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Objective This clinical study used autologous intravesical platelet-rich plasma (PRP) injections to treat patients with recurrent urinary tract infection (rUTI). Changes in urothelial proliferation, cytoskeleton, and barrier function protein expression after treatment were investigated. Materials All patients underwent 4-monthly intravesical PRP injections with 1-year follow-up. Successful treatment was defined as ≤2 UTI episodes within the preceding 1 year. Bladder biopsies were performed at the first and fourth PRP injection, and specimens were investigated by Western blot for the proteins sonic hedgehog (Shh), CD34, cytokeratin 5 (CK5), CK14, CK20, zonula occludens-1 (ZO-1), E-cadherin, inflammatory proteins tryptase and p38, apoptotic protein BAX (BCL2-associated X protein) and caspase-3, functional proteins M2 (muscarinic receptor 2) and M3, and beta-adrenoceptor-3, with glyceraldehyde phosphate dehydrogenase used as normalizing protein for quantification. Results The study enrolled 22 patients with rUTI and 17 controls, with successful outcome in 14 of 22 (63.6%) patients. Compared with controls, Western blot quantification results showed that rUTI patients had lower CD34, CK20, M3, and ZO-1, but higher CK5, BAX, and caspase-3 at baseline. The reduced CD34, CK20, M2, and M3 expressions at baseline were significantly increased after repeat PRP injections. Patients with a successful outcome had significant increase of CD34, Shh, CK20, M2, and M3 expressions after PRP injections. Conclusion Intravesical PRP repeat injections improve the urothelial cell proliferation and increase the CK 20 expression in umbrella cells. PRP repeat injections have a beneficial effect on bladder urothelium-associated changes in rUTI. Thus, PRP injection may restore urothelial health and prevent UTI recurrence in intractable rUTI.

Objectives Recurrent urinary tract infection (rUTI) is a common infectious disease in women. This study investigated the urothelial cell proliferation, the cytoskeleton, barrier proteins, and inflammatory protein expression in women with rUTIs. Methods Female patients with recurrent or persistent UTIs were recruited. Bladder mucosal specimens were investigated by Western blot and immunohistochemical staining for the urothelial cytoskeleton proteins cytokeratin 5 (CK5), CK14, and CK20; proteins involved in cellular proliferation, including CD34, sonic hedgehog (SHH), and tumor protein 63 (TP63); barrier proteins zonula occludens 1 (ZO-1) and E-cadherin; inflammatory proteins p38 and tryptase; and proapoptotic proteins Bcl2-associated agonist of cell death protein (BAD), Bcl2-associated X protein (BAX), and caspase-3. Women with stress urinary incontinence without bladder symptoms served as controls. Bladder specimens from 18 recurrent UTI patients with rUTIs and 12 persistent UTIs, and 17 controls were analyzed, and protein expressions were compared between the three groups. Results Cell proliferation protein expression for CD34, SHH, and TP63 was significantly lower in the urothelium of patients with rUTIs than in controls. Expression of CK5 increased, whereas CK20 decreased significantly in rUTIs compared with those of controls. Apoptotic proteins BAD, BAX, and caspase-3 were significantly higher in patients with rUTIs. However, barrier proteins ZO-1 and E-cadherin, and tryptase were not significantly lower in patients with rUTIs. Conclusion Deficits in expression of proteins involved in urothelial cell proliferation, cytoskeleton, and barrier function were noted in patients with rUTIs. These urothelial deficits may be due to deficient proliferation and differentiation resulting in inadequate urothelial barrier function and further in rUTIs.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.

IL-4 is crucial in many helminth infections, but its role in urogenital schistosomiasis, infection withSchistosoma haematobiumworms, remains poorly understood due to a historical lack of animal models. The bladder pathology of urogenital schistosomiasis is caused by immune responses to eggs deposited in the bladder wall. A range of pathology occurs, including urothelial hyperplasia and cancer, but associated mechanisms and links to IL-4 are largely unknown. We modeled urogenital schistosomiasis by injecting the bladder walls of IL-4 receptor-alpha knockout(Il4ra−/−) and wild type mice withS. haematobiumeggs. Readouts included bladder histology andex vivoassessments of urothelial proliferation, cell cycle and ploidy status. We also quantified the effects of exogenous IL-4 on urothelial cell proliferationin vitro, including cell cycle status and phosphorylation patterns of major downstream regulators in the IL-4 signaling pathway. There was a significant decrease in the intensity of granulomatous responses to bladder-wall injectedS. haematobiumeggs inIl4ra−/−versus wild type mice.S. haematobiumegg injection triggered significant urothelial proliferation, including evidence of urothelial hyperdiploidy and cell cycle skewing in wild type but notIl4ra−/−mice. Urothelial exposure to IL-4in vitroled to cell cycle polarization and increased phosphorylation of AKT. Our results show IL-4 signaling is required for key pathogenic features of urogenital schistosomiasis, and that particular aspects of this signaling pathway may exert these effects directly on the urothelium. These findings point to potential mechanisms by which urogenital schistosomiasis promotes bladder carcinogenesis.

