Cyclophosphamide (CP) causes extensive cystitis, which is ameliorated with concomitant treatment with mesna. We investigated the protective mechanisms of mesna in the expression of uroplakin (UP), a strong mucosal barrier against toxic materials, in CP-induced rat cystitis. A total of 54 SD female rats received a single intraperitoneal injection of 200 mg of CP/kg. Six CP-treated, 6 CP + mesna (120 mg/kg)-treated rats, and 6 negative controls were sequentially sacrificed at 12, 24, and 72 h post-CP injection. The bladders were harvested. The levels of UPIa, Ib, II, and III mRNA on real-time PCR, the UPII and III expressions on immunoblotting, and the UPII expression on immunolocalization study in the harvested bladder were maximally suppressed within 12-24 h, whereas partially or completely recovered at 24-72 h post-CP injection. In addition, the responses in UPs after a CP insult were heterogeneous (i.e., markedly suppressed in UPII and lesser destructive in UPIII). Even though the mesna-treated rats also showed transient and small reductions in the mRNA levels of all UPs, mesna clearly preserved the UP expressions of mRNA and protein in CP-induced urinary bladder mucosa. In conclusion, this study suggests that CP transiently reduces the expression of UPs and mesna protects the urinary bladder mucosa through the preservation of UPs protein.

OBJECTIVE

To construct a dual specific vector which contains prostate stem cell antigen enhancer (PSCAE) and uroplakin II (UPII) promoter targeted bladder cancer.

METHODS

UPII promoter and PSCAE were amplified by polymerase chain reaction (PCR). Luciferase gene (LUC) was obtained from plasmid pBK-CMV-LUC. PSCAE, UPII promoter and LUC were inserted into shuttle plasmid to create Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. Rp-UPII-LUC and Rp-PSCAE-UPII-LUC were cotransfected with pCMV-beta-gal into various cell lines at the presence or absence of androgen receptor agonist R1881 and androgen receptor antagonist flutamide. Luminescence was detected with luciferase assay kit and counted on liquid scintillation counter.

RESULTS

Bladder cancer cells showed higher LUC activity than non-bladder cancer cells after transfected with plasmids Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. PSCAE could improve the LUC activity in both AR positive and AR negative bladder cancer cells but not in non-bladder cancer cells and normal human urothelial (NHU) cells. R1881 could increase the LUC activity in AR positive bladder cancer cells but not in AR negative bladder cancer cells and non-bladder cancer cells. Flutamide could not inactivate PSCAE in bladder cancer cells.

CONCLUSIONS

PSCAE can improve target gene expression in bladder cancer cells but not in non-bladder cancer cells and NHU cells. PSCAE maintains a certain level of androgen independent activity in bladder cancer cells. PSCAE is active in both AR positive and AR negative bladder cancer cells. The results suggest that combination of PSCAE with UPII promoter is feasible in constructing bladder cancer-specific vectors.

The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.

BACKGROUND

Nearly 50% of urothelial carcinoma patients with lymph node-negative invasive cancers recur after radical surgery. In many cases, occult local or lymph node disease may be present but undetectable by current approaches. Reverse-transcriptase polymerase chain reaction (RT-PCR)-detectable mRNA of Uroplakin II (UPII), a urothelial-specific gene mRNA, was evaluated in perivesical and lymph node samples removed at radical surgery as a predictor of clinical recurrence.

METHODS

From November 1999 to August 2002, 46 patients with cTa-T4N0M0 urothelial bladder cancer enrolled in a prospective clinical trial and underwent radical cystectomy and pelvic lymphadenectomy. RT-PCR for UPII was performed on biopsies of the external surface of the bladder specimen and lymph nodes. Results were compared with conventional pathology. Patients were followed every 6 months for tumor recurrence.

RESULTS

Pathologically node-negative patients had a UPII RT-PCR perivesical positivity of 27% and a lymph node positivity rate of 33%. All 22 UPII RT-PCR node-negative patients were pathologically node-negative and all 13 with pathologically positive nodes had positive UPII RT-PCR lymph node signals. In all, 46% of UPII RT-PCR lymph node-positive patients were pathologically node-negative and 5% of pathologically node-negative/UPII RT-PCR node-negative patients had disease recurrence, whereas 91% of pathologically node-negative/UPII RT-PCR node-positive patients (P < .001) recurred. UPII RT-PCR node positivity was a significant predictor of tumor recurrence in multivariate analysis

CONCLUSIONS

Molecular determination of lymph node metastases by UPII RT-PCR node positivity apparently identifies patients with a poor prognosis and may be more predictive of disease recurrence than conventional pathologic analysis.

