Normal Human Urothelium Research Articles

Structurally diverse peroxisome proliferator‐activated receptor agonists induce apoptosis in human uro‐epithelial cells by a receptor‐independent mechanism involving store‐operated calcium channels

Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation. Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARgamma), ragaglitazar (PPARalpha/gamma), fenofibrate (PPARalpha) and L165041 (PPARbeta/delta). NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARgamma-mediated apoptosis was unaffected following pre-treatment with PPARgamma antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365. Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.

S05: Effects of PPAR agonists on proliferation and differentiation in the urothelium

Systemic treatment of rats with PPAR agonists (mainly of dual α/γ activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response (Cohen SM, Toxicological Sciences 2005; 87 (2): 322–7), there is other evidence to indicate a direct signalling effect on the urothelium (Egerod FL, Nielsen HS, Iversen L, et al., Biomarkers 2005; 10 (4): 295–309) and hence the full implications for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor receptor (EGFR). We have shown that specific activation of PPARγ will result in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3 and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of EGFR. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARγ agonists promoting differentiation. Our data indicates that human urothelium is a target tissue for PPARγ signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferative/differentiation balance.

Regulation and Function of Aquaporins in Human Urothelium

PURPOSE We have previously provided molecular evidence that aquaporins (AQP) are expressed by human urothelium, suggesting a potential role in water and solute transport (Rubenwolf et al, 2008). The aim of this work was to investigate factors that regulate AQP expression in human urothelium and to develop functional assays to examine the potential role in urothelial physiology. MATERIAL AND METHODS Normal Human Urothelial (NHU) cell lines were established and the effect on AQP protein expression of exposing NHU cell cultures to osmotic stress was examined by immunochemistry. The barrier properties of differentiated urothelial constructs were assessed by measuring transepithelial electrical resistance (TER) and permeability coefficients for 3H water and 14C urea. HgCl2 was used as a non-competitive AQP channel blocker. RESULTS AQP 3 protein expression was shown to be up-regulated by up to 13-fold in response to changing the culture medium osmolality from 295 to 500 mosm/kg. Differentiated urothelium revealed a significant barrier function (average TER: 3680 Ω.cm2); average diffusive water and urea permeability coefficients (Pd) were 10.4x10-5 and 3.7x10-5 cm/sec, respectively. Mean osmotic permeability coefficients (Posm) were 1.8-fold higher than corresponding diffusive permeability coefficients. A significant, dose-dependent (up to 4-fold) decrease of both water and urea flux across urothelium was observed upon application of HgCl2. CONCLUSIONS Our results demonstrate for the first time that expression of AQP 3 is regulated by osmolality, which suggests that urothelium is capable of reabsorbing water in response to the body's hydration status. The low, but finite, permeability of cultured urothelium to water and urea, which was reduced upon AQP inhibition, is also highly supportive of a previously unrecognised role for AQPs in body fluid regulation with potential implications on urinary tract physiology and urinary continence.

Regulation And Function Of Aquaporins In Human Urothelium

PURPOSE We have previously provided molecular evidence that aquaporins (AQP) are expressed by human urothelium, suggesting a potential role in water and solute transport (Rubenwolf et al, 2008). The aim of this work was to investigate factors that regulate AQP expression in human urothelium and to develop functional assays to examine the potential role in urothelial physiology. MATERIAL AND METHODS Normal Human Urothelial (NHU) cell lines were established and the effect on AQP protein expression of exposing NHU cell cultures to osmotic stress was examined by immunochemistry. The barrier properties of differentiated urothelial constructs were assessed by measuring transepithelial electrical resistance (TER) and permeability coefficients for 3H water and 14C urea. HgCl2 was used as a non-competitive AQP channel blocker. RESULTS AQP 3 protein expression was shown to be up-regulated by up to 13-fold in response to changing the culture medium osmolality from 295 to 500 mosm/kg. Differentiated urothelium revealed a significant barrier function (average TER: 3680 Ω.cm2); average diffusive water and urea permeability coefficients (Pd) were 10.4x10-5 and 3.7x10-5 cm/sec, respectively. Mean osmotic permeability coefficients (Posm) were 1.8-fold higher than corresponding diffusive permeability coefficients. A significant, dose-dependent (up to 4-fold) decrease of both water and urea flux across urothelium was observed upon application of HgCl2. CONCLUSIONS Our results demonstrate for the first time that expression of AQP 3 is regulated by osmolality, which suggests that urothelium is capable of reabsorbing water in response to the body's hydration status. The low, but finite, permeability of cultured urothelium to water and urea, which was reduced upon AQP inhibition, is also highly supportive of a previously unrecognised role for AQPs in body fluid regulation with potential implications on urinary tract physiology and urinary continence.

