Human Urothelial Cell Cultures Research Articles

Barrier-Forming Potential of Epithelial Cells from the Exstrophic Bladder

Bladder exstrophy (BEX) is a rare developmental abnormality resulting in an open, exposed bladder plate. Although normal bladder urothelium is a mitotically quiescent barrier epithelium, histologic studies of BEX epithelia report squamous and proliferative changes that can persist beyond surgical closure. The current study examined whether patient-derived BEX epithelial cells in vitro were capable of generating a barrier-forming epithelium under permissive conditions. Epithelial cells isolated from 11 BEX samples, classified histologically as transitional (n = 6) or squamous (n = 5), were propagated in vitro. In conditions conducive to differentiated tight barrier formation by normal human urothelial cell cultures, 8 of 11 BEX lines developed transepithelial electrical resistances of more than 1000 Ω.cm2, with 3 squamous lines failing to generate tight barriers. An inverse relationship was found between expression of squamous KRT14 transcript and barrier development. Transcriptional drivers of urothelial differentiation PPARG, GATA3, and FOXA1 showed reduced expression in squamous BEX cultures. These findings implicate developmental interruption of urothelial transcriptional programming in the spectrum of transitional to squamous epithelial phenotypes found in BEX. Assessment of BEX epithelial phenotype may inform management and treatment strategies, for which distinction between reversible versus intractably squamous epithelium could identify patients at risk of medical complications or those who are most appropriate for reconstructive tissue engineering strategies.

Mutational signature modelling in vitro recapitulates bladder cancer initiation

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

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

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

180 Primary human urothelial cell culture on a matrix of spider silk: A novel technique in bladder reconstruction

Various diseases of the genitourinary tract can cause damage to the bladder and result in urinary retention or incontinence. Treatment options of affected bladders include augmentation cystoplasty, in which the bladder is enlarged by using a piece of intestine. Since the use of intestine in the urinary tract can cause serious adverse effects, new (bio)materials are being tested for bladder reconstruction. However, the ideal (bio)material for reconstruction of the bladder has not yet been discovered, mostly due to mechanical failure in vivo. A promising natural biomaterial is spider silk, which is outstanding in its slow degradation rate and mechanical properties. In this study, we assess the in vitro potential of spider silk as a suitable biomaterial for bladder reconstruction by cultivating primary human urothelial cells (HUCs) on native spider silk matrices. Here, we manufactured dental wire frames which were woven with the dragline silk of female Nephila Clavipes spiders under standardized conditions. Upon sterilization of spider silk frames, HUCs were cultivated on spider silk matrices and evaluated for adhesion, expansion, viability and cellular differentiation. Analysis of cell morphology and filamentous actin in HUCs revealed abundant adhesion sites and bridging of HUCs to spider silk matrices. In addition, qRT-PCRbased analysis of adhesion molecules present in HUCs revealed down-regulation of certain adhesion molecules when HUCs were cultured on spider silk. Expansion of HUCs on spider silk matrices was determined over time using Presto Blue and DAPI cell count in fields of vision and showed that spider silk supports expansion of HUCs. In vitro biocompatibility of spider silk was assessed by culturing HUCs in an extract of spider silk and revealed no cytotoxic effects on HUCs. This is supported by live/dead staining which demonstrated that HUCs were viable when cultured on spider silk matrices. QRT-PCR-based analysis of epithelial-mesenchymal transition, fibrosis and cellular differentiation markers expressed in HUCs when cultured on spider silk showed, besides down-regulation of CD44, no differences compared to control. Flow cytometry-based analysis of urothelial cellular differentiation markers CD90, CD44 and CD49f demonstrated a small subpopulation lower in CD44 expression at protein level when HUCs were cultured on spider silk. These results indicate that spider silk causes HUCs to mature slightly faster, but has no major effects on cellular differentiation of HUCs. Together, results describe a novel biomaterial which supports adhesion, expansion and viability of HUCs. Our study gives new understanding of cellular mechanisms occurring in HUCs when cultured on spider silk and demonstrates that spider silk is a promising biomaterial for bladder reconstruction.