OBJECTIVE

Urothelial carcinoma is a frequent and aggressive cancer. We wanted to gain better insight into the early molecular mechanisms of bladder carcinogenesis by evaluating Aurora-A gene expression, which is implicated in genomic stability and essential for mitosis.

METHODS

This study, using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), analyzed the expression levels of three selected genes in dissected tissues from normal bladder, noninvasive cancers, and muscle-invasive bladder carcinomas (n = 49). We compared gene expression levels of three genes (Aurora-A, and as control uroplakin II (UPII) and TBP, respectively) at different stages of bladder cancer. We used multivariate analysis, receiver operating characteristic curves and the nonparametric Mann-Whitney test.

RESULTS

The expression of Aurora-A gene studied was significantly deregulated, with an increasing level in cancer versus normal tissue Aurora-A. This development was linear. Aurora-A was already deregulated in early stages of carcinogenesis (pTa/pT1) (P = 0.0004) and displayed even more deregulation in muscle-invasive stages (pT2 to pT4). Immunohistochemistry performed on the same samples using Aurora-A antibody confirmed results of RT-PCR, with statistically significant values when comparing m-RNA expression and immunohistochemical values (P = 0.0001).

CONCLUSIONS

This study highlights the fact that Aurora-A gene expression is already strongly deregulated in early stages of urothelial carcinoma with abnormal expression, and might be considered a biomarker of tumor aggression. The increase in Aurora-A expression might provide further information regarding the behavior of bladder cancer in daily practice.

Distinguishing between invasive urothelial carcinoma from other genitourinary lesions such as prostatic and renal carcinomas can be difficult, and may require highly sensitive immunohistochemical markers. GATA-binding protein 3 (GATA3) has been reported in a high percentage of urothelial and breast carcinomas. Mouse monoclonal uroplakin II (UPII) and p40 antibodies have recently been developed and demonstrated high specificity in urothelial carcinoma. This study evaluated the immunohistochemical staining sensitivities of UPII, GATA3, p40, and p63 in the detection of invasive urothelial carcinoma. UPII, GATA3, and p40 were further tested for specificity in lung, breast, colon, kidney, and prostate cancers. In all invasive urothelial carcinoma cases, UPII, GATA3, p40, and p63 exhibited sensitivities of 77.7%, 83.5%, 85.4%, and 80.6%, respectively. The combination of UPII, GATA3, and p40 antibodies stained 94.2% (97/103) of all invasive urothelial carcinoma cases, including 92.2% (71/77) of grade 2-3 urothelial carcinomas. In addition, GATA3 and UPII showed negative staining in lung squamous cell carcinomas and p40 showed negative staining in breast infiltrating ductal carcinomas. The combination of UPII, GATA3, and p40 showed negative staining in lung adenocarcinoma, colon adenocarcinoma, and renal carcinomas. In conclusion, UPII, GATA3, and p40, when used in combination, are highly sensitive in the differential diagnosis of invasive urothelial carcinoma.

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed approximately 1,900 unique genes differentially expressed >> or =3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers.

Epidemiologic and clinical data suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. We determined the chemopreventive efficacy of licofelone, a dual COX-lipoxygenase (LOX) inhibitor, in a transgenic UPII-SV40T mouse model of urothelial transitional cell carcinoma (TCC). After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized, and urinary bladders were collected to determine urothelial tumor weights and to evaluate histopathology. Results showed that bladders of the transgenic mice fed control diet weighed 3 to 5-fold more than did those of the wild-type mice due to urothelial tumor growth. However, treatment of transgenic mice with licofelone led to a significant, dose-dependent inhibition of the urothelial tumor growth (by 68.6%-80.2%, P < 0.0001 in males; by 36.9%-55.3%, P < 0.0001 in females) compared with the control group. The licofelone diet led to the development of significantly fewer invasive tumors in these transgenic mice. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; P < 0.01) and female mice (41%-44%; P < 0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, and caspase3) with a decrease in proliferation, inflammation, and angiogenesis markers (proliferating cell nuclear antigen, COX-2, 5-LOX, prostaglandin E synthase 1, FLAP, and VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC.

