Bladder Transitional Cell Carcinoma Cells Research Articles

Nuclear Protein 1 Expression Is Associated with PPARG in Bladder Transitional Cell Carcinoma.

The Nuclear protein 1 gene was first discovered in acute pancreatitis and functions as an oncogene in cancer progression and drug resistance. However, the role of Nuclear protein 1 in bladder transitional cell carcinoma (BTCC) is still unclear. The Cancer Genome Atlas database and immunohistochemical analysis were adopted to evaluate Nuclear protein 1 expression in BTCC. We applied lentivirus-mediated small-interfering RNA to down-regulate the expression of Nuclear protein 1 in BTCC cell lines. We further performed an Affymetrix microarray and Gene Set Enrichment Analysis (GSEA) to assess the genes and signaling pathways related to Nuclear protein 1. We found that Nuclear protein 1 expression was up-regulated in BTCC and positively related to the degree of BTCC malignancy. Compared with Caucasian patients with BTCC, Nuclear protein 1 expression was attenuated in Asian patients. The Affymetrix microarray showed that lipopolysaccharide was the upstream regulatory factor of Nuclear protein 1 in BTCC. The GSEA indicated that Nuclear protein 1 expression was associated with signaling pathways in cancer, peroxisome proliferator-activated receptor (PPAR) pathways, and RNA degradation. The expression of Nuclear protein 1 was negatively correlated with PPARG (R = -0.290, P < 0.001), but not with PPARA (R = 0.047, P = 0.344) and PPARD (R = -0.055, P = 0.260). The study findings indicate that Nuclear protein 1 is positively associated with the malignancy degree of BTCC and that Nuclear protein 1 expression is negatively correlated with PPARG.

Corrigendum] MicroRNA‑497 inhibits the proliferation, migration and invasion of human bladder transitional cell carcinoma cells by targeting E2F3.

Subsequently to the publication of the above article, the authors have realized that Fig.4E in their paper contained errors. The image selected to represent the experiment showing the UM‑UC‑3 cells of the siE2F3 group (0h) was chosen incorrectly during the figure compilation process. A corrected version of Fig.4 is shown opposite. Note that this error affected neither the interpretation of the data nor the reported conclusions of this work, and all the authors agree to this Corrigendum. The authors apologize to the Editor and the readership of the Journal for this unintentional error, and for any inconvenience caused. [the original article was published in Oncology Reports 36: 1293‑1300, 2016; DOI: 10.3892/or.2016.4923].

Effects of Epigallocatechin Gallate (EGCG) on Urinary Bladder Urothelial Carcinoma-Next-Generation Sequencing and Bioinformatics Approaches.

Background and objectives: Bladder urothelial carcinoma is the most common type of genitourinary cancer. Patients with bladder cancer may have limited treatment efficacy related to drug toxicity, resistance or adverse effects, and novel therapeutic strategies to enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea leaves, and a potential anti-cancer agent in various cancer types through modulating and regulating multiple signaling pathways. The current study aimed to explore the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma. Materials and Methods: The BFTC-905 cells, human urinary bladder transitional cell carcinoma (TCC) cell line, were treated with EGCG or water for 24 hours, and the expression profiles of mRNAs and microRNAs were analyzed using next generation sequencing (NGS). The enriched biological functions were determined using different bioinformatics databases. Results: A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, which were mainly involved in nicotinamide adenine dinucleotide (NAD) biogenesis, inflammatory response and oxidation-reduction metabolism. Moreover, several microRNA-mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p- ARRB1 (arrestin beta 1), miR-3116- MGAT5B (alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B), miR-31-5p-TNS1 (tensin 1), miR-642a-5p-TNS1, miR-1226-3p- DLG2 (discs large homolog 2), miR-484-DLG2, and miR-22-3p- PPM1K (protein phosphatase 1K). Conclusions: The current findings provide insights into novel therapeutic targets and underlying mechanisms of action of EGCG treatment in bladder cancer.

Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro.

PurposeReceptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR) and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX)-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt) and COX-2 signaling pathways in bladder cancer cells.MethodsThe expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB) analysis in human and canine bladder transitional cell carcinoma (TCC) cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2) levels.ResultsAll tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c-Kit receptors. RTKIs increased the expression of COX-2 in h-5637 and K9TCC#1Lillie cells. Co-treatment of indomethacin inhibited AB1010-induced COX-2 expression leading to an additive effect in inhibition of cell viability and PGE2 production in tested TCC cells.ConclusionCo-treatment of RTKIs with indomethacin inhibited cell viability and AB1010-induced COX-2 expression resulting in decreased PGE2 production in tested TCC cells. Thus, COX inhibition may further potentiate RTKIs therapies in bladder cancer.

Abstract 4739: The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro

Abstract Several types of cancer, including transitional cell carcinoma (TCC) overexpress several receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR), c-kit receptor, epidermal growth factor receptor (EGFR), as well as the vascular endothelial growth factor receptor (VEGFR). Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with high expression of RTKs. Cyclooxygenase-2 (COX-2) is highly expressed in bladder cancer as well other types of cancers and is one of the key proteins during tumorigenesis. In this study, we validated the effects of RTKIs, Masitinib® (AB1010) and Axitinib on cell proliferation of human (UMUC-3 and T24) and canine (K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) bladder TCC cells. Using WB analysis, tested human and canine TCC cells, except T24 cells, expressed PDGFR and c-kit receptors. There was no detectable EGFR and VEGFR expressions in any of tested cells by WB analysis. The K9TCC#1Lillie and K9TCC#5Lilly cells had a high expression of COX-2, however K9TCC#2Dakota and human T24 had moderate expression of COX-2, and no COX-2 expression was detected in UMUC3 cells by WB analysis. Both RTKIs, Masitinib® and Axitinib inhibited cell proliferation of the tested human and canine bladder TCC cells in a dose-dependent manner by MTS and apoptosis assays. Interestingly, both tested RTKIs, Masitinib® and Axitinib increased COX-2 protein expressions in tested canine TCC cells. Combined treatment of RTKIs with COX-2 inhibitors decreased cell proliferation of tested TCC cells more effectively as either treatments alone. Our results indicate a possible role of COX-2 signaling pathway in developed RTKIs-resistance in bladder cancer cells in vitro. Co-treatment of RTKIs with COX-2 inhibitors might indicate better clinical outcomes in treatments of patients diagnosed with bladder cancer. Citation Format: Jennifer Bourn, Maria Cekanova. The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4739.

MicroRNA-497 inhibits the proliferation, migration and invasion of human bladder transitional cell carcinoma cells by targeting E2F3.

Accumulating evidence indicates that microRNAs (miRNAs) play critical roles in regulating cellular processes, such as cell growth and apoptosis, as well as cancer progression and metastasis. Low expression of miR-497 has been observed in breast, colorectal and cervical cancers. Human bladder transitional cell carcinoma (BTCC) progression typically follows a complex cascade from primary malignancy to distant metastasis, but whether the aberrant expression of miR-497 in BTCC is associated with malignancy, metastasis or prognosis remains unknown. In the present study, we found that miR-497 was markedly downregulated in BTCC tissue samples when compared with that noted in adjacent normal tissues, and low expression of miR-497 was correlated with poor prognosis in BTCC patients. We also found that overexpression of miR-497 inhibited the proliferation, migration and invasion of bladder cancer cells by downregulating E2F3 (an miR-497 target gene) mRNA and protein and that siRNA against E2F3 inhibited cell proliferation, migration and invasion, which was similar to the effect of miR-497 overexpression in the BTCC cells. Our experimental data indicated that miR-497 mediates the invitro proliferation, migration and invasion of BTCC cells. Together, these results suggest that miR-497 may represent a novel prognostic indicator, a biomarker for the early detection of metastasis and a target for gene therapy of BTCC.