Emetine Dihydrochloride: A Novel Therapy for Bladder Cancer

Current cisplatin based therapies for stage IV bladder cancer show 4% to 20% 5-year survival, underscoring the need to develop novel therapies for these patients. In the 1970s the natural alkaloid emetine dihydrochloride demonstrated modest anticancer efficacy as a single agent in clinical trials but this was not pursued. Groups recently reported that emetine induced apoptosis in leukemia cell lines, which was enhanced by cisplatin. We determined the antiproliferative effects of emetine with and without cisplatin in bladder cancer cells. Human bladder cancer cell lines and normal human urothelial cell cultures were treated with emetine and/or cisplatin. We measured cell proliferation and evaluated synergy using the Chou-Talalay method. The combination index was calculated. Cell cycle analysis was done and caspase activation was evaluated to assess growth arrest and apoptosis. Emetine and cisplatin individually inhibited bladder cancer cell proliferation. When combined, emetine and cisplatin acted synergistically to inhibit tumor cell proliferation with combination index values reflecting moderate to strong synergy. Normal urothelial cells were relatively resistant to this treatment. Emetine alone and combined with cisplatin appeared to primarily induce tumor cell growth arrest and not apoptosis. To our knowledge this study demonstrates for the first time that emetine has invitro antiproliferative activity against bladder cancer cell lines at nanomolar concentrations but little effect on normal urothelial cells. Moreover, emetine and cisplatin worked synergistically to inhibit tumor cell proliferation. Results suggest that combined emetine and cisplatin based chemotherapy may benefit patients with bladder cancer.

Aquaporin Expression Contributes to Human Transurothelial Permeability In Vitro and Is Modulated by NaCl

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm2), with mean diffusive water and urea permeability coefficients of 6.33×10−5 and 2.45×10−5 cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.

Toll-Like Receptor Responses of Normal Human Urothelial Cells to Bacterial Flagellin and Lipopolysaccharide

We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5. Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results. Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA. Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.

Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 microM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 microM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.

Primary Cultures of Human Urothelial Cells for Genotoxicity Testing

A cell culture system with human-derived urothelial cells was established based upon previous experience with cultures of porcine urinary bladder epithelial cells. Human tissue specimens used were derived from urinary bladders (n = 17) or ureters (n = 50) of patients undergoing urological operations. The epithelial origin and differentiation status was evaluated by an immunohistochemical staining for cytokeratins 7, 8, 18, 19, and 20 for isolated and cultured cells. Specimens from human ureters were better suited for primary cell cultures of the urothelium than specimens from human urinary bladders. Successful attachment and proliferation were reached by 98% of the ureter specimens (urinary bladder: 71%) and confluency was reached by 78% of the ureter cultures (urinary bladder: 18%). In the first 14 d of culture the cytokeratin patterns of cultured cells were comparable to those of native mucosa cells. During prolonged cell culture the cytokeratin patterns of the human urothelial cells (HUC) changed into a beginning dedifferentiation: Cytokeratin (CK) 18 was only detectable in cell cultures cultured for more than 29 d, whereas CK 19 was not detectable at d 29. Cell cultures of primary human urothelial cells may be used for in vitro testing of cytotoxic or genotoxic effects. The authors thank Dr. M. Albrecht for providing samples from pediatric surgery. Present address for Dr. Angelika Flieger is Federal Office of Civil Protection and Disaster Assistance, Bonn Federal Office of Civil Protection and Disaster Assistance, Provinzialstr. 93, D-53127 Bonn, Germany. Prof. Harald Schulze is deceased.

Immunoreactivity of p63 in Monolayered and In Vitro Stratified Human Urothelial Cell Cultures Compared with Native Urothelial Tissue

ObjectiveTo investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs. Methodsp63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated. ResultsIn native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen. Conclusionsp63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.

Editorial Comment on: Immunoreactivity of p63 in Monolayered and In Vitro Stratified Human Urothelial Cell Cultures Compared with Native Urothelial Tissue

Editorial Comment on: Immunoreactivity of p63 in Monolayered and In Vitro Stratified Human Urothelial Cell Cultures Compared with Native Urothelial Tissue

FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial and smooth muscle cells in the lower urinary tract

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

Epidermal Growth Factor Receptor Regulates Normal Urothelial Regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1) a specific tyrosine kinase inhibitor (AG1478) and (2) a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-α and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.

Functions of epidermal growth factor-like growth factors during human urothelial reepithelialization in vitro and the role of erbB2.