O.114 Ex vivo gene therapy of hydrocephalus: Intrathecal implantation of mesenchymal stem cells producing the neuroprotective peptide Glucagon like peptide-1

Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated.To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs.AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs.Qualitative study.Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro.This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

Expression and Localisation of Aquaporin Water Channels in Human Urothelium In Situ and In Vitro

BackgroundUrothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. ObjectiveTo investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. Design, setting, and participantsAQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. MeasurementsQualitative study. Results and limitationsTranscripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. ConclusionsThis is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

FOXA1 and IRF-1 intermediary transcriptional regulators of PPARγ-induced urothelial cytodifferentiation

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that has been implicated in the induction of differentiation of various cell types, including human uroepithelial cells. PPARgamma-mediated differentiation of normal human urothelial (NHU) cells in vitro requires coinhibition of epidermal growth factor receptor (EGFR) signalling and is characterised by de novo expression of late/terminal differentiation-associated genes, including uroplakins (UPK), over a 6-day period. We used gene microarrays to identify intermediary transcription factors induced in direct response to PPARgamma activation of EGFR-inhibited NHU cells. FOXA1 and IRF-1 contained consensus cognate binding sites in UPK1a, UPK2, and UPK3a promoters and transcripts were induced within 12 h of PPARgamma activation; transcription complex formation was confirmed by electromobility shift assays. In urothelium in situ, both FOXA1 and IRF-1 were nuclear and expressed in a differentiation-associated pattern. Knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARgamma-induced uroplakin expression in vitro. This is the first evidence that ligand activation of PPARgamma induces expression of intermediary transcription factors that mediate an epithelial differentiation programme and represents a new paradigm for understanding differentiation, regenerative repair and inflammation in epithelial tissues.

Effects of PPAR agonists on proliferation and differentiation in human urothelium

Systemic treatment of rats with peroxisome proliferator-activated receptor (PPAR) agonists (mainly of dual α/ γ activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response, there is other evidence to indicate a direct signalling effect on the urothelium and hence the full implication for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor (EGF) receptor. We have shown that specific activation of PPAR γ results in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3, and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of the EGF receptor. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPAR γ agonists promoting differentiation. Our data indicate that human urothelium is a target tissue for PPAR γ signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferation/differentiation balance.

Prostate stem cell antigen enhancer and uroplakin II promoter based bladder cancer targeted tissue-specific vector

Purpose To construct a dual specific vector which contains prostate stem cell antigen enhancer (PSCAE) and uroplakin II (UPII) promoter targeted bladder cancer. Methods UPII promoter and PSCAE were amplified by polymerase chain reaction (PCR). Luciferase gene (LUC) was obtained from plasmid pBK-CMV-LUC. PSCAE, UPII promoter and LUC were inserted into shuttle plasmid to create Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. Rp-UPII-LUC and Rp-PSCAE-UPII-LUC were cotransfected with pCMV-β-gal into various cell lines at the presence or absence of androgen receptor agonist R1881 and androgen receptor antagonist flutamide. Luminescence was detected with luciferase assay kit and counted on liquid scintillation counter. Results Bladder cancer cells showed higher LUC activity than non-bladder cancer cells after transfected with plasmids Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. PSCAE could improve the LUC activity in both AR positive and AR negative bladder cancer cells but not in non-bladder cancer cells and normal human urothelial (NHU) cells. R1881 could increase the LUC activity in AR positive bladder cancer cells but not in AR negative bladder cancer cells and non-bladder cancer cells. Flutamide could not inactivate PSCAE in bladder cancer cells. Conclusions PSCAE can improve target gene expression in bladder cancer cells but not in non-bladder cancer cells and NHU cells. PSCAE maintains a certain level of androgen independent activity in bladder cancer cells. PSCAE is active in both AR positive and AR negative bladder cancer cells. The results suggest that combination of PSCAE with UPII promoter is feasible in constructing bladder cancer-specific vectors.

