Urothelial Cell Cultures Research Articles

IMMUNOREGULATORY POTENTIAL OF UROTHELIUM: CHARACTERIZATION OF NF-κB SIGNAL TRANSDUCTION

To characterize the basal and activated form of nuclear factor-kappaB (NF-kappaB) complex in normal urothelial cell cultures after stimulation with tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and double-stranded ribonucleic acid (dsRNA). Human urothelial cells cultured from normal bladder specimens underwent immunohistochemical staining and cellular extracts were prepared for Electrophoretic Mobility Shift Assays (EMSA), Western blot analyses, RNA isolation and Northern Blot analyses before and after stimulation with TNF-alpha, LPS, and dsRNA. In normal human urothelial cells, activation of the NF-kappaB complex in response to stimulation with TNF-alpha, LPS, and dsRNA was detected by immunohistochemical methods and EMSA. Depending on the stimulus, a specific NF-kappaB complex was activated as seen by supershift experiments in EMSA. By Western blot, the inhibitor of NF-kappaB complex, IkappaB-alpha, degraded in response to stimulation. Northern blot analysis from total RNA revealed subsequent inducible interleukin-8 (IL-8) mRNA expression of normal urothelial cells when treated with TNF-alpha, LPS, and dsRNA. Normal human urothelial cells contain basal NF-kappaB complexes in an inactivated state. When these cells are challenged by different agents such as TNF-alpha, LPS, and dsRNA, the cells respond by activation of the NF-kappaB signal transduction pathway, degradation of its inhibitor, IkappaB-alpha, and translocation of this primary factor into the nucleus to induce specific genetic responses such as IL-8 expression.

The long-term culture of porcine urothelial cells and induction of urothelial stratification.

To assess porcine urothelial cell cultures and the in vitro induction of urothelial stratification in long-term cultures, to study their morphological, functional and genetic behaviour, and thus provide potential autologous urothelium for tissue-engineered substitutes for demucosalized gastric or colonic tissue. Primary cultures of porcine urothelium were established and the cells passaged thereafter. Cell specificity was confirmed by cytokeratin analysis, cell membrane stability assessed using lactate dehydrogenase leakage, cell de-differentiation by gamma-glutamyl transferase activity and genomic stability by karyotype investigations. Histology and scanning electron microscopy were performed to study the cultured cells and the stratified constructs. Furthermore, collagen matrices were tested as cell scaffolds. The cells were cultured for 180 days; 10 subcultures were established during this period. Stratification was induced in a culture flask and on a collagen matrix. Cytokeratins 7, 8, 17 and 18 were expressed in all cultures, and cell membranes were stable, with no evident de-differentiation. The cultures were stable in their genotype and no chromosomal aberrations were found. The histology and immunohistochemistry of the stratified porcine constructs, and cell membrane stability and cell de-differentiation, were compared with those in the human system. Pig and human urothelial cells can be cultured over a long period with no signs of senescence. Urothelial stratification can be induced in vitro. The collagen matrix seems to be an excellent scaffold, allowing cell adherence and growth.

Effects of short-chain fatty acids on primary urothelial cells in culture: Implications for intravesical use in enterocystoplasties

The presence of inflammatory changes and mucopus production in an enterocystoplasty may be similar to the condition of diversion colitis and starvation diarrhea caused by a lack of luminal short-chain fatty acids (SCFAs). We postulate a therapeutic role for intravesical SCFA. Because this treatment will also contact the urothelium, we have assessed the effect on cellular proliferation by utilizing primary urothelial cells in culture. Primary urothelial cells were grown from biopsy samples of normal urothelium obtained intraoperatively. A cocktail of SCFA used in the treatment of diversion colitis was incubated with these cells for time intervals ranging from 30 minutes to 72 hours at drug concentrations ranging from 0.04 to 20 mmol/L butyrate equivalent (BE). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the residual viable biomass to assess growth inhibition. These experiments were repeated on cells grown on matrigel substrate. The human urothelial cancer line RT112 was likewise exposed to SCFAs to assess selectivity between primary and transformed cells. Primary urothelial cells in culture undergo growth inhibition when exposed to SCFAs. The concentration of SCFAs required to reduce the general biomass by 50% or more (IC ≥ 50) was 20 mmol/L BE when exposure was for 2 hours or less. When drug exposure was prolonged for 72 hours, the IC ≥ 50 was 2.5 mmol/L BE. Cells grown on matrigel had their growth similarly inhibited. The IC ≥ 50 for the RT112 cell line was 2.5 mmol/L BE after 72 hours of drug incubation. Primary urothelial cells in culture undergo a time- and dose-dependent growth inhibition when exposed to SCFAs. This inhibition is particularly apparent at the higher doses similar to those in use in clinical practice. Cells grown on a matrigel substrate suffer growth attenuation similar to that affecting cells grown on polystyrene plates. In vivo assessment in a rodent intravesical model is advisable before considering instillations in patients.