Downregulation of fatty acid synthase complex suppresses cell migration by targeting phospho-AKT in bladder cancer

The aim of the present study was to investigate the effect of fatty acid synthase complex (FASN) on the migration capacity of bladder transitional cell carcinoma (BTCC) cells and the involvement of matrix metalloproteinase‑9 (MMP‑9) via targeting of phospho‑AKT (p‑AKT). FASN‑specific small‑interfering RNA (FASN‑siRNA) was used to inhibit FASN gene expression in the 5637 and 253J BTCC cell lines. The knockdown efficiency of FAM‑conjugated FASN‑siRNA was confirmed by fluorescence microscopy. The migratory abilities of BTCC cells were assessed using a Transwell assay. Furthermore, protein and mRNA expression of FASN, p‑AKT, AKT, and migration‑associated protein MMP‑9 were detected by western blot analysis. Treatment with FASN inhibitor Cer and FASN‑siRNA decreased the migratory capacity of bladder cancer cells and reduced the levels of p‑AKT as well as the expression of MMP‑9. These results indicated that FASN inhibition suppressed the migratory capacity of BTCC cells through suppressing AKT activation and consequently reducing MMP‑9 expression. Targeting FASN may represent a promising novel therapeutic strategy for BTCC.

Effects of sunitinib malate on growth of human bladder transitional cell line T24 in vitro.

To investigate the growth-inhibitory effect of sunitinib malate on human bladder transitional cell carcinoma (TCC) in vitro. Human bladder TCC cell line T24 was cultured and exposed to graded concentrations of sunitinib malate for 72 hours in vitro to determine the sensitivities to drug. Cell viability was measured by MTT assay. Cell apoptotic morphology was observed by fluorescence microscope following DAPI staining. Band expressions of Fas, Fas ligand, poly (ADP-ribose) polymerase (PARP) and β-actin were analyzed by Western blot. Wound healing process of T24 cells exposed to sunitinib malate was assayed. Sunitinib malate exerted a concentration-dependent and time-dependent inhibitory effect on the T24 cell lines. Fluorescence microscopy showed that small vacuoles appeared in the nuclei of T24 cells and the vacuoles were bigger with higher drug concentrations. The expressions of Fas ligand and PARP in T24 cells treated with sunitinib malate exhibited a concentration-dependent increase. Moreover sunitinib malate suppressed the wound healing process in a concentration-dependent manner. Sunitinib malate exerted marked inhibitory activity against bladder cancer cell line T24.

Quantitation of rare circulating tumor cells by folate receptor α ligand-targeted PCR in bladder transitional cell carcinoma and its potential diagnostic significance.

Numerous attempts for detection of circulating tumor cells (CTC) have been made to develop reliable assays for early diagnosis of cancers. In this study, we validated the application of folate receptor α (FRα) as the tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR) and assessed its utility for diagnosis of bladder transitional cell carcinoma (TCC). Immunohistochemistry for FRα was performed on ten bladder TCC tissues. Enzyme-linked immunosorbent assay (ELISA) for FRα was performed on both urine and serum specimens from bladder TCC patients (n = 64 and n = 20, respectively) and healthy volunteers (n = 20 and n = 23, respectively). Western blot analysis and qRT-PCR were performed to confirm the expression of FRα in bladder TCC cells. CTC values in 3-mL peripheral blood were measured in 57 bladder TCC patients, 48 healthy volunteers, and 15 subjects with benign urologic pathologies by the folate receptor α ligand-targeted PCR. We found that FRα protein was overexpressed in both bladder TCC cells and tissues. The levels of FRα mRNA were also much higher in bladder cancer cell lines 5637 and SW780 than those of leukocyte. Values of FRα were higher in both serum and urine specimens of bladder TCC patients than those of control. CTC values were also higher in 3-mL peripheral blood of bladder TCC patients than those of control (median 26.5 Cu/3 mL vs 14.0 Cu/3 mL). Area under the receiver operating characteristic (ROC) curve for bladder TCC detection was 0.819, 95 % CI (0.738-0.883). At the cutoff value of 15.43 Cu/3 mL, the sensitivity and the specificity for detecting bladder cancer are 82.14 and 61.9 %, respectively. We concluded that quantitation of CTCs through FRα ligand-PCR could be a promising method for noninvasive diagnosis of bladder TCC.