Most mouse kidney stone models induce nephrocalcinosis rather than urolithiasis. The aim of our study was to find an accelerated experimental model in order to study the early events of stone formation, that is, at the time of crystal binding to intrarenal urothelium. C57B6 mice exposed to vitamin D supplements and water containing hydroxyl-L-proline, ammonium chloride and calcium chloride were studied for 42 days. A group receiving urothelial cell mitogen Fibroblast Growth Factor 7 (FGF7) was compared to control group receiving saline. Calcium oxalate monohydrate (COM) crystals were detected in urines by day 2 and within urinary spaces in specialized fornix areas in both groups as soon as day 14 with enhanced deposits in FGF7 group compared to controls at day 21. Urothelial cells proliferation, uroplakin III downregulation and de novo expression of osteopontin receptor CD44 detected in FGF7 group, were delayed in the control group (day 42). Crystal aggregates within specialized fornix areas by day 42 were located in urinary spaces but also within and under a multilayered metaplastic urothelium, simultaneous to macrophages influx. Point of note, administration of a normal diet by day 21 was responsible for a spontaneous crystal clearance. Our data show that under supersaturation conditions, urothelial cell proliferation and calcium oxalate crystal retention occur within specialized fornix areas. Enhanced crystal deposits following FGF7 administration suggest that urothelium proliferation would be a relevant trigger for renal stone formation.

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Of currently available therapies, prophylactic 2-mercaptoethanesulfonic acid (MESNA) has limited efficacy and cannot treat pre-existing lesions. Therefore, novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis haematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansonieggs), is superior to 3 doses of MESNA in alleviating hemorrhagic cystitis. Based on prior reports by others on S. mansoni IPSE and additional work by our group, we reasoned that H-IPSEH06 mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. haematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared to systemic administration of IPSEH06. We demonstrate herein that H-IPSEH03 is a promising therapeutic for the treatment of voiding dysfunction and bladder pain in hemorrhagic cystitis. Namely, it attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia. Finally, H-IPSEH03 drives increased urothelial cell proliferation. This indicates that IPSE induces bladder healing mechanisms, which suggests that it may be a novel non-opioid analgesic to treat bladder pain syndromes.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3’-hydroxy cotinine and N’-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 μM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.

To study the protection offered by empty liposomes (LPs) alone against acrolein-induced changes in urothelial cell viability and explored uptake of LPs by primary (rat) urothelial cells. Acrolein was used as a means to induce cellular damage and reduce urothelial cellular viability. The effect of acrolein or liposomal treatment on cellular proliferation was studied using 5-bromo-2′-deoxy-uridine assay. Cytokine release was measured after urothelial cells were exposed to acrolein. Temperature-dependent uptake study was carried out for fluorescent-labeled LPs using confocal microscopy. Liposome pretreatment protected against acrolein-induced decrease in urothelial cell proliferation. LPs also significantly affected the acrolein-induced cytokine (interferon-gamma) release offering protection to the urothelial cells against acrolein damage. We also observed a temperature-dependent urothelial uptake of fluorescent-labeled LPs occurred at 37 °C (but not at 4 °C). Empty LPs alone provide a therapeutic efficacy against acrolein-induced changes in urothelial cell viability and may be a promising local therapy for bladder diseases. Hence, our preliminary evidence provides support for liposome-therapy for urothelial protection and possible repair.

Tobacco smoking is a major risk factor for multiple human cancers including urinary bladder carcinoma. Tobacco smoke is a complex mixture containing chemicals that are known carcinogens in humans and/or animals. Aromatic amines a major class of DNA-reactive carcinogens in cigarette smoke, are not present at sufficiently high levels to fully explain the incidence of bladder cancer in cigarette smokers. Other agents in tobacco smoke could be excreted in urine and enhance the carcinogenic process by increasing urothelial cell proliferation. Nicotine is one such major component, as it has been shown to induce cell proliferation in multiple cell types in vitro. However, in vivo evidence specifically for the urothelium is lacking. We previously showed that cigarette smoke induces increased urothelial cell proliferation in mice. In the present study, urothelial proliferative and cytotoxic effects were examined after nicotine treatment in mice and rats. Nicotine hydrogen tartrate was administered in drinking water to rats (52 ppm nicotine) and mice (514 ppm nicotine) for 4 weeks and urothelial changes were evaluated. Histopathologically, 7/10 rats and 4/10 mice showed simple hyperplasia following nicotine treatment compared to none in the controls. Rats had an increased mean BrdU labeling index compared to controls, although it was not statistically significantly elevated in either species. Scanning electron microscopic visualization of the urothelium did not reveal significant cytotoxicity. These findings suggest that oral nicotine administration induced urothelial hyperplasia (increased cell proliferation), possibly due to a mitogenic effect of nicotine and/or its metabolites.

Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. 2,3-Dimercaptopropane-1-sulfonic acid (DMPS), a chelating agent, is capable of reducing pentavalent arsenicals to the trivalent state and binding to the trivalent species, and it has been used in the treatment of heavy metal poisoning in humans. Therefore, we investigated the ability of DMPS to inhibit the cytotoxicity and regenerative urothelial cell proliferation induced by arsenate administration in vivo. Female rats were treated for 4 weeks with 100 ppm AsV. DMPS (2800 ppm) co-administered in the diet significantly reduced the AsV-induced cytotoxicity of superficial cells detected by scanning electron microscopy (SEM), and the incidence of simple hyperplasia observed by light microscopy and the bromodeoxyuridine (BrdU) labeling index. It also reduced the total concentration of arsenicals in the urine and the methylation of arsenic. There were no differences in oxidative stress as assessed by immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8OHdG) of the bladder urothelium. No differences were detected in urine sediments between groups. These data suggest that DMPS has the ability to inhibit both arsenate-induced acute toxicity and regenerative proliferation of the rat bladder epithelium, most likely by decreasing exposure of the urothelium to trivalent arsenicals excreted in the urine. These data provide additional evidence that the effects of arsenate exposure in vivo do not appear to be related to oxidative effects on dG in DNA.

In normal urothelium, superficial umbrella cells express four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa, which compose urothelial plaques. In the apical plasma membrane, urothelial plaques form microridges. During neoplastic changes, microridges are replaced by microvilli, while uroplakin expression is retained. We correlated individual uroplakin expression with apical plasma membrane structure, cytokeratin 20 expression, and urothelial cell proliferation (Ki-67). Male Wistar rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, which caused flat hyperplasia with mild dysplasia, low-grade papillary urothelial carcinoma, invasive low- and high-grade papillary urothelial carcinoma and invasive squamous cell carcinoma with extensive keratinization, grade 2. During urothelial carcinogenesis, UPII expression was the most decreased in all urothelial lesions, while UPIa, UPIb, and UPIIIa expression was differently altered in different types of lesions. Superficial cells were covered with microvilli and ropy ridges, while microridges were disappearing. The expression of cytokeratin 20 was decreased and limited to superficial urothelial cells. Proliferation indices were increased, except for invasive squamous cell carcinoma with extensive keratinization. Our results indicate that during urothelial carcinogenesis the expression of UPII is diminished, suggesting that UPIb/UPIIIa heterodimer can still be formed, while heterodimer UPIa/UPII formation is disrupted. Correlation between decreased level of UPII expression and changed apical plasma membrane structure suggests that diminished expression of UPII hinders the urothelial plaque formation.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which belong to the nuclear receptor superfamily. Some PPARgamma agonists, such as pioglitazone, and dual PPARgamma/PPARalpha agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyuridine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPARgamma agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat-specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

We reported previously that both subtypes of estrogen receptors, ERalpha and ERbeta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERalpha and ERbeta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERbeta, but immortalized and cancer cells express much higher amounts of ERalpha than primary cells. Higher levels of ERalpha mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERalpha-selective agonist PPT, the ERbeta-selective agonist DPN, and specific small interfering RNA against ERalpha or ERbeta, we found that ERbeta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERalpha predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17beta-estradiol (E(2)) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E(2)-induced urothelial cell proliferation. E(2) up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERalpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo. To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.

Interstitial cystitis (IC)/painful bladder syndrome/bladder pain syndrome (BPS) is a chronic hypersensory condition of unknown etiology. Moreover, the optimal modality for diagnosing IC remains disputed. Several urinary markers have been investigated that may have potential utility in the diagnosis or confirmation of IC/BPS. Thus, inflammatory mediators, proteoglycans, urinary hexosamines, proliferative factors, nitric oxide (NO), BK polyomavirus family, and urothelial proinflammatory gene analysis have been found to correlate with varying degrees with the clinical diagnosis or cystoscopic findings in patients with IC/BPS. The most promising urinary biomarker for IC/BPS is antiproliferative factor, a sialoglycopeptide that has demonstrated inhibitory effects on urothelial cell proliferation and a high sensitivity and specificity for IC/BPS symptoms and clinical findings. In this article, we review the urinary markers, possible future therapies for IC/BPS, and the clinical relevance and controversies regarding the diagnosis of IC/BPS.

Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-alpha, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.