Culture of Human Urothelial Cells on a Cell-Free Dermis for Autotransplantation

Objective: Techniques for in vitro culturing and autotransplantation have been developed for a variety of human cells and are used today in several fields of medicine. In reconstructive surgery within the genitourinary tract, autologous urothelial cells cultured in vitro could be of considerable value but have not yet been used clinically. The aim of this study was to facilitate transplantation of cultured urothelium by establishing a reliable method for culturing urothel on an immunologically inert and biodegradable structure. Methods: Normal human urothelial cells were cultured in vitro using a feeder-cell system. To achieve an optimal carrier structure, cells were removed enzymatically from a split thickness skin graft. Human urothelial cells were then seeded on the cell-free dermis and incubated in vitro. The seeded dermis samples were investigated histologically and with immunohistochemical methods at days 7, 14 and 21. Results: The human urothelial cells incubated in vitro reached confluence after 7–10 days and the cells could be cultured through 9 passages with preserved proliferative potential. When the cells were seeded on a cell-free dermis they attached, formed colonies and became confluent and stratified up to three cell layers after 21 days of incubation. The urothelial origin of the cells was confirmed by immunohistochemical staining against cytokeratin. Conclusion: The advantages of culturing the urothelial cells on a cell-free dermis include a short time lag until grafts are available, probably facilitated transplantation procedure, transplantation of undifferentiated cells and the formation of a vascularised base under the new urothelium. The method described in this study may be of great value in providing autologous urothelium for reconstructive surgery in the genitourinary tract.

Reconstitution of human urothelium from monolayer cultures.

We established a 3-dimensional organ culture model of urinary tract tissue in which to study the effects of seeding cultured urothelial cells onto de-epithelialized urothelial stroma. Normal human urinary tract tissues were placed in organ culture or used to establish urothelial cell cultures. At passage 2 cell cultures were harvested and used to reconstitute autologous organ cultures by seeding onto de-epithelialized stroma. Organ cultures were harvested at intervals and analyzed by immunohistology with a panel of antibodies against differentiation associated antigens, cytokeratins, cell adhesion molecules, extracellular matrix components and proliferation associated antigens. Human urothelial tissues were maintained in organ culture for at least 18 weeks and they retained a transitional epithelial morphology with expression of normal in situ antigenic characteristics. Within 2 weeks of reconstitution recombined organ cultures formed a stratified, polarized, transitional-like neo-epithelium that expressed many of the phenotypic and differentiated characteristics of normal tissue. Basement membrane formed at sites of direct contact between urothelial cells and stroma. After an initial stabilization period the proliferation rate of the urothelium of intact and reconstituted organ cultures decreased to the low turnover rate characteristic of normal urothelium in situ, indicating that the cells were responsive to normal growth regulatory controls. Normal human urothelial cells, which express a proliferative nondifferentiated phenotype in monolayer culture, retain the capacity to differentiate and reform a slow turnover, stratified transitional epithelium.