Calcium signalling in wound-responsive normal human urothelial cell monolayers

Epithelial tissue repair requires coordination of migratory and proliferative activity both adjacent to and remote from the wound edge. Although calcium signalling is implicated, the specific mechanisms are poorly understood. This study characterises the calcium signal invoked in response to scratch wounding of normal human urothelial (NHU) cells and relates it to the localised cellular response. Immediately after wounding of confluent NHU cell monolayers, cells adjacent to the wound edge showed a sustained (>30 min) rise in [Ca(2+)](i), while there was an independent, but simultaneous calcium wave that propagated out from the wound edge. The transient signal involved release of calcium from intracellular stores and was not mediated via gap junctions, but by diffusion of extracellular agonists. We demonstrated that ATP was partially responsible for the initiation and propagation of the calcium wave and showed that the calcium release mechanism was mediated in part via activation of inositol-1,4,5-triphosphate (IP(3)) receptors. By contrast, the sustained calcium signal originated from the extracellular milieu and correlated with an increased rate of migration by these cells. The work presented here provides supportive evidence that the calcium signature, defined by its temporal and amplitude characteristics, is important in co-ordinating the response of cells within an epithelial cell monolayer after wounding.

Cancer-specific enhancement of cisplatin-induced cytotoxicity with triptolide through an interaction of inactivated glycogen synthase kinase-3β with p53

To improve conventional chemotherapeutic efficacy, a combination use of traditional medicines is effective but detailed mechanisms have been rarely elucidated. In the this study, we attempted to clarify how triptolide (PG490), an oxygenated diterpene derived from a Chinese herb, enhances the cisplatin (CDDP)-induced cytotoxicity in urothelial cancer cells. Our results showed that a combined CDDP/triptolide therapy induced apoptosis in urothelial cancer cell lines with wild-type p53, but not in those with mutant-type p53 or normal human urothelium. As the mechanism, triptolide suppressed CDDP-induced p53 transcriptional activity, leading to p21 attenuation, which promoted apoptosis via the activation of c-Jun N-terminal kinase (JNK) and Bax. We further demonstrated that the functional regulation of p53 by triptolide was mediated by an intranuclear association of p53 with glycogen synthase kinase-3beta (GSK3beta), which was inactivated by protein kinase C (PKC). This modulation of the PKC-GSK3beta axis by triptolide was observed in a cancer-specific manner. A mouse xenograft model also showed that a combined CDDP/triptolide therapy completely suppressed tumor growth without any side effects. We expect that cancer-specific enhancement of CDDP-induced cytotoxicity with triptolide may effectively overcome the resistance to a CDDP-based conventional chemotherapy as a treatment for urothelial cancer.

Trans-Species Comparison of PPAR and RXR Expression by Rat and Human Urothelial Tissues

Because some investigational peroxisome proliferator-activated receptors (PPAR) agonists cause tumors in the lower urinary tract of rats, we compared normal human and rat urothelium in terms of PPAR and retinoid X receptor (RXR) expression and proliferation-associated phenotypes. In situ, few human but most rat urothelial cells were Ki67 positive, indicating fundamental differences in cell cycle control. Rat and human urothelia expressed all 3 PPAR and the RXRalpha and RXRbeta isoforms in a predominantly nuclear localization, indicating that they may be biologically active. However, immunolocalization differences were observed between species. First, whereas PPARalpha and PPARbeta/delta were expressed throughout the human bladder or ureteric urothelium, in the rat urothelium PPARalpha was primarily, and PPARbeta/delta exclusively, restricted to superficial cells. Second, RXRbeta was restricted to intermediate and superficial layers of the human urothelium but tended to be absent from the rat superficial cells. Third, PPARgamma expression was present throughout the urothelia of both species but was most intense in the superficial human urothelium. Species differences were also observed in the expression of PPAR and RXR isoforms between cultured rat and human urothelial cells and in the smooth muscle. Our findings highlight the unique coexpression of multiple PPAR and RXR isoforms by urothelium and suggest that species differences in PPAR function between rat and human urothelia may be explored in an in vitro setting.