Inorganic arsenic is a known human carcinogen of the skin and respiratory tract. Epidemiologic evidence indicates that it is also carcinogenic to the urinary bladder and other internal organs. Lack of an animal model has limited progress on understanding the mechanism of arsenic carcinogenesis. It was recently reported that high doses of an organic arsenical, dimethylarsinic acid (DMA), increased urinary bladder tumors in rats when administered in the diet or in the drinking water for 2 years, with the female being more sensitive than the male. We previously showed that high doses of DMA (40 or 100 ppm of the diet) fed for 10 weeks increased urothelial cell proliferation in the rat. Treatment with DMA also increased renal calcification and increased urinary calcium concentration. In 2 experiments, we examined the urothelial proliferative effects of treatment with 100 ppm DMA in the diet in female F344 rats for 2 and 10 weeks and for 6 and 24 h, and 3, 7, and 14 days. Cytotoxic changes in the urothelium were evident by SEM as early as 6 h after treatment was begun. Foci of cellular necrosis were detected after 3 days of treatment, followed by widespread necrosis of the urothelium after 7 days of treatment. The bromodeoxyuridine (BrdU) labeling index was not increased until after 7 days of treatment, suggesting that administration of DMA results in cytotoxicity with necrosis, followed by regenerative hyperplasia of the bladder epithelium. Although the rat provides an animal model to study the urothelial effects of DMA, the relevance of this finding to inorganic arsenic carcinogenesis in humans must be extrapolated cautiously, due to the high doses of DMA necessary to produce these changes in the rat and the differences in metabolism of arsenicals in rodents, especially rats, compared to humans.

Background: It was observed in the early 1970s that saccharin produced bladder cancer in rats. However, it has been unclear whether sodium saccharin when consumed by humans poses a substantial carcinogenic hazard. Numerous epidemiologic studies have not shown any evidence of increased urothelial proliferation associated with ingestion of sodium saccharin. Purpose: Our purpose was to determine the effects of long-term feeding of sodium saccharin to three species of nonhuman primates. Methods: Twenty monkeys of three species (six African green, seven rhesus, six cynomolgus, and one hybrid [of rhesus male and cynomolgus female parentage]) were treated with sodium saccharin (25 mg in the diet/kg body weight daily for 5 days a week) beginning within 24 hours after birth and continuing for up to 24 years. Sixteen monkeys (seven rhesus and nine cynomolgus) served as controls. During their last 2 years of life, urine was collected from selected treated and control animals and evaluated for various urinary chemistries and for the presence of calculi, microcrystalluria, and precipitate. Urinary bladders were examined by light microscopy and by scanning electron microscopy. Results: Sodium saccharin treatment had no effect on the urine or urothelium in any of these monkeys. There was no evidence of increased urothelial cell proliferation, and there was no evidence of formation of solid material in the urine. Conclusion: Although the dose of sodium saccharin administered to these monkeys was only five to 10 times the allowable daily intake for humans, the results provide additional evidence that sodium saccharin is without a carcinogenic effect on the primate urinary tract. [J Natl Cancer Inst 1998;90:19‐25]

Purpose: Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Experimental Design: Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors. Results: Significantly higher (P = 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P = 0.01), grade (P = 0.03), and invasion status (P = 0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P = 0.0012), invasion (P = 0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P = 0.04, EGFR; P = 0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P = 0.03) and invasion (P = 0.04) was also evident. Conclusions: Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC. Clin Cancer Res; 19(17); 4621–33. ©2013 AACR.

Purpose: To determine the effect of keratinocyte growth factor (KGF), a mesenchymally derived epithelial growth factor that can cause proliferation of pulmonary, gastrointestinal and mammary epithelia, on urothelium.Materials and Methods: Recombinant human KGF was systemically administered to rats and Rhesus monkeys, and the proliferative effects on the bladder were evaluated.Results: Keratinocyte growth factor causes proliferation of transitional epithelial cells. Proliferating cell nuclear antigen (PCNA) expression in rat bladder is dramatically increased along the basal layer of urothelium 1, 3, 7 and 14 days after daily injections of KGF. Incorporation of 5-bromodeoxyuridine (BrdU) at 7 and 14 days in the urothelium of KGF-treated rats parallels PCNA immunoreactivity and confirms that KGF increases DNA synthesis in urothelial cells. Urothelial cell proliferation is accompanied histologically by an increase in mitotic activity. Keratinocyte growth factor-induced PCNA expression is reversible up...

Secreted protein acidic and rich in cysteine (SPARC) has been implicated in multiple aspects of human cancer. However, its role in bladder carcinogenesis and metastasis are unclear,with some studies suggesting it may be a promoter and others arguing the opposite. Using a chemical carcinogenesis model in Sparc-deficient mice and their wild-type littermates, we found that loss of SPARC accelerated the development of urothelial preneoplasia (atypia and dysplasia), neoplasia, and metastasis and was associated with decreased survival. SPARC reduced carcinogen-induced inflammation and accumulation of reactive oxygen species as well as urothelial cell proliferation. Loss of SPARC was associated with an inflammatory phenotype of tumor-associated macrophages and fibroblasts, with concomitant increased activation of urothelial and stromal NF-κB and AP1 in vivo and in vitro. Syngeneic spontaneous and experimental metastasis models revealed that tumor- and stroma-derived SPARC reduced tumor growth and metastasis through inhibition of cancer-associated inflammation and lung colonization. In human bladder tumor tissues, the frequency and intensity of SPARC expression were inversely correlated with disease-specific survival. These results indicate that SPARC is produced by benign and malignant compartments of bladder carcinomas where it functions to suppress bladder carcinogenesis, progression, and metastasis.