Reconstitution of Human Urothelium From Monolayer Cultures

Purpose We established a 3-dimensional organ culture model of urinary tract tissue in which to study the effects of seeding cultured urothelial cells onto de-epithelialized urothelial stroma. Materials and Methods Normal human urinary tract tissues were placed in organ culture or used to establish urothelial cell cultures. At passage 2 cell cultures were harvested and used to reconstitute autologous organ cultures by seeding onto de-epithelialized stroma. Organ cultures were harvested at intervals and analyzed by immunohistology with a panel of antibodies against differentiation associated antigens, cytokeratins, cell adhesion molecules, extracellular matrix components and proliferation associated antigens. Results Human urothelial tissues were maintained in organ culture for at least 18 weeks and they retained a transitional epithelial morphology with expression of normal in situ antigenic characteristics. Within 2 weeks of reconstitution recombined organ cultures formed a stratified, polarized, transitional-like neo-epithelium that expressed many of the phenotypic and differentiated characteristics of normal tissue. Basement membrane formed at sites of direct contact between urothelial cells and stroma. After an initial stabilization period the proliferation rate of the urothelium of intact and reconstituted organ cultures decreased to the low turnover rate characteristic of normal urothelium in situ, indicating that the cells were responsive to normal growth regulatory controls. Conclusions Normal human urothelial cells, which express a proliferative nondifferentiated phenotype in monolayer culture, retain the capacity to differentiate and reform a slow turnover, stratified transitional epithelium.

Evidence for Altered Proliferative Ability of Progenitors of Urothelial Cells in Interstitial Cystitis

Secondary cultures of basal urothelial cells isolated from patients with stress incontinence (7 patients), neurogenic bladder (2 patients), interstitial cystitis (IC) (27 patients), bladder rupture (1 patient) and bacterial cystitis (3 patients) grew under growth restricting conditions. All groups displayed reproducible colony size distribution, reflecting the proliferative potential distribution in the population of progenitor cells seeded. The percentage of large colonies (>6 cells/colony), progeny of basal cells with high proliferative potential, was low in cultures from control patients with stress incontinence, neurogenic bladder or bladder rupture. Exposure of cultures from control patients with stress incontinence to lipoteichoic acid from Streptococcus faecalis, in vitro, increased the percentage of large colonies to levels statistically indistinguishable from those in untreated IC cultures. This supported the possibility that exposure of progenitors of urothelial cells to infection in vivo may cause the persistent increase in the percentage of large colonies in 80% of the IC patients tested. Given these findings, it was not surprising that the percentage of large colonies was also high in cultures from patients with acute bacterial cystitis. In conclusion, the present findings support the theoretical model for the etiology of IC we proposed based on our studies in normal urothelial cells (Elgavish et al., Journal of Cellular Physiology 169: 42-51, 52-65, 66-77, 1996): (1) The proliferative ability of a subpopulation of progenitors of urothelial cells is increased in IC; and (2) This change may be the result of recurrent exposure of progenitors of urothelial cells to injury due, possibly but not exclusively, to infection and chronic inflammation. We propose to use this change as a diagnostic tool for IC.

Expression of microfibrillar proteins by bovine bladder urothelium

Objectives To determine the occurrence and potential function of proteins composing elastic microfibrils in the developing bovine bladder. Methods Monospecific antibodies, generated against two well-characterized microfibrillar proteins, microfibril-associated glycoprotein (MAGP) and fibrillin-1 (FBN1), were used in immunohistochemical analysis of full-thickness frozen sections of fetal bovine bladder. The localization of these two antibodies was compared with that of anti-type IV collagen antibody. Adjacent serial sections were stained for routine light microscopy. Cultured urothelial cells were fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 before immunoanalysis. Control reactions used either preimmune serum or a monoclonal antibody to a nonmatrix protein. Poly(A+) ribonucleic acid was isolated from cultured urothelial cells and subjected to Northern analysis using specific complementary deoxyribonucleic acid probes for MAGP and FBN1. Results Both MAGP and FBN1 are expressed by the urothelium and are found in association with the underlying basement membrane, as visualized by their co-localization with type IV collagen. Furthermore, urothelial cells in culture continue to express both microfibrillar proteins. Conclusions The developing bovine urothelium expresses major microfibrillar protein components. The role of these microfibrils in the urothelium remains to be determined, but they may have an important anchoring function.