Cholera toxin, a typical protein kinase A activator, induces G1 phase growth arrest in human bladder transitional cell carcinoma cells via inhibiting the c-Raf/MEK/ERK signaling pathway

The biotoxin cholera toxin has been demonstrated to have anti-tumor activity in numerous types of cancer, including glioma. However, the role of cholera toxin in the tumorigenesis of transitional cell carcinoma (TCC), the most common malignant tumor of the bladder, remains to be elucidated. To address this, in the present study, two TCC cell lines, T24 and UM-UC-3, were treated with cholera toxin [protein kinase A (PKA) activator] and KT5720 (PKA inhibitor). Cell survival and proliferation, cell cycle alterations and apoptosis were analyzed using Hoechst staining, the MTT assay, fluorescence microscopy and flow cytometry. Western blot analysis was used to detect the expression of proteins involved in cell cycle regulation. The results revealed that cholera toxin significantly induced G1 arrest and downregulated the expression of cyclin D1 and cyclin-dependent kinase 4/6 in the TCC cell lines, and this was rescued by KT5720. Furthermore, it was demonstrated that cholera toxin downregulated the activation of the c-Raf/Mek/Erk cascade, an important mediator of tumor cell proliferation, via the PKA-dependent c-Raf phosphorylation at Ser-43. Furthermore, inhibition of Mek activity with UO126 mimicked the effects of cholera toxin. In conclusion, these results confirmed that cholera toxin specifically inhibited proliferation and induced G1 phase arrest in human bladder TCC cells. This effect was due to PKA-dependent inactivation of the c-Raf/Mek/Erk pathway. This suggested that cholera toxin may be a viable therapeutic treatment against tumorigenesis and proliferation in bladder cancer.

Carcinogenicity Risk Assessment Supports the Chronic Safety of Dapagliflozin, an Inhibitor of Sodium–Glucose Co-Transporter 2, in the Treatment of Type 2 Diabetes Mellitus

IntroductionDapagliflozin is a selective inhibitor of the sodium–glucose co-transporter 2 (SGLT2) that increases urinary glucose excretion to reduce hyperglycemia in the treatment of type 2 diabetes mellitus. A robust carcinogenicity risk assessment was undertaken to assess the chronic safety of dapagliflozin and SGLT2 inhibition.MethodsGenotoxicity potential of dapagliflozin and its metabolites was assessed in silico, in vitro, and in vivo. Dapagliflozin was administered daily by oral gavage to mice, rats, and dogs to evaluate carcinogenicity risks, including the potential for tumor promotion. SGLT2−/− mice were observed to evaluate the effects of chronic glucosuria. The effects of dapagliflozin and increased glucose levels on a panel of human bladder transitional cell carcinoma (TCC) cell lines were also evaluated in vitro and in an in vivo xenograft model.ResultsDapagliflozin and its metabolites were not genotoxic. In CD-1 mice and Sprague–Dawley rats treated for up to 2 years at ≥100× human clinical exposures, dapagliflozin showed no differences versus controls for tumor incidence, time to onset for background tumors, or urinary bladder proliferative/preneoplastic lesions. No tumors or preneoplastic lesions were observed in dogs over 1 year at >3,000× the clinical exposure of dapagliflozin or in SGLT2−/− mice observed over 15 months. Transcription profiling in Zucker diabetic fatty rats showed that 5-week dapagliflozin treatment did not induce tumor promoter-associated or cell proliferation genes. Increasing concentrations of glucose, dapagliflozin, or its primary metabolite, dapagliflozin 3-O-glucuronide, did not affect in vitro TCC proliferation rates and dapagliflozin did not enhance tumor growth in nude mice heterotopically implanted with human bladder TCC cell lines.ConclusionA multitude of assessments of tumorigenicity risk consistently showed no effects, suggesting that selective SGLT2 inhibition and, specifically, dapagliflozin are predicted to not be associated with increased cancer risk.Electronic supplementary materialThe online version of this article (doi:10.1007/s13300-014-0053-3) contains supplementary material, which is available to authorized users.