Generation of a Functional, Differentiated Porcine Urothelial Tissue In Vitro

BackgroundThe primary function of urothelium is to serve as a physical urinary barrier. This function is dependent on features expressed at the molecular level that are acquired during cytodifferentiation. Urothelial cells lose differentiated and functional characteristics when propagated in vitro. ObjectiveTo investigate methods of inducing molecular and functional differentiation of normal porcine urothelial (NPU) cells in vitro. Design and MeasurementsNPU cells were isolated from normal porcine bladders and propagated in a low-calcium keratinocyte serum-free medium. Effects of 5% fetal bovine serum (FBS) and exogenous calcium were investigated. Molecular differentiation was assessed by immunolabelling for urothelial differentiation-associated proteins (UPIIIa, CK20, ZO-1), and barrier function was assessed by measurement of transepithelial electrical resistance (TER). ResultsNPU cell cultures grew as monolayers in low-calcium, serum-free medium. Supplementation with 5% FBS and/or physiological calcium resulted in stratification into basal, intermediate, and superficial cell zones. Superficial cells were positive for UPIIIa, CK20, and ZO-1. TER measurement showed that NPU cells grown with FBS had significantly enhanced barrier function (6720ohms·cm2±1312 SD) compared with cells grown without FBS (102ohms·cm2±34 SD; p<0.001). LimitationsImportantly, our study demonstrates that expression of differentiation-associated immunohistochemical markers by cultured urothelial cells can be regarded as evidence of only morphological differentiation and does not represent a surrogate marker of function. ConclusionsWe have shown that normal porcine bladder urothelium has many cell biological properties equivalent to normal human urothelium, making it an excellent research substitute for difficult-to-obtain tissue. A differentiated, functional barrier urothelium has been produced from porcine bladder urothelial cells propagated in vitro and displays molecular and functional properties equivalent to native urothelium. This tissue has application in developing tissue-engineered bladders with urinary barrier properties and as a research tool for understanding the relationship between molecular and functional tissue differentiation.

Expression of Aquaporins by normal human Urothelium

PURPOSE It has long been assumed that the bladder is an impermeable storage organ and that the kidney is solely responsible for regulating the composition and volume of voided urine. This assumption has been recently challenged by our preliminary bioinformatics and microarray findings which revealed transcripts of aquaporin (AQP) 3 and 7 in human urothelium. AQPs play a crucial role in water and solute transport across membranes, with 13 isoforms being identified in man. This study was designed to obtain molecular evidence of AQP expression by urothelium. MATERIAL AND METHODS To investigate AQP transcript expression in normal human urothelium, reverse-transcribed polymerase chain reaction was used. The expression screen was carried out on multiple samples of freshly isolated urothelium from different surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Immunolabelling and immunoblotting techniques were employed to study AQP protein expression and localisation within native urothelium and cultured urothelial cells, respectively. Tissue engineered urothelial constructs were established and investigated for AQP expression in preparation for physiological studies. RESULTS Transcripts for AQP 3,4,7,9 and 11 were expressed consistently by freshly-isolated urothelium as well as in cultures of NHU cells. AQP 0, 1, 2, 5, 6, 8, 10 and 12 were not expressed. Immunochemistry confirmed expression of AQP 3 at the protein level. AQP 3 was shown to be localised to basolateral borders in both urothelium in situ and differentiated tissue constructs in vitro. CONCLUSIONS This study provides the first evidence of AQP expression in human urothelium raising the possibility that the bladder might be capable of modifying the composition and volume of urine stored within it. The in vitro models provide a platform for determining the role of AQP in bladder (patho)physiology.

PPARγ‐regulated tight junction development during human urothelial cytodifferentiation

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation-associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1-5 and 7. Following induction of differentiation by pharmacological activation of PPARgamma and blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation-associated development of TJ formation in vitro reflected the stage-related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARgamma and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme.