I. A subpopulation of human urothelial cells is stimulated to proliferate by treatment in vitro with lipoteichoic acid, a cell wall component of Streptococcus faecalis

Urinary tract infection with gram-positive bacteria is common. Avenues for ingress of bacteria into the bladder include luminal and suburothelial infection. Terminally differentiated superficial urothelial cells lining the lumen of the bladder are often shed in response to infection. In contrast, infection-induced altered function of progenitors of urothelial cells residing in the basal layer of the urothelium is likely to have long lasting effects on the structure and function of the urothelium. The main objective of the present studies was to investigate in vitro the possibility that exposure to lipoteichoic acid, a cell wall component of the gram-positive Streptococcus faecalis (LT-2), stimulates basal urothelial cells to proliferate. To simulate conditions that restrict proliferation and inhibit terminal differentiation of urothelial cells in the basal layer, secondary cultures of urothelial cells (UT) were grown on collagen or fibronectin-coated substrate in medium containing low levels of Ca2+ (0.2 mM) and growth factors (0.005% bovine pituitary extract [BPE]). Under these conditions, UT cultures displayed a highly reproducible colony size distribution, possibly due to the fact that colonies were progeny of basal cells with various proliferative potentials, retained in vitro. In cultures grown under growth-restricting conditions, the majority of progenitors appeared to be quiescent, just like stem cells in the basal layer of the urothelium. Thus, the population of large colonies (more than six cells/colony), was small when a steady state of growth was achieved, 3–7 days after seeding. Growth factors (0.005–0.5% BPE) caused a dose-dependent increase in this population of large colonies. Moreover, treatment of UT grown under growth-restricting conditions (0.005% BPE) with LT-2 increased steady-state levels of the population of large colonies to levels obtained in cultures growing under optimal conditions with respect to growth factors. These results indicated that the subpopulation of progenitors, quiescent under normal conditions, could be stimulated to proliferate. Two lines of evidence were consistent with the possibility that treatment with LT-2 stimulated proliferation of the subpopulation of progenitors and that large colonies were the progeny of this subpopulation of single cells: (1) treatment with LT-2 increased the percentage of single cells that incorporated bromodeoxyuridine (i.e., proliferated) in a time-dependent manner; (2) An increase in the percentage of large colonies was found following LT-2-triggered proliferation of single cells. We propose that, under normal conditions, cells produced in response to LT-2-triggered proliferation of stem cells are removed from the system due to an increased rate of differentiation followed by apoptosis. Recurrent infection and inflammation may not allow these processes to proceed effectively, resulting in chronic injury to the bladder. Moreover, under conditions in which stem cells accumulate mutations that incapacitate their progeny to undergo apoptosis, LT-triggered proliferation could be a contributing factor to tumorigenesis. © 1996 Wiley-Liss, Inc.

A subpopulation of human urothelial cells is stimulated to proliferate by treatment in vitro with lipoteichoic acid, a cell wall component of Streptococcus faecalis.

