were infected with bacteria and incubated (3–72 h) in the presence of synthetic urine and amikacin to prevent extracellular bacterial growth. During the incubation the number of bacteria (CFU/ml) inside epithelial cells and the intensity of crystallization were established. Crystallization was determined as an amount of a calcium radioisotope. The chosen strains of uropathogens were able to invade both types of epithelial cells but the Hu 609 cells were invaded to a higher extent. However, crystallization occurred only in the presence of P. mirabilis strains which were invasive and urease-positive. The highest intensity of cell-associated crystallization was observed when the number of bacteria within the urothelium remained stable during the time of incubation. These results show that P. mirabilis has an ability to form crystals inside the host cells. Under these conditions bacteria are protected from antibiotic killing, which leads to persistent and recurrent infections. We also suspect that this phenomenon may be an important stage of kidney stones formation.

Human milk oligosaccharides protect bladder epithelial cells against uropathogenic E. coli and Streptococcus agalactiae infections (38.5)

Urinary tract infection (UTI) is primarily caused by uropathogenic E. coli (UPEC) and sometimes Streptococcus agalactiae (GBS). Recurrent infection that can progress to life‐threatening renal failure has remained as a serious global health concern in infants. Human milk oligosaccharides (HMOs) have been detected in urine of breast‐fed, but not formula‐fed neonates. We investigated the antimicrobial properties and mechanisms HMOs deploy to elicit protection in human bladder epithelial cells. We found HMO‐pretreated epithelial cells were significantly more resistant to invasion by UPEC CFT073, a prototypic strain causing urosepsis. However, HMO pre‐treatment had no significant effect in interfering with UPEC adhesion. Conversely, HMO pre‐treated cells suffer significantly less GBS COH‐1 adhesion and invasion. This result was recapitulated in vivo, in which significantly less GBS was recovered from the bladder of mice treated with HMOs. While HMOs do not play a role in maintaining host cell viability during GBS infection, we found HMOs treatment rapidly UPEC‐mediated host cell cytotoxicity. Moreover, the sialic acid‐containing fraction of HMOs reduced UPEC‐mediated MAPK and NF‐κB activation. Collectively, we showed that HMOs protect the urinary tract from microbial infection, and may be a contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast‐fed infants.Grant Funding Source: Canadian Institutes of Health Research (CIHR)

Multiresistant uropathogenic extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are susceptible to the carbon monoxide releasing molecule-2 (CORM-2)

Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum β-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 μM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli.

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization

ObjectivesTo identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC).MethodsUsing an SDS–PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone–usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined.ResultsN-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili.ConclusionsWe have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone–usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.

Cell-type-specific modelling of intracellular calcium signalling: a urothelial cell model

Calcium signalling plays a central role in regulating a wide variety of cell processes. A number of calcium signalling models exist in the literature that are capable of reproducing a variety of experimentally observed calcium transients. These models have been used to examine in more detail the mechanisms underlying calcium transients, but very rarely has a model been directly linked to a particular cell type and experimentally verified. It is important to show that this can be achieved within the general theoretical framework adopted by these models. Here, we develop a framework designed specifically for modelling cytosolic calcium transients in urothelial cells. Where possible, we draw upon existing calcium signalling models, integrating descriptions of components known to be important in this cell type from a number of studies in the literature. We then add descriptions of several additional pathways that play a specific role in urothelial cell signalling, including an explicit ionic influx term and an active pumping mechanism that drives the cytosolic calcium concentration to a target equilibrium. The resulting one-pool model of endoplasmic reticulum (ER)-dependent calcium signalling relates the cytosolic, extracellular and ER calcium concentrations and can generate a wide range of calcium transients, including spikes, bursts, oscillations and sustained elevations in the cytosolic calcium concentration. Using single-variate robustness and multivariate sensitivity analyses, we quantify how varying each of the parameters of the model leads to changes in key features of the calcium transient, such as initial peak amplitude and the frequency of bursting or spiking, and in the transitions between bursting- and plateau-dominated modes. We also show that, novel to our urothelial cell model, the ionic and purinergic P2Y pathways make distinct contributions to the calcium transient. We then validate the model using human bladder epithelial cells grown in monolayer cell culture and show that the model robustly captures the key features of the experimental data in a way that is not possible using more generic calcium models from the literature.

Human Milk Oligosaccharides Protect Bladder Epithelial Cells Against Uropathogenic Escherichia coli Invasion and Cytotoxicity

The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.