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

Comparing normal human urothelial (NHU) cells to a panel of six representative urothelial cell carcinoma (UCC)-derived cell lines, we showed that while TRAIL receptor expression patterns were similar, susceptibility to soluble recombinant crosslinked TRAIL fell into three categories. 4/6 carcinoma lines were sensitive, undergoing rapid and extensive death; NHU and 253J cells were partially resistant and HT1376 cells, like normal fibroblasts, were refractory. Both normal and malignant urothelial cells underwent apoptosis via the same caspase-8/9-mediated mechanism. Rapid receptor downregulation was a mechanism for evasion by some UCC cells. TRAIL resistance in malignant urothelial cells was partially dependent on FLIP(L) and was differentially mediated by p38(MAPK), whereas in normal cells, resistance was mediated by NF-kappaB. Importantly, extensive killing of UCC cells could be induced using noncrosslinked TRAIL after prolonged exposure, with no damage to their homologous, normal urothelial cell counterparts.

The effect of low-intensity pulsed ultrasound on repair of epithelial cell monolayers in vitro

Low intensity pulsed ultrasound (LIPUS) is widely used to accelerate tissue regeneration following injury, but the biological mechanisms of this effect are poorly understood. An in vitro model of epithelial wound healing was used to investigate the effect of LIPUS on the reepithelialization of scrape wounds in normal human urothelial (NHU) cell monolayers. The effects of clinical doses of ultrasound treatment on NHU cell growth and migration were investigated in cells grown under optimal conditions, without growth supplements and in media containing low vs. physiological calcium concentrations. No differences in cell growth or migration were observed. We conclude that there is no direct effect upon uro-epithelial regeneration by therapeutic ultrasound in vitro and suggest that any stimulation of epithelial wound repair in vivo may occur indirectly, for example by modulating the extracellular matrix composition and/or production of paracrine factors by the stroma.

Experimental models of human bladder carcinogenesis

Bladder cancer is the fifth most common cancer in the UK, yet human bladder carcinogenesis remains poorly understood and the response of bladder tumours to radio- and chemo-therapy is unpredictable. The aims of this article are to review human bladder carcinogenesis and appraise the different in vitro and in vivo approaches that have been developed to study the process. The review considers how in vitro models based on normal human urothelial (NHU) cells can be applied to human bladder cancer research. We conclude that recent advances in NHU cell culture offer novel approaches for defining urothelial tissue-specific responses to genotoxic and non-genotoxic carcinogens and elucidating the role of specific genes involved in the mechanisms of bladder carcinogenesis and malignant progression.

Alternative Splicing of Fibroblast Growth Factor Receptor 3 Produces a Secreted Isoform That Inhibits Fibroblast Growth Factor–Induced Proliferation and Is Repressed in Urothelial Carcinoma Cell Lines

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 Delta8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 Delta8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 Delta8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 Delta8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered.

In vitro Antiproliferative Activity of Psoralenes and Mytomicyn C on Reconstituted Bladder Mucosa

In this study, we used a new experimental model, proposed by our group, based on in vitro reconstitution of the human neo-bladder to improve pharmacological studies targeted at optimizing endocavity therapy of superficial bladder cancer. In the light of this, we evaluated the antiproliferative activity on neoplastic and normal origin human urothelium of two psoralen derivatives, 4-methyl-11-dimethilaminopropoxy-benzopsoralen (G50-E) and 8-methoxypsoralen (8-MOP), which are photosensitized by UVA radiation (photochemotherapy PUVA). The pharmacological treatment effects were evaluated by cytotoxic assay based on intramitochondrial capture of MTT, and by using scanning electron microscopy (SEM). Moreover, the cytotoxic effects exerted by the two psoralen derivatives on the neo-bladders were compared to those of mytomicin C, a reference drug for the treatment and prophylaxis of superficial carcinoma bladder relapse. Qualitatively, these investigations confirmed the cytotoxic activity of both psoralen derivatives tested, but, nevertheless, they did not allow a quantitative evaluation of cellular damage.