Urinary tract infection with gram-positive bacteria is common. Avenues for ingress of bacteria into the bladder include luminal and suburothelial infection. Terminally differentiated superficial urothelial cells lining the lumen of the bladder are often shed in response to infection. In contrast, infection-induced altered function of progenitors of urothelial cells residing in the basal layer of the urothelium is likely to have long lasting effects on the structure and function of the urothelium. The main objective of the present studies was to investigate in vitro the possibility that exposure to lipoteichoic acid, a cell wall component of the gram-positive Streptococcus faecalis (LT-2), stimulates basal urothelial cells to proliferate. To simulate conditions that restrict proliferation and inhibit terminal differentiation of urothelial cells in the basal layer, secondary cultures of urothelial cells (UT) were grown on collagen or fibronectin coated substrate in medium containing low levels of Ca2+ (0.2 mM) and growth factors (0.005% bovine pituitary extract [BPE]). Under these conditions, UT cultures displayed a highly reproducible colony size distribution, possibly due to the fact that colonies were progeny of basal cells with various proliferative potentials, retained in vitro. In cultures grown under growth-restricting conditions the majority of progenitors appeared to be quiescent, just like stem cells in the basal layer of the urothelium. Thus, the population of large colonies (more than six cells/colony), was small when a steady state of growth was achieved, 3-7 days after seeding. Growth factors (0.005-0.5% BPE) caused a dose-dependent increase in this population of large colonies. Moreover, treatment of UT grown under growth-restricting conditions (0.005% BPE) with LT-2 increased steady-state levels of the population of large colonies to levels obtained in cultures growing under optimal conditions with respect to growth factors. These results indicated that the subpopulation of progenitors, quiescent under normal conditions, could be stimulated to proliferate. Two lines of evidence were consistent with the possibility that treatment with LT-2 stimulated proliferation of the subpopulation of progenitors and that large colonies were the progeny of this subpopulation of single cells: (1) treatment with LT-2 increased the percentage of single cells that incorporated bromodeoxyuridine (i.e., proliferated) in a time-dependent manner. (2) An increase in the percentage of large colonies was found following LT-2-triggered proliferation of single cells. We propose that, under normal conditions, cells produced in response to LT-2-triggered proliferation of stem cells are removed from the system due to an increased rate of differentiation followed by apoptosis. Recurrent infection and inflammation may not allow these processes to proceed effectively, resulting in chronic injury to the bladder. Moreover, under conditions in which stem cells accumulate mutations that incapacitate their progeny to undergo apoptosis, LT-triggered proliferation could be a contributing factor to tumorigenesis.

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.

Cultured urothelium in sheep bladder augmentation

In search of alternatives for urothelial-lined augmentation or reconstruction of the urinary bladder, this study combined the application of seromuscular gastrointestinal (GI) segments with the use of in-vitro cultured, autologous urothelial cells in a sheep model. A cell culture system was set up for establishment and expansion of urothelial cells out of small biopsies from bladder mucosa. A biodegradable carrier made of lactidcaprolactoncopolymer was introduced, allowing upside-down transplantation of cell cultures in vivo. Bladder mucosal biopsies were taken from 14 sheep (mean weight 13.3 kg) with an average yield of 3.5×10(5) viable cells/cm(2) after trypsinization. Primary low-density cultures grew to confluence within 5-7 days. Secondary cultures were established on the biodegradable film and were available a week later. They were transplanted onto demucosalized segments of stomach (group 1) or colon (group 2) in 5 animals each, followed by bladder incorporation in clam fashion. The earliest specimens, demonstrating survival and some proliferation of the cultured urothelium in both groups, were obtained 13 days postoperatively. To exclude urothelial regrowth, a temporary pouch grafted with cultured urothelium was created in 2 more sheep of each group. Biopsies were taken after 2 and 3 weeks, respectively, when the reopened pouch was integrated into the bladder (delayed augmentation). In these pouches, adherence and proliferation of urothelial cells could not be demonstrated. Limited radiologic and urodynamic investigations after 5-6-month follow-up revealed good shape, capacity, and compliance of the primarily augmented bladders only. The results indicate that urothelial cell cultures can be established and applied in vivo. Despite upside-down transplantation, they are able to survive on seromuscular segments in an autologous setting. The bladder environment is necessary to promote complete covering of the seromuscular segments. Based on our histologic findings, the share of both resident bladder urothelium and transplanted cells in the formation of the final urothelial lining remains uncertain. Morphologic and urodynamic follow-up data indicate that this process can be accelerated by the transplanted urothelial cells, reducing fibrotic changes of the GI segments. The functional quality of the augmented bladder seemed to improve compared to results after seromuscular augmentation alone.

Expression of p53 in urothelial cell cultures from tumour-bearing and tumour-free patients