Inhibition of Fatty-acid Synthase Suppresses P-AKT and Induces Apoptosis in Bladder Cancer

To investigate the role of fatty acid synthase (FASN) in bladder transitional cell carcinoma (BTCC). FASN expression was investigated in non-muscle-invasive BTCC tissue specimens by immunohistochemistry and BTCC cell lines by Western blot. After treatment with FASN-siRNA or FASN inhibitor cerulenin (Cer), the proliferation and apoptosis of BTCC cell lines 5637 and 253 J were determined by cell counting Kit-8 (CCK8) assay and flow cytometry respectively. The expression of p-AKT, cyclin D1 (CCND1), and apoptosis-related proteins were detected by Western blot. High levels of FASN expression were observed in 59% (32/54) of non-muscle-invasive BTCC tissue specimens, and FASN expression was associated with histologic grade (P < .05) and recurrence (P < .05). FASN expression was high in 6 BTCC cell lines. FASN inhibitor Cer and FASN-siRNA produced the increased apoptosis and decreased proliferation of bladder cancer cells, and caused inactivity of AKT and downregulation of CCND1. Furthermore, treatment of BTCC cell lines with Cer resulted in apoptosis via the caspase-dependent pathway involving inactivation of antiapoptotic bcl-2 protein. Our data suggest that FASN plays an important role in BTCC development. Targeting FASN may be a new therapeutic strategy for BTCC.

Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand

ObjectiveTo determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.Experimental DesignThe binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.ResultsPLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.Conclusions and Clinical RelevancePLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

Expression of hypoxia inducible factor-1alpha and glucose transporter protein 1 in renal and bladder cancers and the clinical significance thereof

To investigate the protein expression of hypoxia inducible factor-1alpha (HIF-1alpha) and glucose transporter protein 1 (Glut-1) in renal cancer and bladder cancers and the clinical significance thereof. TransAMTMELISA was used to detect the protein expression of HIF-1alpha in the tissue sections from 25 patients with bladder transitional cell carcinoma (TCC) and 16 patients with renal clear cell carcinoma resected during operation and small amounts of normal renal and bladder tissues far from the cancer tissue resected simultaneously. Immunohistochemistry was used to detect the protein expression of Glut-1 in tissue sections from 58 patients with bladder carcinoma transitional cell carcinoma and 38 patients with renal clear cell carcinoma resected during operation. 16 specimens of normal bladder and 16 specimens of normal renal tissue were obtained from the patients with other diseases who underwent operation during the same period to be used as controls. Follow-up was conducted for more than 2 years for all patients. The protein expression of HIF-1alpha in the tissue of renal clear cell carcinoma was 3.38 +/- 1.71 microg/well, significantly higher than that in the tissue near the cancer (2.23 +/- 1.07 microg/well, P < 0.01). The protein expression of HIF-1alpha in the bladder TCC tissue was 2.69 +/- 1.34 microg/well, not significantly different from that in the tissue near the cancer (248 +/- 1.28 microg/well, P = 0.60). Protein expression of Glut-1 was not detected in the normal renal and bladder tissues, however, was significantly higher in the bladder TCC and renal clear cell carcinoma tissues. The positive rate of Glut-1 in the bladder TCC tissue was 77.90% (45/58). The positive rates of Glut-1 in the tissues of bladder TCC tissue, grades G1, G2, and G3 were 66.7%, 89.1%, and 53.3%. (P = 0.29), showing the correlation of Glut-1 expression with the grading of cancer. The positive rate of Glut-1 in the superficial TCC was 83.9%, not significantly different from that of the invasive TVV (70.4%, P = 0.90), showing that the Glut-1 is not correlated with the cancer staging. Nineteen of the 31 cases of superficial TCC showed recurrence within 2 years, however, the protein expression of Glut-1 rate was not significantly different between those with recurrence and those without recurrence (P = 0.90), showing that the protein expression of Glut-1 is not correlated with the recurrence of TCC. The protein expression of Glut-1 in the renal clear cell carcinoma was at a rate of 86.9% (33/38), however, it was not correlated with the grade and stage of the cancer. The protein expression of HIF-1alpha is strongly associated with the neoplastic progression of renal clear cell carcinoma, but its role in the development of bladder TCC is not clear yet. The protein expression of Glut-1 is strongly associated with the neoplastic progression of bladder TCC and renal clear cell carcinoma.