Arsenite (As(III)), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As(III) and dimethylarsinous acid (DMA(III)) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as l-ascorbate and N-acetylcysteine, did not inhibit As(III)-induced cytotoxicity but they were more effective at inhibiting DMA(III)-induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100ppm As(III). Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As(III) treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As(III)-induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

We have previously shown that estrogen stimulates cell proliferation in both normal and transformed urothelial cells mainly through activation of the two primary estrogen receptors (ERs), ERα and ERβ. A growing body of evidence suggests that estrogen also initiates nongenomic effects that cannot be explained by activation of primary ERs. In the present study, we observed that urothelial cells express high amounts of GPR30, a G protein-coupled receptor recently identified as a candidate for membrane-associated estrogen binding. Membrane- impermeable bovine serum albumin-conjugated 17β-estradiol and the specific GPR30 agonist G-1 both inhibited urothelial cell proliferation in a concentration-dependent manner. Transient overexpression of GPR30 inhibited 17β-estradiol (E2)-induced cell proliferation. Decreased GPR30 expression caused by specific small interfering RNA increased E2-induced cell proliferation. These results indicate that membrane-associated inhibitory effects of E2 on cell proliferation correlate with abundance of GPR30. Although E2 induced a significant increase in caspase-3/7 activity, G-1 did not, suggesting that the GPR30-mediated inhibitory effect on cell proliferation was not caused by apoptosis. Furthermore, we found that G-1 failed to induce c-fos, c-jun, and cyclin D1 expression, and GPR30 overexpression abolished E2-induced c-fos, c-jun, and cyclin D1 expression. However, inactivation of GPR30 by small interfering RNA increased c-fos, c-jun, and cyclin D1 expression. These results suggest that GPR30-mediated inhibition of urothelial cell proliferation is the result of decreased cyclin D1 by down-regulation of activation protein-1 signaling.

BackgroundSteroid hormones influence mitogenic signaling pathways, apoptosis, and cell cycle checkpoints, and it has long been known that incidence of bladder cancer (BC) in men is several times greater than in women, a difference that cannot be attributed to environmental or lifestyle factors alone. Castration reduces incidence of chemically-induced BC in rodents. It is unclear if this effect is due to hormonal influences on activation/deactivation of carcinogens or a direct effect on urothelial cell proliferation or other malignant processes. We examined the effect of castration on BC growth in UPII-SV40T transgenic mice, which express SV40 T antigen specifically in urothelium and reliably develop BC. Furthermore, because BC growth in UPII-SV40T mice is exophytic, we speculated BC growth was dependent on angiogenesis and angiogenesis was, in turn, androgen responsive.MethodsFlat panel detector-based cone beam computed tomography (FPDCT) was used to longitudinally measure exophytic BC growth in UPII-SV40T male mice sham-operated, castrated, or castrated and supplemented with dihydrotestosterone (DHT). Human normal bladder and BC biopsies and mouse bladder were examined quantitatively for thrombospondin-1 (TSP1) protein expression.ResultsMice castrated at 24 weeks of age had decreased BC volumes at 32 weeks compared to intact mice (p = 0.0071) and castrated mice administered DHT (p = 0.0233; one-way ANOVA, JMP 6.0.3, SAS Institute, Inc.). Bladder cancer cell lines responded to DHT treatment with increased proliferation, regardless of androgen receptor expression levels. TSP1, an anti-angiogenic factor whose expression is inhibited by androgens, had decreased expression in bladders of UPII-SV40T mice compared to wild-type. Castration increased TSP1 levels in UPII-SV40T mice compared to intact mice. TSP1 protein expression was higher in 8 of 10 human bladder biopsies of normal versus malignant tissue from the same patients.ConclusionFPDCT allows longitudinal monitoring of exophytic tumor growth in the UPII-SV40T model of BC that bypasses need for chemical carcinogens, which confound analysis of androgen effects. Androgens increase tumor cell growthin vitroandin vivoand decrease TSP1 expression, possibly explaining the therapeutic effect of castration. This effect may, in part, explain gender differences in BC incidence and implies anti-androgenic therapies may be effective in preventing and treating BC.

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR)alpha/gamma agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPARalpha, PPARdelta, PPARgamma, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPARgamma agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPARgamma agonism. Urothelial cell PPARalpha and gamma expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPARalpha, delta, or gamma mRNA or protein expression, PPARalpha- or gamma-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPARgamma-regulated gene expression. These results support the contention that urine acidification does not prevent PPARgamma agonist-induced bladder tumors by altering PPARalpha, gamma, or EGFR expression or PPAR signaling in rat bladder urothelium.