An explant culture technique was used to culture normal urothelium from patients with muscle-invasive bladder cancer (transitional cell carcinoma, TCC) (n = 11) and from non-tumour-bearing patients (n = 60). Cell cultures were examined for expression of p53 using the monoclonal antibody p53-240. There was a statistically significant increase in p53 expression in normal urothelial cell cultures from patients with TCC (P < 0.0005). Normal urothelial cultures from patients with TCC also showed more rapid proliferation in vitro when compared with non-tumour-bearing patients (P < 0.0005). A subgroup of non-tumour-bearing patients (n = 14) showed > 5% of cells expressing p53. p53 expression in this subgroup was found to correlate with cell proliferation in vitro (r2 = 0.766). None of these urothelial specimens was observed to express p53 when paraffin-embedded preparations were stained with p53-D07 antibody prior to culture. The rate of cellular proliferation in this subgroup did not differ from that of normal urothelium from TCC patients. Twenty-two paraffin-embedded, muscle-invasive TCC specimens were also evaluated for p53 expression using p53-D07. The expression of p53 in these tumours did not differ from that observed in normal urothelial cell cultures from patients with TCC (P = 0.26). This study identifies an overexpression of p53 in normal urothelial cells from patients with TCC and in proliferating cultures from a significant subgroup of patients without malignant disease. Increased p53 expression in normal cultured urothelial cells from patients with bladder cancer implies a global change in the mechanisms controlling urothelial cell division. This may represent an early step in the pathway to carcinogenesis.

Expression and in vitro regulation of integrins by normal human urothelial cells.

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

An in vitro model of urothelial regeneration: effects of growth factors and extracellular matrix proteins.

Although the cellular turnover of resting urothelium is very low, its regenerative capacity is known to be outstanding. In organotypic mouse urothelial cultures closely mimicking the differentiation and multilayering of normal urothelium, we examined the cell biological mechanisms underlying urothelial regeneration and the specific role of growth factors and several extracellular matrix (ECM) components. Exposure to epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) and culture on laminin resulted in enhanced expansion of the urothelium. Microscopy and assessment of proliferative activity revealed that enhanced urothelial expansion due to EGF could be attributed to increased proliferative activity and an increase in cell numbers, whereas aFGF-stimulated expansion must be considered the consequence of increased cellularity and migration. Laminin-enhanced urothelial expansion was shown to be the result of spreading of the entire urothelial organotypic culture. This was associated with a considerable decrease in the number of cell layers. A synergistic effect of growth factors and laminin was not found. This organotypic urothelial cell culture model seems to be very useful in studying strategies to improve urothelial regeneration.

Short-term culture of exfoliated cells from the urine of patients with bladder tumors.

This report concerns the short-term culture of urothelial cells from the urine sediment of over 100 patients with bladder tumors. Primary cell outgrowth was obtained in approximately 60% of the cultures initiated. Culture outcome was not related to tumor grade, patient age, or volume of the urine sample. Around 85% of the proliferating cultures were successfully transferred into multi-compartment chamber/slides. These results suggest that the culture system may be a useful tool for the study of urothelial cells using patient material obtained by non-invasive means.

Evaluation of the actions and interactions of retinoic acid and epidermal growth factor on transformed urothelial cells in culture: Implications for the use of retinoid therapy in the treatment of bladder cancer patients

The effects and interactions were investigated of the two growth regulatory molecules s retinoic retinoic acid (RA), and epidermal growth factor (EGF) on the in vitro expression by bladder cancer cell lines of the transformed phenotype (anchorage-independent growth in soft agar). When tested individually, the two molecules had opposite effects: RA (10 11 to 10 5 M) caused a dose-related reduction in anchorage-independent growth, whereas EGF (0.1 to 50 ng/ml) caused a dose-dependent increase. These effects were observed with both cell lines tested: RT112, a human papillary, non-metastatic bladder cancer cell line and RU-CL2, a rat metastatic bladder carcinoma cell line. When the effect of EGF (2.5 ng/ml) was tested against the growth inhibition produced by a range of doses of RA, EGF stimulated growth and reduced the degree of inhibition produced by RA at all dose levels. Conversely, a single dose of 10 8 M RA tested against a range of EGF concentrations reduced the dose-related EGF-induced increase in anchorage-independent growth. The two cell lines responded similarly to these combinations of RA and EGF in vitro, regardless of their different biological potentials in vivo. These experiments provide no evidence that RA potentiates EGF-induced growth, as has been observed by others using mesenchymal cells. RA could, therefore, theoretically be used to inhibit or delay bladder tumour recurrences. Trials would show whether oral doses of RA, or of synthetic retinoids metabolized to RA, would reach therapeutic levels and be chemopreventive in bladder cancer patients.