Kappa carrageenan-g-poly (acrylic acid)/SPION nanocomposite as a novel stimuli-sensitive drug delivery system

In this article, a novel triple-stimuli hydrogel was prepared by simultaneous formation of super paramagnetic iron oxide nanoparticles (SPION) and crosslinking of poly (acrylic acid) grafted onto kappa carrageenan (κC-g-PAA). The structure, thermal stability, surface morphology, and magnetic property of the κC-g-PAA/SPION hydrogel were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy with energy dispersive X-ray analysis (SEM–EDAX), transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM). Moreover, swelling capacity of the obtained hydrogel was measured at different temperature, pH, and magnetic-field to assess the sensitivity of κC-g-PAA/SPION hydrogel. This synthetic hydrogel was also examined as a controlled drug delivery system and defrasirox release was investigated at different temperature, pH, and magnetic-field. The in vitro antibacterial activity of κC-g-PAA/SPION hydrogel was studied against Escherichia coli and Staphylococcus aureus bacteria where the results showed no antibacterial activity of this new hydrogel. In vitro biocompatibility experiments were undertaken using human bladder epithelial cell line HTB 5637. These results indicated the synthesized κC-g-PAA/SPION hydrogel are nontoxic that will be useful for biomedical applications.

Alterations of Histone H1 Phosphorylation During Bladder Carcinogenesis

There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid chromatography-mass spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by Western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions.

Abstract 655: Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma .

Abstract Urothelial Cell Carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that lead to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 months of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found 26 genes were up-regulated and 22 genes were down-regulated after 6 months of CSE exposure of HUC cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were down-regulated in the transformed cells while AKT1, AKT2, HRAS, RAC1 were up-regulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE treated urothelial cells when compared to non-CSE exposed cells. The methylation status of these genes was validated using Quantitative Methylation Specific PCR (QMSP), confirming an increase in methylation of CSE treated urothelial cells compared untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking induced cancer. Citation Format: Enrico Munari, Mariana Brait, Cynthia LeBron, Maartje G. Noordhuis, Shahnaz Begum, Christina Michailidi, Nilda Gonzalez-Roibon, Leonel Maldonado, Tanusree Sen, Rafael Guerrero-Preston, Leslie Cope, Paola Parrella, Vito Michele Fazio, Patrick K. Ha, George Netto, David Sidransky, Mohammad O. Hoque. Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2013-655

Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma

Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.

Characterization and Zoonotic Potential of Uropathogenic Escherichia coli Isolated from Dogs

The aim of this study was to investigate the characteristics of canine uropathogenic Escherichia coli (UPEC) and the interaction between canine UPEC and human bladder epithelial cells. Ten E. coli isolates collected from dogs with cystitis were analyzed for antimicrobial resistance patterns, the presence of virulence factors, and biofilm formation. The ability of these isolates to induce cytotoxicity, invade human bladder epithelial cells, and stimulate an immune response was also determined. We observed a high rate of antimicrobial resistance among canine UPEC isolates. All virulence genes tested (including adhesins, iron acquisition, and protectin), except toxin genes, were detected among the canine UPEC isolates. We found that all isolates showed varying degrees of biofilm formation (mean, 0.26; range, 0.07 to 0.82), using a microtiter plate assay to evaluate biofilm formation by the isolates. Cytotoxicity to human bladder epithelial cells by the canine UPEC isolates increased in a time-dependent manner, with a 56.9% and 36.1% reduction in cell viability compared with the control at 6 and 9 h of incubation, respectively. We found that most canine UPEC isolates were able to invade human bladder epithelial cells. The interaction between these isolates and human bladder epithelial cells strongly induced the production of proinflammatory cytokines such as IL-6 and IL-8. We demonstrated that canine UPEC isolates can interact with human bladder epithelial cells, although the detailed mechanisms remain unknown. The results suggest that canine UPEC isolates, rather than dogspecific pathogens, have zoonotic potential.