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.

We have previously reported that estrogen induced proliferation of urothelial cells is modulated by nerve growth factor (NGF). In this study we investigated whether progesterone induces urothelial cell proliferation and whether this effect is modulated by NGF or by epidermal growth factor (EGF).Experiments were performed using human urothelial cells immortalized by human papillomavirus E6. Cell proliferation was determined using the alamarBlue (Trek Diagnostic, Westlake, New York) assay. Human papillomavirus were seeded in 48-well plates. They were incubated with 5% alamarBlue and different concentrations of progesterone, EGF or NGF in the presence or absence of neutralizing EGF or NGF antibody, K252a (an inhibitor of trkA, the high affinity receptor for NGF), Ru-486 (an antagonist of progesterone and glucocorticoid receptor) or ZK 137 316 (a specific antagonist of progesterone receptor). Immunoblotting was performed using specific antibodies for progesterone receptor, glucocorticoid receptor or EGF receptor. EGF content in conditioned medium was determined by enzyme-linked immunosorbent assay.In the presence of 10 nM to 1 microM progesterone urothelial cell proliferation was significantly increased 8.6% to 51.1%. This effect was abolished by ZK137 316 or by Ru-486. Hydrocortisone also induced urothelial cell proliferation. This effect was blocked by Ru-486 but not by ZK137 316. In addition, progesterone stimulated urothelial cell proliferation was inhibited by neutralizing EGF antibody but not by NGF antiserum or K252a. We also found that EGF synthesis and release by urothelial cells was increased by exogenous progesterone. This effect of progesterone was inhibited by ZK 137 316.These findings indicate that progesterone has the capacity to induce urothelial cell proliferation through its cognate receptor and this effect is mediated by EGF but not by NGF.

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-alpha 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.

Fibroblast growth factor-7 (FGF-7, keratinocyte growth factor, KGF) is a 163 amino acid glycoprotein synthesized and secreted by mesenchymal cells (e.g. fibroblasts/ fibrocytes) in epithelial organs, thereby functioning as a paracrine mediator of epithelial cell proliferation. In the urinary bladder, FGF-7 is transported from the lamina propria across the urothelial basement membrane to where it ultimately binds to splice variants of the FGFR2 receptor present on the basolateral surface of transitional epithelial cells. We administered 100 μg/ml (LP.) recombinant FGF-7 (rFGF-7) to RAG1-deficient mice (n=3) for 7 days and observed a striking expansion of the urinary bladder urothelium. This expansion was characterized by a layer of stratified urothelium >20 cells thick and by positive immunostaining for the proliferation marker Ki-67. In contrast, RAG1-deficient mice (n=3) that received only buffer injection did not exhibit detectable urothelial expansion. rFGF-7 was detected by immunoblot analyses in the serum, but not in the urine, from RAG1-deficient mice that received the recombinant protein. Mice that have a targeted disruption in the gene encoding the V(D)J recombination activation gene RAG1 have small lymphoid organs with no mature B and T lymphocytes, due to the inability of cell progenitors to perform V(D)J recombination. The biological activity of FGF-7 in RAG-1 mice indicates that immuno-dependent mechanisms are not required for the induction of urothelial cell proliferation by this epithelial cell-specific growth factor.

Bladder carcinoma accounts for 26% of reported human malignancies in Egypt, and has been strongly associated with urinary schistosomiasis. Nevertheless, the immediate role of schistosomal egg proteins in bladder carcinogenesis is unexplored. We investigated the effects of crude soluble egg antigens (SEA) of Schistosoma hematobium on urothelial cell proliferation. The proliferation of bovine endothelial Endo, human urothelial J82 and smooth muscle SMC cell lines was assessed by low-density growth assays. SEA induced proliferation of both J82 and Endo cells in a dose-dependent fashion, but not SMC. Preboiling or proteinase K treatment of SEA abolished its effect. In addition, SEA enhanced urothelial expression of B-cell translocation protein (BTG1) and human proliferating cell nuclear antigen (PCNA) mRNAs. Given the strong correlation between cell proliferation and carcinogenesis, the findings suggest that crude SEA may play some role in schistosomal bladder carcinogenesis.