Cyclic AMP response in SV40 immortalized human bladder cells.

Forskolin-mediated increase in cyclic AMP and subsequent activation of protein kinase A were evaluated in SV40-immortalized human urothelial cells. This cell line is being used to evaluate the multistep carcinogenic process. Forskolin elicited a time- and dose-dependent increase in cyclic AMP. Increases in intracellular cyclic AMP preceded media increases in cyclic nucleotide. Large increases in intracellular cyclic AMP occurred within 5 min of forskolin addition. The lowest effective concentration of forskolin was between 0.1 and 1.0 microM. Cyclic AMP increases as large as 20- to 100-fold were observed in cells and media following forskolin addition. A 60 min preincubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) did not reduce the magnitude of the forskolin increase in cyclic AMP. TPA has been shown to affect cyclic AMP metabolism in many types of cells including primary and secondary cultures of urothelial cells. In the latter, preincubation with TPA reduces the magnitude of the forskolin increase. A 4.2-fold increase in protein kinase A activity was observed within 0.5 min of forskolin addition, while only small increases in cyclic AMP (1.6-fold) were detected within 1 min. Much more cyclic AMP is synthesized than is needed to maximally activate protein kinase A. While results demonstrate a forskolin-responsive cyclic AMP system, this system does not appear to be regulated by TPA. Lack of regulation of this second messenger system by TPA may be part of the immortalization process.

Activating missense mutations in Ha‐ras‐1 genes in a malignant subset of Bladder lesions induced by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine or N‐[4‐(5‐nitro‐2‐furanyl)‐2‐thiazolyl]formamide

Urothelial cell cultures generated from urinary bladders from a series of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)- or N-[4-(5-nitro-2-furanyl)-2-thiazolyl]formamide (FANFT)-treated Fischer 344 rats were examined for activating missense mutations in Ha-ras-1 genes. Our overall objective was to identify oncogene-activating mutations in this system and to determine what altered biological properties correlate with such genetic changes. The urinary bladders from the treated animals showed a spectrum of histopathologies, from simple hyperplasia to transitional cell carcinoma (TCC). Using restriction analysis, oligonucleotide hybridization, and DNA sequencing, we found that approximately 20% (3/14) of the bladder cell cultures had acquired oncogenic single-base substitutions in codon 61 of Ha-ras-1 genes (CAA----AAA or CGA). The donor bladder lesions for these three cultures, which also harbored the same ras-activating mutations, were all classified as stage A or B TCCs. However, four other TCCs also arising in this series were found to have normal Ha-ras genes. Whereas approximately half of the bladder cultures derived from the carcinogen-treated rats were nontumorigenic in athymic mice, the three cultures containing ras oncogenes were all highly tumorigenic (forming tumors within 5 wk of injection into athymic mice). These cultures also displayed a high degree of anchorage-independent growth and NIH 3T3-transforming activity in gene transfer assays. The nontumorigenic cultures were derived from bladder lesions that included three hyperplasias and three stage A TCCs. We conclude that ras-activating missense mutations were present in a malignant subset of bladder lesions induced by BBN or FANFT, but most of the lesions in this system appeared to involve genetic alterations elsewhere. Thus other oncogenes besides activated Ha-ras may apparently be associated with the same bladder histopathologies and transformation markers.

Establishment and characterization of long-term primary mouse urothelial cell cultures.

Long-term mouse urothelial cell cultures were routinely established from explants of neonatal mouse bladders. Foci of proliferating cells could be observed one week after the initiation of the explant cultures. These persisted throughout the culture period and up to one year. Expression of keratin proteins confirmed the epithelial nature of the cultured cells. Morphologic analysis of nuclei sorted after DNA flow cytometry revealed a population of DNA-tetraploid and octoploid cells with large nuclei and prominent nucleoli in addition to a DNA-diploid cell population. Both cell populations showed DNA replicative activity as reflected by bromodeoxyuridine incorporation studies and mitotic activity. These long-term primary mouse urothelial cell cultures may prove useful for studies on urothelial cell kinetics and bladder carcinogenesis.