Soluble Fas—A promising novel urinary marker for the detection of recurrent superficial bladder cancer

The objective of this study was to test the hypothesis that elevated urinary levels of soluble Fas (sFas) would aid in the surveillance of patients with a past history of nonmuscle-invasive transitional cell carcinoma (TCC) of the urinary bladder. sFas levels were determined in cell lysates and supernatants from 2 human bladder cancer cell lines (T24 and TCC-SUP) and in voided urine from 188 consecutive patients who were at risk for TCC recurrence, 31 patients who had noncancerous urologic conditions, and 10 healthy individuals. The authors also obtained barbotage cytology and voided nuclear matrix protein 22 (NMP22) levels. sFas was analyzed continuously and categorically on the basis of its quintile distribution. sFas was present in cell lysates and conditioned media from both cell lines. sFas levels were found to be higher in the TCC group (n = 122 patients) compared with the control group (P < .001). Higher levels of sFas were associated with positive cytology assay results (P < .001), higher NMP22 levels (P < .001), NMP22 levels > 10 U/mL (P < .001), and tumor stage > or = T1 (P < .001). The areas under the receiver operating characteristics (ROC) curves of sFas and NMP22 for bladder cancer detection were 0.757 (95% confidence interval, 0.694-0.819) and 0.704 (95% confidence interval, 0.637-0.772), respectively. In the > 75% sensitivity region of the ROC curves, sFas was consistently more specific than NMP22. In multivariate analyses, sFas, NMP22, and cytology all were found to be associated with the presence of bladder cancer (P values < or = .009), but only sFas and cytology were associated with tumor stage > or = T1 (P values < or = .026). sFas was produced and released by bladder TCC cells. Urine sFas was an independent predictor of bladder cancer recurrence and invasiveness in patients who had a past history of nonmuscle invasive bladder TCC, and it outperformed NMP22.

CG0070, a Conditionally Replicating Granulocyte Macrophage Colony-Stimulating Factor–Armed Oncolytic Adenovirus for the Treatment of Bladder Cancer