Transitional epithelium of the urinary bladder can be damaged during, for example, catheterization, overstretching due to obstructed voiding, or partial resection. The subsequent repair process can be stimulated by specific proteins such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha). However, little is known about the role of EGF-like growth factors and their respective receptors in human urothelial repair. In this study, we examined the effects of EGF, TGFalpha, amphiregulin and heregulin-alpha (HRGalpha) on proliferation, wound closure, and the expression of their receptors c-erbB1-c-erbB4 in primary cultures of human urothelial cells in vitro. Under conditions representing intact urothelium, all EGF-like growth factors except HRGalpha induced proliferation. TGFalpha induced proliferation up to four times. Amphiregulin increased expression of c-erbB1. Treatment with either TGFalpha or amphiregulin resulted in higher c-erbB1 activation and c-erbB3 levels. None of the growth factors affected the constitutive expression of c-erbB2 and c-erbB4. In the repair model, both EGF and TGFalpha stimulated the wound closure most strongly. This was mainly achieved by increased cellular migration. Receptor expression was not affected by the addition of exogenous growth factor. The role of c-erbB2 in wound healing was further investigated with the use of antisense DNA. Wound closure could be delayed up to 50% by antisense c-erbB2 but not by mismatched or sense oligonucleotides. Excessive production (e.g. in bladder tumors) or application of EGF, TGFalpha or amphiregulin, but not HRGalpha may lead to either hyperplasia or a faster repair of damaged urothelium in vivo. These effects seem to be mediated not only via c-erbB1 but also via c-erbB2. Our results suggest that modified members of the EGF-EGFR family are potential targets for future therapies for bladder wound healing and malignancy.

Estrogen-induced proliferation of urothelial cells is modulated by nerve growth factor.

Both nerve growth factor (NGF) and estrogen have been shown to stimulate proliferation of various cell types. Human urothelial cells (HUC) express the alpha- and beta-subtypes of the estrogen receptor (ER(alpha) and ER(beta)) as well as tyrosine kinase A (trkA), the high-affinity receptor for NGF. We investigated interactions between estrogen and NGF relative to cell proliferation using primary cultures of HUC. 17 beta-estradiol (E2) stimulated NGF synthesis by HUC, and E2 (50 nM), the ER(alpha) agonist 16 alpha-iodo-17 beta-estradiol (10 nM), or the ER(beta) agonist genistein (50 nM) each stimulated HUC proliferation, an effect that was abolished by the estrogen antagonist ICI-182,780 (100 nM). NGF (1-100 ng/ml) stimulated HUC proliferation, and this was abolished by NGF antiserum (0.1 microl/ml) or the trkA antagonist K252a (100 nM). HUC proliferation stimulated by E2 was also abolished by NGF antiserum or K252a. Finally, we observed that treatment of HUC with NGF (50 ng/ml) or E2 (50 nM) stimulated trkA phosphorylation, and this was abolished by K252a (100 nM) or NGF antiserum (0.1 microl/ml). These data indicate that the effects of ER activation on HUC proliferation at least partly involve activation of trkA by NGF.

In vitro biocompatibility assessment of naturally derived and synthetic biomaterials using normal human urothelial cells

The reconstruction of urinary tissues often employs various types of biomaterials, and adequate material biocompatibility is essential for the successful reconstruction of urinary tissues. In this study we utilized a primary normal human urothelial cell culture system to evaluate the in vitro biocompatibility of a number of naturally derived biomaterials [i.e., bladder submucosa, small intestinal submucosa, collagen, and alginate] and polymeric biomaterials [i.e., poly(glycolic acid), poly(L-lactic acid), poly(lactic-co-glycolic acid), and silicone] that are either experimentally or clinically used in urinary reconstructive surgery. To determine the cytotoxic and bioactive effects of these biomaterials, the cell viability, metabolic activity, apoptotic properties, and DNA-synthesis activity were measured with four types of assays [Neutral Red, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, apoptotic activity, and tritiated thymidine incorporation assays] using extract and direct contact methods. Most of the biomaterials tested did not induce significant cytotoxic effects and exhibited normal metabolic function and cell growth in vitro. This normal primary human urothelial cell culture model is suitable for in vitro biocompatibility assessments and is able to provide information on the cell–biomaterial interactions and the ability of biomaterials to support bioactive cell functions. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res 55: 33–39, 2001

In vitro biocompatibility assessment of naturally derived and synthetic biomaterials using normal human urothelial cells.

The reconstruction of urinary tissues often employs various types of biomaterials, and adequate material biocompatibility is essential for the successful reconstruction of urinary tissues. In this study we utilized a primary normal human urothelial cell culture system to evaluate the in vitro biocompatibility of a number of naturally derived biomaterials [i.e., bladder submucosa, small intestinal submucosa, collagen, and alginate] and polymeric biomaterials [i.e., poly(glycolic acid), poly(L-lactic acid), poly(lactic-co-glycolic acid), and silicone] that are either experimentally or clinically used in urinary reconstructive surgery. To determine the cytotoxic and bioactive effects of these biomaterials, the cell viability, metabolic activity, apoptotic properties, and DNA-synthesis activity were measured with four types of assays [Neutral Red, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, apoptotic activity, and tritiated thymidine incorporation assays] using extract and direct contact methods. Most of the biomaterials tested did not induce significant cytotoxic effects and exhibited normal metabolic function and cell growth in vitro. This normal primary human urothelial cell culture model is suitable for in vitro biocompatibility assessments and is able to provide information on the cell-biomaterial interactions and the ability of biomaterials to support bioactive cell functions.