OBJECTIVE

To construct urothelium-specific recombinant adenovirus and investigate its inhibition in bladder cancer cell.

METHODS

RT-PCR analysis was used to determine expression patterns of hUPII and coxsackie adenovirus receptor on multiple cell lines. Transient transfection and luciferase detecting assay were used to detect tissue specificity of the hUPII promoter. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed. Restrictive enzyme digestion assay and PCR confirmed the correct construction. The adenovirus E1A protein expressed in BIU-87 was tested by Western blot after cells were infected with recombinant adenovirus. Recombinant adenovirus Ad-UPII-E1A was tested for its inhibition in bladder cancer cell line BIU-87.

RESULTS

HUPII and CAR were expressed and the hUPII promoter is highly active in bladder cancer cell line BIU-87. Using homologous recombination in bacteria technology, the hUPII promoter and E1A gene were inserted into the genome of type 5 recombinant adenovirus. The E1A protein was markedly positive in the samples of BIU-87 cells infected with recombinant adenovirus Ad-UPII-E1A. MTT assay demonstrated recombinant adenovirus Ad-UPII-E1A inhibited bladder cancer cell BIU-87 growth.

CONCLUSIONS

The hUPII promoter shows high tissue specificity. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed and confirmed. Recombinant adenovirus Ad-UPII-E1A is effective in inhibition in bladder cancer cell line BIU-87.

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.

OBJECTIVE

To establish reliable noninvasive in vivo methods to detect, measure, and monitor experimentally induced urothelial tumors in mice.

METHODS

UPII-SV40T transgenic mice reliably develop bladder tumors by expression of simian virus 40 large T antigen specifically in bladder urothelium through the use of the uroplakin II promoter. Two wild-type and 10 UPII-SV40T transgenic mice were monitored for microhematuria two to three times weekly using dipstick analysis. A unique flat panel detector-based cone beam computed tomography (FPD-CBCT) system imaged the urinary tracts of anesthetized mice after tail vein injection of an iodinated contrast agent (Omnipaque) that is excreted in urine. Within 10 seconds, the FPD-CBCT system acquired 290 two-dimensional images, which produced three-dimensional volumes with true isotropic resolution (180 microm)3 using a filtered back projection-based modified Feldkamp reconstruction algorithm. Amira, version 3.1.1-1, for MacOSX was used for data analysis and advanced visualization of the three-dimensional reconstructed FPD-CBCT images.

RESULTS

Hematuria was present in UPII-SV40T transgenic mice at 32 days of age; the wild-type animals exhibited no hematuria. Filling defects, associated with histologically confirmed tumors, in the bladders of the UPII-SV40T transgenic mice were visualized in the reconstructed FPD-CBCT images 1 to 45 minutes after contrast agent injection. Longitudinal FPD-CBCT imaging sessions showed the tumor position, volume, and growth.

CONCLUSIONS

The combination of early detection of hematuria and high-resolution in vivo FPD-CBCT imaging of murine bladder tumors enabled accurate longitudinal assessment of tumor growth and progression in individual animals. This approach could provide an important alternative to serial sacrifice experimental designs, while refining statistical power and reducing animal use.

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.

The high frequency of recurrence and poor survival rate of bladder cancer demand exploration of novel strategies. Gene therapy via adenovirus has shown promising potential for the treatment of tumors. We constructed a bladder cancer-specific adenovirus carrying E1A-androgen receptor (AR) under the control of UPII promoter and prostate stem cell antigen enhancer (PSCAE), designated as Ad/PSCAE/UPII/E1A-AR, and investigated its antitumor effects in vitro and in vivo. We demonstrated that Ad/PSCAE/UPII/E1A-AR could be selectively replicated in bladder tumor cell lines (5637, BIU87, EJ and T24) when compared with control adenovirus Ad/PSCAE/UPII/Luc. However, there was no evidence of cytotoxicity for normal human bladder cell line SV-HUC-1 and hepatoma cell line SMMC7721. AR agonist R1881 could strengthen the oncolytic effect of Ad/PSCAE/UPII/E1A-AR in bladder cancer cells. In addition, we demonstrated that intratumoral injection of Ad/PSCAE/UPII/E1A-AR into established subcutaneous human EJ tumors in nude mice could significantly regress the growth of tumor and markedly prolong survival for tumor-bearing mice; on the other hand, saline-treated tumors continued to grow rapidly. Our studies indicate that Ad/PSCAE/UPII/E1A-AR could effectively treat bladder cancer in vitro and in vivo. Furthermore, our findings provide a promising therapeutic modality for the treatment of bladder cancer.