The purpose of this study was to examine the tumor specificity, cytotoxicity, and granulocyte macrophage colony-stimulating factor expression of CG0070, a conditionally replicating oncolytic adenovirus, in human bladder transitional cell carcinoma (TCC) cell lines and determine its antitumor efficacy in bladder TCC tumor models. Virus yield and cytotoxicity assays were used to determine tumor specificity and virus replication-mediated cytotoxicity of CG0070 in a panel of human bladder TCC cell lines and primary cells in vitro. Two s.c. and one orthotopic bladder TCC xenograft tumor models were used to assess antitumor activity of CG0070. In a matched isogenic pair of cell lines with differing retinoblastoma (Rb) pathway status, CG0070 showed selective E1a and granulocyte macrophage colony-stimulating factor (GM-CSF) expression in Rb pathway-defective cells. CG0070 replicated in Rb-defective bladder TCC cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. CG0070 was up to 1,000-fold more cytotoxic in Rb pathway-defective bladder TCC cells in comparison with normal human cells. Antitumor activity of CG0070 was shown in two bladder TCC s.c. xenograft tumor models following intratumoral injections and intravesical treatment in an orthotopic xenograft tumor model when compared with PBS treatment. In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production, and antitumor efficacy of CG0070 in several bladder TCC models, suggesting a potential utility of this oncolytic agent for the treatment of bladder cancer. Further studies are warranted to show the role of human GM-CSF in the antitumor efficacy of CG0070.

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective: Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma (BTCC). However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determine a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cells from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods: Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results: That RNA integrity was good after LCM was confirmed by control experiment I; By control experiment II, the correlation between the number of LCMshooting and RNA quantity under arranged conditions was preliminarily confirmed. About 0.5-2.5 kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion: Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.

Restoration of Bone Morphogenetic Protein Receptor Type II Expression Leads to a Decreased Rate of Tumor Growth in Bladder Transitional Cell Carcinoma Cell Line TSU-Pr1

Bone morphogenetic proteins (BMPs), potential regulators of cellular growth and metastasis that signal through an interaction with plasma membrane receptors, have been suggested to be important regulators of malignant cells. The present study was carried out to evaluate the potential role of BMP receptor (BMP-R) types IA, IB, and II in bladder transitional cell carcinoma (TCC) cells. Initially, we investigated the expression of these BMP-Rs in 30 archival tissues of human bladder TCC using immunohistochemistry; 10 benign bladder specimens were used for comparison. The results demonstrated that the expression of BMP-Rs is localized preferentially to the transitional epithelium and that there was a significant association between loss of BMP-RII expression and tumor grade. To find a cell line that can serve as a model system for clinical observation, we subsequently examined sensitivity to BMP-4 and expression of BMP-RII, BMP-RIA, and BMP-RIB in three human bladder cancer cell lines, TCC-Sup, RT4, and TSU-Pr1. Of the three cell lines, TSU-Pr1 exhibited a decreased level of BMP-RII expression and was resistant to the growth-inhibitory effect of BMP-4. Overexpression of BMP-RII in TSU-Pr1 cells not only restored BMP-4 responsiveness but also significantly decreased tumorigenicity in vivo. Taken together, these results demonstrate that human bladder TCC tissues have a frequent loss of BMP-RII expression and that overexpression of BMP-RII leads to restoration of BMP signaling and decreased tumor growth in the human bladder TCC cell line TSU-Pr1.

Inactivation ofMMAC1 in bladder transitional-cell carcinoma cell lines and specimens

We recently limited the location of a candidate tumor suppressor gene in invasive (T3a/b) bladder transitional-cell carcinoma (TCC) to a 2.5-cM region at chromosome 10q23.3. This region harbors the MMAC1/PTEN/TEP1 gene (referred to hereafter as MMAC1), a dual-phosphatase tumor-suppressor gene frequently inactivated in variety of malignant tumors. In the present study, we examined whether MMAC1 is a target for inactivation by mutations and deletions in bladder TCC cell lines and specimens. MMAC1 was inactivated by homozygous deletions and mutations in three (27%) of 11 bladder cancer cell lines. One cell line, UC-3, had homozygous deletions, and two other cell lines, T-24 and UC-9, had missense mutations. T-24 had also a nonsense mutation. However, none of the 33 bladder TCC specimens examined had a mutation or deletion in the coding region. These results suggest that MMAC1 is not the primary target for inactivation in bladder TCC and that another gene, in close proximity to the MMAC1 locus, within this region of frequent allelic losses, may be the target for inactivation.