32 A SIGNALING NETWORKING EVOKED BY THE INTERSTITIAL CYSTITIS-ASSOCIATED FRIZZLED 8-RELATED ANTIPROLIFERATIVE FACTOR

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research (1)1 Apr 201332 A SIGNALING NETWORKING EVOKED BY THE INTERSTITIAL CYSTITIS-ASSOCIATED FRIZZLED 8-RELATED ANTIPROLIFERATIVE FACTOR Sungyong You, Jennifer Anger, Tack Lee, Susan Keay, Michael Freeman, and Kim Jayoung Sungyong YouSungyong You Los Angeles, CA More articles by this author , Jennifer AngerJennifer Anger Los Angeles, CA More articles by this author , Tack LeeTack Lee Incheon, Korea, Republic of More articles by this author , Susan KeaySusan Keay Baltimore, MD More articles by this author , Michael FreemanMichael Freeman Los Angeles, CA More articles by this author , and Kim JayoungKim Jayoung Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1407AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic idiopathic visceral pain syndrome that mostly affects women, is one of the most bothersome conditions among bladder diseases. A sialoglycopeptide urinary biomarker is antiproliferative factor (APF), which has been detected in urine from approximately 95% of IC/PBS patients, is a small glycosylated peptide with 100% of homology to frizzled-8 (a receptor of Wnt signaling). A series of publications from our laboratory has previously shown that APF suppresses the proliferation rate of normal bladder epithelial cells through a mechanism that involves p53 and beta-catenin in vitro and in vivo, leading to inflammation, growth arrest, and urothelial permeability. The objectives of this study were (1) to identify the signaling networks altered in response to APF treatment in bladder epithelial cells, and (2) to understand the global network perturbed in IC/PBS in vivo by computational approaches. METHODS Chemically synthesized APF (as-APF) was used in combination with the APF-responsive hTERT-immortalized human bladder epithelial cell line, TRT-HU1. Biochemical and functional analysis including western blot, proliferation assay, immunoprecipitation and immunoflourescence staining were performed to identify the regulators of altered gene expression upon APF treatment. RESULTS Our in vitro experiments using TRT-HU1 cells showed that as-APF reduced the level of the deubiquitinase USP2a, leading to an increase in p53 expression and growth arrest. These responses to as-APF were recovered by enforced expression of USP2a, which resulted in MDM2 deubiquitination and subsequent p53 inactivation. Computational analysis of publicly available IC/PBS data sets also suggested the NF-κB pathway as an additional important regulator of IC/PBS. CONCLUSIONS Our findings suggest that as-APF functions similarly to native APF, and highlight a new mechanism by which the USP2a-MDM2-p53 signaling pathway plays an important role in the APF network. Targeting USP2a, MDM2, p53 and NF-κB may be relevant in the development of novel therapeutic approaches to block APF signaling. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e12-e13 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sungyong You Los Angeles, CA More articles by this author Jennifer Anger Los Angeles, CA More articles by this author Tack Lee Incheon, Korea, Republic of More articles by this author Susan Keay Baltimore, MD More articles by this author Michael Freeman Los Angeles, CA More articles by this author Kim Jayoung Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of Enteroaggregative Escherichia coli Virulence Factors in Uropathogenesis

A multiresistant clonal Escherichia coli O78:H10 strain qualifying molecularly as enteroaggregative Escherichia coli (EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenic E. coli.

Selective hydride generation- cryotrapping- ICP-MS for arsenic speciation analysis at picogram levels: analysis of river and sea water reference materials and human bladder epithelial cells.

An ultra sensitive method for arsenic (As) speciation analysis based on selective hydride generation (HG) with preconcentration by cryotrapping (CT) and inductively coupled plasma- mass spectrometry (ICP-MS) detection is presented. Determination of valence of the As species is performed by selective HG without prereduction (trivalent species only) or with L-cysteine prereduction (sum of tri- and pentavalent species). Methylated species are resolved on the basis of thermal desorption of formed methyl substituted arsines after collection at -196°C. Limits of detection of 3.4, 0.04, 0.14 and 0.10 pg mL-1 (ppt) were achieved for inorganic As, mono-, di- and trimethylated species, respectively, from a 500 μL sample. Speciation analysis of river water (NRC SLRS-4 and SLRS-5) and sea water (NRC CASS-4, CASS-5 and NASS-5) reference materials certified to contain 0.4 to 1.3 ng mL-1 total As was performed. The concentrations of methylated As species in tens of pg mL-1 range obtained by HG-CT-ICP-MS systems in three laboratories were in excellent agreement and compared well with results of HG-CT-atomic absorption spectrometry and anion exchange liquid chromatography- ICP-MS; sums of detected species agreed well with the certified total As content. HG-CT-ICP-MS method was successfully used for analysis of microsamples of exfoliated bladder epithelial cells isolated from human urine. Here, samples of lysates of 25 to 550 thousand cells contained typically tens pg up to ng of iAs species and from single to hundreds pg of methylated species, well within detection power of the presented method. A significant portion of As in the cells was found in the form of the highly toxic trivalent species.