The effects of dietary fatty acids on the proliferation of normal human urothelial cells in vitro.

Little is known of the mechanisms by which dietary fatty acids (FAs) may affect normal epithelial cell physiology and thereby directly or indirectly influence tumour incidence and progression. In this study, we have used normal human urothelial cell cultures to investigate whether FAs may modify proliferation of normal human epithelial cells in vitro. FAs were presented as albumin complexes in serum-free medium and the effects on proliferation over a concentration range of 1-100 microM were assayed by thymidine incorporation. Saturated FAs had no effect. At lower concentrations (1-10 microM), mono-unsaturated FAs (MUFAs) and n-3 polyunsaturated FAs (PUFAs) were slightly stimulatory. Concentrations of unsaturated FAs above 10 microM were growth inhibitory in a dose-dependent manner. Oleic acid showed least cytostatic effect, whereas gamma-linolenic acid induced irreversible growth arrest. Although marked morphological effects were observed in irreversibly growth-inhibited cells, the cells remained attached to the substratum and showed no evidence of nuclear pyknosis or apoptosis. The inhibitory effects of single PUFAs could be reduced, but not abolished, by the addition of saturated FAs or MUFAs. Mixtures of different PUFAs were inhibitory in an additive manner. These data suggest that PUFAs have a cytostatic effect on rapidly proliferating epithelial cells which appears unrelated to malignant transformation.

Characteristics of bradykinin and TPA increases in the PGE2 levels of human urothelial cells.

Prostaglandins play a potential key role in the pathogenesis of urinary bladder cancer. Bradykinin and TPA increases in prostaglandin (PG)E2 levels were compared in primary cultures of human urothelial cells. Increased PGE2 levels were dependent upon the dose of TPA and were not apparent until 30-60 min after addition of TPA, with larger increases occurring between 60 and 120 min. Stimulation was inhibited by cycloheximide. Addition of arachidonic acid to TPA-stimulated cells increased PGE2 to a level similar to that seen in arachidonic acid-stimulated controls, and this level was not altered by cycloheximide. In contrast to TPA, the bradykinin-increased PGE2 levels were maximal at 5 min (the earliest time-point assessed) and were not inhibited by cycloheximide. Increases in PGE2 levels by both TPA and bradykinin required calcium. Excessive stimulation by TPA resulted in a desensitization to subsequent stimulation by TPA, but not bradykinin. Combination of TPA with bradykinin produced at least an additive effect on PGE2 levels. Both agonists increased the release of [3H]arachidonic acid over a time-course similar to their PGE2 response. Bradykinin and TPA appear to increase PGE2 levels by enhancing arachidonic acid availability through separate phospholipase pathways. Thus, human urothelial cells exhibit similar, but yet distinct profiles for prostaglandin stimulation by TPA and bradykinin.

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells.

As a prelude to study the promotion with TPA of in vitro transformation of human urothelial cells (HUC) in culture, we characterized tumor promoter TPA receptors in primary cultures of HUC. [3H]TPA bound specifically to intact living HUC; maximum specific binding was attained in approximately 30 min at 37 degrees C. [3H]TPA bound to HUC in a saturable and competitive manner. Scatchard analysis of specific binding to intact cells displayed a single slope corresponding to an equilibrium dissociation constant (Kd) of 0.56 nM; at saturation TPA-binding capacity was 2.37 pmol/10(6) HUC (1.43 X 10(6) sites per cell). [3H]TPA bound specifically and with high affinity to the particulate fractions of HUC; binding was both saturable and reversible. Saturation of the specific binding of [3H]TPA occurred at approximately 1 nM at 4 degrees C. Scatchard analysis of specific binding to the particulate fraction displayed a single slope corresponding to a Kd of 1.08 nM; at saturation TPA-binding capacity was 2.05 pmol/mg protein (750 000 molecules per HUC). [3H]TPA binding was inhibited by the biologically active phorbol ester, phorbol didecanoate, whereas inactive phorbol did not compete for TPA binding. Binding was not affected by sodium saccharin, epidermal growth factor, retinoic acid or dexamethasone. [3H]TPA bound specifically to the HUC cytosolic fraction but only in the presence of calcium and phosphatidylserine. Calcium-activated and phospholipid-sensitive protein kinase activity was detected in HUC fractions. These results indicate the presence of high-affinity specific receptors for TPA in HUC.