BACKGROUND

The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials.

METHODS

Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific UroplakinII(UPII) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay.

RESULTS

General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5x10(8) pfu or higher dose (5x10(9) pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5x10(9) pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay.

CONCLUSIONS

Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5x10(7) pfu and 5x10(8) pfu intratumorally injection in mice, without any discernable effects on general health and behavior.

Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs) are the most primitive of those isolated from other post-natal tissue source. The hUCMSCs possess the capability of differentiating along multi-lineage. This study aimed to investigate whether hUCMSCs can differentiate into urothelium-like cells. The hUCMSCs were isolated from fresh human umbilical cord postpartum and expanded at least to passage 3 in vitro. Subsequently, they were cultured with conditioned medium from urothelial cells (UC-CM) supplemented with 20 ng/ml exogenous epidermal growth factor (EGF). Urothelial cell specific marker uroplakin II (UPII) and cytokeratins were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence technology. During culture, hUCMSCs started to express UPII and cytokeratins weakly at 7 days and were significantly up-regulated at 2 weeks post-induction. Additionally, morphology of hUCMSCs changed from spindle-shape to a polygonal epithelial-shape similar to that of urothelial cells after 7 days. The study results indicated that hUCMSCs can differentiate into urothelium-like cells in a defined micro-environment in vitro constituted by UC-CM and exogenous EGF.

Bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, carrying E1A gene regulated by human Uroplakin II (UPII) promoter and prostate stem cell antigen enhancer (PSCAE), could kill bladder tumor cells preferentially. The aim of this study was to examine the effects of Ad/PSCAE/UPII/E1A combined with cisplatin on human bladder cancer cells and to identify the underlying mechanisms. The combined effects of Ad/PSCAE/UPII/E1A and cisplatin on EJ, 5637, and BIU-87 bladder cancer cells were evaluated by MTT cell proliferation assay. Cell apoptosis was detected by flow cytometry with fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) and propidium iodide staining. The activation of the caspase pathway and the expression of Bcl-2 family proteins were determined by western blot assay. Ad/PSCAE/UPII/E1A adenovirus vector could infect bladder cancer cell lines selectively and induce growth inhibition effectively. Of note, the combination treatment of cisplatin and Ad/PSCAE/UPII/E1A could inhibit the proliferation of bladder cancer cells significantly compared with the "alone" treatment. Furthermore, Ad/PSCAE/UPII/E1A plus cisplatin combined treatment resulted in enhanced apoptosis in bladder cancer cells. The enhanced antitumor effects in vitro elicited by Ad/PSCAE/UPII/E1A plus cisplatin were closely related to the increased Fas expression and cleavage of caspase-8 and Bid and decrease in the ratio of anti- to pro-apoptotic proteins followed by activation of caspase-9 and caspase-3, which may contribute to the activation of extrinsic and intrinsic apoptotic pathways. Our results indicate that the combination of Ad/PSCAE/UPII/E1A with cisplatin exerts a synergistic antitumor effect on human bladder cancer cells and is a potential combined treatment strategy for bladder cancer.