The chronic toxicity and carcinogenicity of Wingstay 100 (W 100), a rubber antioxidant/antiozonant, were studied in Fischer 344 (F 344) rats in two chronic studies. Earlier genetic studies indicated that the product had weak activity in a bacterial mutation assay, but lacked activity in chromosomal aberration assays. In an one year study, both genders of F 344 rats were exposed to 53, 310 or 1900 ppm in NIH-07 diet for 52 weeks, and sacrifices were made at 38, 52 and 64 weeks. No test substance related deaths occurred, although the high dose of 1900 ppm caused a decrease in body weight gain and food consumption in both genders. Red blood cell mean corpuscular volume was significantly increased at 38 weeks, accompanied by a significant decrease in mean corpuscular hemoglobin concentration. At 52 weeks, the red blood cell count and hemoglobin values were also significantly decreased in high dose animals of both genders. Total bilirubin and cholesterol were increased in high dose animals of 38 and 52-week sacrifices. During the 3 month recovery, hematology parameters, bilirubin and cholesterol returned to control values. Total protein was reduced in high dose animals of both genders, throughout the entire exposure and recovery periods. W 100 also produced increases in relative liver, spleen, heart and kidney weights in high dose animals. Both genders of all W 100 groups exhibited significant increases in urothelial cell proliferation (measured by PCNA) and adaptive hyperplasia. No regenerative hyperplasia, preneoplasia, or neoplasia were present. There was microscopic evidence of extramedullary erythropoiesis in the spleen and liver of high dose animals in both genders, otherwise no other pertinent microscopic finding was evident. In parallel, an accelerated bioassay (ABA) was conducted, which is a mechanistic initiation/promotion carcinogenicity study designed to assess tumor induction and promotion potential of a test substance in major organs of carcinogenesis. The present study was conducted in male F 344 rats for 38 weeks. The target sites chosen for the ABA were liver and urinary bladder and the dose for W 100 was 1900 ppm previously established to be a toxic dose. The liver tumor initiator was diethylnitrosamine (DEN), and the urinary bladder initiator was N-butyl N-(4-hydroxybutyl) nitrosamine (BBN). The initiators were administered during the first 14 weeks followed by the promoters. The promoters, phenobarbital (PB) for the liver and nitrilotriacetate (NTA) for the urinary bladder, were administered during the last 24 weeks of the study after the test substance. The study had 11 test groups including a negative control. The critical comparisons for initiating activity were conducted between groups 3 (PB) and 6 (W 100 + PB) for the liver and groups 8 (NTA) and 11 (W 100 + NTA) for the urinary bladder. The critical comparisons for promoting activity were conducted between groups 2 (DEN) and 5 (DEN + W 100) for the liver and groups 7 (BBN) and 10 (BBN + W 100) for the urinary bladder. There were 26 and 38-week sacrifices. In this study, most body weight reductions were due to DEN. At 26 weeks, significant increases in liver weights were present in all PB-exposed groups. Significant increases in renal weights occurred in all NTA, BBN and DEN groups. A similar organ weight pattern was present at 38 weeks. At 26 weeks, there were hepatocellular (33%) and urothelial (67%) tumors present in positive control groups (DEN/DEN + PB/BBN/BBN + NTA). In contrast, in the DEN + W 100 (5) and the BBN + W 100 (10) groups no tumors were present indicating absence of promotion. In addition, no tumors were present in groups 6 (W 100 + PB) or 11 (W 100 + NTA) indicating absence of initiation. At 38 weeks, the incidences of hepatocellular adenomas and carcinomas in positive control group (DEN) was 44%. The incidence of urothelial adenomas and carcinomas was 67% in group 7 (BBN). In contrast, groups 5 (DEN + W 100) or group 10 (BBN + W 100) had

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p

The proliferation response of rat renal pelvic epithelium, lined by transitional epithelium, to administration of various bladder cancer promoters was investigated. In addition, prostaglandin E 2 (PGE 2 ), lipid peroxide (LPO), malondialdehyde (MDA) and cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were assessed in urine of rats given the non-promoter l -ascorbic acid (AsA) and the promoters sodium l -ascorbate (AsA-Na) or sodium bicarbonate (NaHCO 3 ) for 4 or 8 weeks. DNA synthesis in the renal pelvic epithelium, as assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation, was increased in the groups given AsA-Na, an extremely high dose of sodium chloride (NaCl), tert -butylhydroquinone (TBHQ) or ethoxyquin (EQ). Moreover, with the exception of AsA-Na, ail treatments that induced an elevation of DNA synthesis also caused morphological epithelial alterations as observed by scanning electron microscopy (SEM). Treatment with butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) did not result in any proliferative response of the rat renal pelvis. No treatment-related changes in urinary PGE 2 and cyclic AMP were noted, although AsA-Na and AsA but not NaHCO 3 reduced levels of LPO and MDA in the urine. The results indicate that while the response of renal pelvic epithelium to certain bladder cancer promoters is similar to that of the bladder itself, none of the urinary cellular growth or free radical biochemical parameters is directly related to urothelial cell proliferation.