Uroplakins are markers of terminally differentiated urothelium. Uroplakin II (UPII) is a newly described sensitive marker for urothelial carcinoma (UC). The expression profile of UPII in different types of UC and its utility in the diagnostic setting are needed. We evaluated UPII expression in bladder tissue microarrays, including urothelial neoplasm of low malignant potential (n = 8), low-grade papillary UC (n = 72), noninvasive high-grade papillary UC (n = 77), UC in situ (n = 27), and invasive high-grade UC (INVUC) (n = 122). UPII expression in 52 breast carcinomas and 38 high-grade prostate adenocarcinomas was also assessed. UPII expression was compared with GATA binding protein 3 (GATA3) and estrogen receptor for its role in facilitating the differential diagnosis of the above 3 types of malignancy. UPII labeling was seen in 83.0% of UC overall, including 95.7% of noninvasive UC and 65.6% of INVUC. UPII labeling was not found in any breast and prostate carcinomas. In comparison, GATA3 labeling was seen in 91.6% of all UCs, including 96.4% of noninvasive UCs and 85.1% of INVUC, with stronger intensity and extent compared with UPII (P < .005). GATA3 labeled 2 (5%) of 38 high-grade prostate adenocarcinoma. Estrogen receptor nuclear labeling was seen in 13.0% of UCs and 12.5% of prostate carcinomas. UPII was highly specific (100%) but only moderately sensitive for UC and can therefore be a potentially useful marker to identify urothelial lineage and help distinguish UC from prostate cancer or, in conjunction with GATA3, from metastatic breast cancer.

OBJECTIVE

To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model.

METHODS

Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous beta-galactosidase activity and p16(INK4a) mRNA expression.

RESULTS

Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor alpha (TGF alpha) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16(INK4a) mRNA expression and beta-galactosidase activity increased on day 25, giving signs of senescence.

CONCLUSIONS

This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.

The objective of this study was to report the initial anatomic, radiographic, and genetic evaluations of a novel form of spontaneous pelvic organ prolapse (S-POP) in mice. We observed S-POP in a colony of UPII-SV40T transgenic mice developed for studies on bladder cancer. We utilized magnetic resonance imaging and necropsy to characterize this finding. We have established a breeding colony to identify inheritance patterns and for future studies. Selective breeding isolated the S-POP phenotype from the transgene. In contrast to other animal models, the S-POP mouse does not require an obligatory antecedent event to manifest pelvic organ prolapse. Necropsy and imaging demonstrate significant displacement of the pelvic organs distal to the pelvic floor in both sexes. The appearance of the POP is similar to that seen in the human female phenotype. Preliminary breeding studies indicate an autosomal dominant inheritance pattern. This mouse may be an effective animal model for the study of POP in humans.

Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.6 kb mUPII promoter was also detected in brain, kidney, and testis in some transgenic mouse lines. Here, we have cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to human erythropoietin (hEPO) or a luciferase gene. pUPII-luciferase expression vectors with various deletions of the promoter region were introduced into mouse fibroblast (NIH3T3), Chinese hamster ovary (CHO), and human bladder transitional carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high levels of luciferase activity in transiently transfected RT4 cells, whereas the 8.8 kb pUPII promoter region displayed only low levels of activity. The pUPII-hEPO expression vector was injected into the pronucleus of zygotes to make transgenic mice. To elucidate the in vivo molecular mechanisms controlling the tissue- and cell-specific expression of the pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII promoter fragments linked to the genomic hEPO gene were generated. An erythropoietin (EPO) assay showed that all nine transgenic lines carrying the 8.8 kb construct expressed recombinant human erythropoietin (rhEPO) only in their urethra and bladder, whereas two transgenic lines carrying the 2.1 kb pUPII promoter displayed hEPO expression in several organs including bladder, kidney, spleen, heart, and brain. These studies demonstrate that the 2.1 kb promoter contains the DNA elements necessary for high levels of expression, but lacks critical sequences necessary for tissue-specific expression. We compared binding sites in the 2.1 and 8.8 kb promoter sequences and found five peroxisome proliferator responsive elements (PPREs) in the 8.8 kb promoter. Our data demonstrated that proliferator-activated receptor (PPAR)-gamma activator treatment in RT4 cells induced the elevated expression of hEPO mRNA under the control of the 8.8 kb pUPII promoter, but not the 2.1 kb promoter. Collectively, our data suggested that all the major trans-regulatory elements required for bladder- and urethra-specific transcription are located in the 8.8 kb upstream region and that it may enhance tissue-specific protein production and be of interest to clinicians who are searching for therapeutic modalities with high efficacy and low toxicity.

While pharmacoepidemiologic and laboratory studies have supported the hypothesis that the antidiabetic drug metformin may be useful in treating or preventing cancer, there is limited evidence to suggest which specific cancer sites may be particularly sensitive. Sensitivity likely is determined both by features of tumor pathophysiology and by pharmacokinetic factors. We used UPII-mutant Ha-ras transgenic mice that develop hyperplasia and low-grade, papillary urothelial cell carcinoma to determine whether metformin has activity in a model of superficial bladder cancer. Metformin significantly improved survival, reduced urinary tract obstruction, reduced bladder weight (a surrogate for tumor volume), and led to clear activation of AMP α kinase and inhibition of mTOR signaling in neoplastic tissue. We investigated the basis of the unusual sensitivity of this model to metformin, and observed that following oral dosing, urothelium is exposed to drug concentrations via the urine that are approximately 240-fold higher than those in the circulation. In addition, we observed that bladder cancer cell lines (RT4, UMUC-3, and J82) with homozygous deletion of either TSC1 or PTEN are more sensitive to metformin than those (TEU2, TCCSUP, and HT1376) with wild-type TSC1 and PTEN genes. Our findings provide a strong rationale for clinical trials of oral metformin in treatment of superficial bladder cancer.

The differential expression of the desired gene product in the target tissue is central for gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic gene expression. UroplakinII (UPII) is a urothelium-specific membrane protein. To investigate the feasibility of targeting gene therapy for bladder cancer, a DNA fragment of 2542-bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. The transient transfection showed that the DNA fragment resulted in preferential expression in bladder carcinoma cells, with negligible expression in nonurothelium cells. Furthermore, the DNA segment located between -2545 and -1608 decided the tissue-specificity of the UPII promoter, the segment located between -328 and -4 being the core promoter of UPII. We generated two recombinant adenoviruses under the control of the UPII promoter: Ad-hUPII-GFP, carrying green fluorescence protein (GFP), and Ad-hUPII-TNF, carrying the tumor necrosis factor alpha (TNFalpha). ELISA revealed that the secretion of TNFalpha by Ad-hUPII-TNF-infected bladder cancer cells was significantly higher than Ad-hUPII-TNF-infected nonurothelium cells. The conditioned medium from Ad-hUPII-TNF-infected bladder cancer cells apparently inhibited the proliferation of L929 cells, a TNFalpha-sensitive cell line, comparing to Ad-hUPII-TNF-infected nonurothelium cells. Intravesical inoculation with Ad-hUPII-TNF inhibited tumor growth in the orthotopic human bladder cancer model. The sustained high level of TNFalpha in urine was identified with ELISA. Taken together, these data suggest that most of the cis elements that confer the bladder-specificity and differentiation-dependent expression of the human UPII gene reside in the 2542-bp sequence, and TNFalpha driven by the human UPII (hUPII) promoter is effective in the specific inhibition of bladder cancer growth both in vivo and in vitro. These results may yield a new therapeutic approach for bladder cancer and provide information on the molecular regulation of urothelial growth, differentiation, and disease.

Mutations of the tumor suppressor p53 and elevated levels of polyamines are known to play key roles in urothelial tumorigenesis. We investigated the inhibition of polyamines biosynthesis and the restoration of p53 signaling as a possible means of preventing muscle invasive urothelial tumors using DFMO, an ODC-inhibiting agent, and CP-31398 (CP), a p53 stabilizing agent. Transgenic UPII-SV40T male mice at 6weeks age (n=15/group) were fed control diet (AIN-76A) or experimental diets containing DFMO (1000 and 2000 ppm) or 150 ppm CP or both. At 40 weeks of age, all mice were euthanized and urinary bladders were evaluated to determine tumor weight and histopathology. Low-dose DFMO had a moderate significant inhibitory effect on tumor growth (38%, P<0.02) and tumor invasion (23%). High-dose DFMO had a 47% tumor inhibition (P<0.0001) and 40% inhibition tumor invasion. There was no significant difference between 1000 and 2000 ppm doses of DFMO (P>0.05). CP at 150 ppm alone had a strong inhibitory effect on tumor growth by 80% (P<0.0001); however, no effect on tumor invasion was observed. Interestingly, the combination of DFMO (1000 ppm) and CP (150 ppm) led to significant decrease in tumor weight (70%, P<0.0001) and tumor invasion (62.5%; P<0.005). Molecular analysis of the urothelial tumors suggested a modulation of polyamine biosynthesis, proliferation, cell cycle regulators resulting from the use of these agents. These results suggest that targeting two or more pathways could be an effective approach for chemoprevention. A combination of CP and DFMO appears to be a promising strategy for urothelial TCC prevention.