Abuse of combination analgesics containing phenacetin, antipyrine (phenazone) and caffeine have been associated with urinary tract tumors. Phenacetin and antipyrine have been shown to be promoters of urinary tract carcinogenesis and antipyrine is also a weak urinary tract carcinogen. Acetaminophen, the main metabolite of phenacetin, is one of the most commonly used analgesics in the USA. In the present study, the dose-related effect on the cell proliferation of the urothelium was evaluated in male Sprague-Dawley rats by autoradiography. Nine groups of twenty, 6-week old rats were treated with 0.5%, 1.0% or 1.5% of acetaminophen, antipyrine or phenacetin in the diet. A tenth group of rats received control diet without added chemicals. Ten rats from each group were killed after each of 6 and 12 weeks of feeding. There was a dose-related increase in the labeling index in the urothelium of the bladder and kidney, particularly after 6 weeks of drug administration. In particular, the 1.0% and 1.5% dose levels of antipyrine and phenacetin showed a marked proliferative effect on the urothelium. In the bladder after 6 weeks, the labeling indices were significantly increased. After 12 weeks, although numerically increased, the indices were not statistically significant. In the renal pelvic urothelium the labeling index was significantly increased in antipyrine and phenacetin treated rats at doses of 1.0% and 1.5%. After 12 weeks the majority of rats treated with 1.5% antipyrine and phenacetin had labeling indices greater than or equal to 2-fold than the control rats both in the kidney and bladder. The increased labeling indices were associated with urothelial hyperplasia, in particular after 6 weeks. In the rats treated with antipyrine there were significant degenerative changes in the urothelial cells expressed as marked vacuolization. The vacuolization is considered to be a toxic effect and the beginning of cell death. Thus cell death with regeneration may be responsible for the increased labeling index in the antipyrine groups. High doses of antipyrine were also associated with renal papillary necrosis in 50% of the rats.

Uracil is known to cause reversible urolithiasis and to induce papillomatosis in the urinary bladder of F344 rats. We examined whether the marked urothelial cell proliferation caused by uracil, given in the middle of the post-initiation stages, enhances the promoting activity of a promoter in the two-stage model or the promoting and/or carcinogenic activity of a low-dose carcinogen in the multistage model of urinary bladder carcinogenesis. Rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks, and then 2% butylated hydroxyanisole (BHA) or 0.002% N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) were given during experimental weeks 4-9 and weeks 12-20. Uracil was given during weeks 9-12 at a level of 3% of the diet. Rats in the control group were treated with BBN and uracil. Rats were killed at weeks 16 and 20. At week 16, higher occurrences of papillary or nodular (PN) hyperplasia and papilloma were observed in uracil-BHA-treated rats than in the controls. At week 20, significantly higher incidences of PN hyperplasia and papilloma were observed in both uracil-EHBN- and uracil-BHA-treated groups, and a summation effect of uracil was observed. These results indicate that uracil given in the middle of the post-initiation stage enhanced the promoting activity of the compound through marked proliferation of the bladder epithelium.

Background The PTEN/Phosphatidylinositol 3'-kinase (PI3-kinase) growth factor signaling pathway plays a critical role in epithelial tumor development in a multitude of tissue types. Deletion of the Pten tumor suppressor gene in murine urothelial cells in vivo results in upregulation of cyclin-dependent kinase inhibitor p21. We have previously shown in mice that p21 expression blocks an increase in urothelial cell proliferation due to Pten deletion. In this study, we utilized human urothelial carcinoma cells UMUC-3 and UMUC-14 to identify the signaling pathways downstream of PI3-kinase that regulate p21. Methods Cells were treated with a combination of PI3-kinase stimulating growth factors and kinase inhibitors, or transfected with exogenous genes in order to identify the signaling events that are necessary for p21 induction. Mice with conditional deletion of Pten in bladder urothelium were also examined for evidence of PI3-kinase pathway signaling events that affect p21 expression. Results When cells were treated with PI3-kinase activating growth factors EGF or PDGF, we found that p21 levels increased, in a manner similar to that observed in mice. We used the inhibitors LY294002, Akti-1/2, and rapamycin, to show that p21 induction is dependent upon PI3-kinase and AKT activity, and partially dependent on mTOR. We treated the cells with proteasome inhibitor MG-132 and found that p21 may be degraded in the proteasome to regulate protein levels. Importantly, our findings show that GSK-3β plays a role in diminishing p21 levels in cells. Treatment of cells with the GSK-3β inhibitor SB-216763 increased p21 levels, while exogenous expression of GSK-3β caused a decrease in p21, indicating that GSK-3β actively reduces p21 levels. We found that a combined treatment of LY294002 and SB-216763 improved the cytotoxic effect against UMUC-3 and UMUC-14 carcinoma cells over LY294002 alone, suggesting potential therapeutic uses for GSK-3β inhibitors. Immunohistochemical staining in bladders from wild-type and Pten-deleted mice indicated that GSK-3β inhibitory phosphorylation increases when Pten is deleted. Conclusion PI3-kinase and AKT cause an upregulation of p21 by suppressing GSK-3β activity and activating mTOR in both cultured human urothelial carcinoma cells and mouse urothelial cells in vivo.