The Institutional Animal Care and Use Committee and Institutional Biosafety Committee of the Cincinnati Children's Research Foundation approved all animal experimental procedures (IACUC protocol #9D09068 and IBC Protocol #2009-0086), and these experiments were carried out in accordance with standards as described in the NIH Guide to the Care and Use of Laboratory Animals.
Preparation of Explant Cultures of Urothelium from Mouse Bladder
The H-2Kb-tsA58 mouse (Immortomouse™), transgenic for the thermosensitive mutant of the SV40 large T antigen (tsA58) expressed by an interferon-inducible MHC Class I promoter, was obtained from Charles River Laboratories, (Wilmington, MA). The tsA58 antigen is ubiquitously expressed, although at the non-permissive body temperature of the mouse (39°C) the protein product of the transgene is unstable and is degraded. Once tissues are removed from the animal, cell lines established from these tissues may be conditionally immortalized under permissive tissue culture conditions of 33°C and in the presence of recombinant mouse interferon gamma (IFN-γ).
nducible) cells were isolated from these mice using a modification of procedures described by Kreft et al 
. Briefly, following CO2
asphyxiation, an incision was made from the symphysis pubis through the sternum, and the urinary bladder was resected in its entirety. Bladders were rinsed with phosphate-buffered saline (PBS), divided sagitally into two equal parts, and mucosa separated from the muscle layer and submucosa. The mucosa was applied to Cyclopore 0.45 µm membrane supports (Becton Dickinson, Franklin Lakes, NJ) in 6 well dishes. Each well contained DMEM:Ham's F12 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 1% penicillin/streptomycin (Invitrogen), amphotericin B 2.5 µg/ml (Sigma, St. Louis, MO), 1% insulin/transferrin/ethanolamine/selenium (ITES) media supplement (Sigma), mouse epidermal growth factor 10 ng/ml (Sigma), and mouse IFN-γ 10 U/ml (Invitrogen). Cultures were incubated at 5% CO2
, 37°C for 24 hours, then incubated at 5% CO2
, 33°C and observed for growth. Media was refreshed from both the inserts and wells twice weekly. Once outgrowth of cells from the mucosal pieces became confluent (at approximately 28 days in culture), the cells were subcultured with 0.25% trypsin and 0.02% EDTA onto 100 mm plastic dishes coated with type I collagen (Becton Dickinson). To drive the ULTI urothelial cells towards a non-transformed phenotype, subcultured cells were grown at 37°C in media identical to that used in the explant experiments, with the exception of the removal of IFN-γ and amphotericin B and the reduction of the FBS concentration to 0.5%.
Established Cell Lines and Reagents
The RT4 transitional cell carcinoma and NIH 3T3 mouse fibroblast cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in cell culture in DMEM:F12 media (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. Conditionally immortalized gastrointestinal epithelial cell lines ImSt (gastric), YAMC (colon), and MSIE (small intestine), also derived from the H-2Kb-tsA58 mouse, were generous gifts from Dr. Robert Whitehead (Vanderbilt University, Nashville, TN) and were maintained in identical media to our derived urothelial cell line. Etoposide, pifithrin-α and sterile filtered DMSO were obtained from Sigma Chemical.
Crystal violet cell proliferation assay
ULTI cells were seeded into 96 well plates at a density of 5000 cells/well in complete media containing FBS at concentrations of 10%, 5%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, and 0%, both with and without IFN-γ. Cultures containing IFN-γ were incubated at 33°C, and cultures lacking IFN-γ were incubated at 37°C, both for 72 hours. Wells were then washed with PBS, fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), washed with ddH2O, and incubated with 0.1% crystal violet (Becton Dickinson) for 30 minutes. Cells were again washed with ddH2O, and treated with 10% glacial acetic acid. Absorbance was then read at 540 nm with a Bio-Rad Benchmark Plus microplate spectrophotometer. The experiment was carried out in triplicate, and to account for minor variation in initial seeding density between experiments, absorbances from each experiment were normalized to that of 10% FBS in both conditions. Student's t-test was applied between absorbances measured from cells cultured at 33°C and from cells cultured at 37°C for each concentration of FBS to determine statistical significance.
Cell cycle analysis by flow cytometry
ULTI cells were seeded into 60 mm plastic dishes at a density of 1×105 cells/dish in complete media containing either 10% or 0.5% FBS, both with and without the addition of 10 U/ml IFN-γ. Cultures containing IFN-γ (with either 10% or 0.5% FBS) were incubated at 33°C, and cultures lacking IFN-γ (with either 10% or 0.5% FBS) were incubated at 37°C, each for 72 hours. Cells were then detached with trypsin-EDTA (Invitrogen), pelleted by centrifugation, resuspended in staining buffer consisting of propidium iodide 50 µg/mL, NP-40 0.3%, and RNAse A 1 mg/mL in PBS, and incubated at 4°C for 30 minutes. Cells were then filtered, analyzed with a Becton-Dickinson FACSCanto II cytometer, and the resulting data was interpreted using FlowJo v8.8.6 (TreeStar, Ashland, OR). Experiments were carried out in triplicate, and the mean percentage of cells in G0/G1, S, and G2/M phases were calculated.
In a separate set of experiments, cells were seeded at a density of 1×105 cells/dish in complete media containing either 10% FBS and 10 U/ml IFN-γ, or 0.5% FBS without IFN-γ. Cultures containing IFN-γ and 10% FBS were incubated at 33°C, and cultures without IFN-γ and 0.5% FBS were incubated at 37°C, each for 72 hours. At 60 hours of incubation, cells were treated with etoposide (a potent inhibitor of topoisomerase II that causes double strand DNA breaks) at a final concentration of 25 µM, or a similar volume of the DMSO vehicle. Cells were then detached, pelleted, stained with propidium iodide, and analyzed in the same manner as the previous experiment.
In a third set of experiments, cells were seeded at a density of 1×105 cells/dish in complete media without IFN-γ containing 0.5% FBS and incubated at 37°C for 72 hours. At 54 hours of incubation, cells were treated with either pifithrin-α (a specific inhibitor of p53) at a final concentration of 20 µM or a similar volume of the DMSO vehicle, then at 60 hours of incubation (without washing) treated with etoposide 25 µM or DMSO vehicle. Cells were then detached, pelleted, stained with propidium iodide, and analyzed in the same manner as the previous experiments.
Western blot analysis
ULTI cells were subcultured onto plastic dishes both in complete media containing 10% FBS as described above, as well as in media lacking IFN-γ and containing 0.5% FBS, at a density of 1.0×106 cells per 100 mm dish. NIH 3T3 fibroblasts were also cultured in standard media as described above. ULTI cultures were either maintained at 33°C in the presence of IFN-γ, or 37°C in the absence of IFN-γ for twenty-four hours, treated with either pifithrin-α 20 µM or DMSO vehicle for six hours, then (without washing) treated with either etoposide 25 µM or DMSO vehicle for twelve hours. Whole cell lysates were then generated by lysis into ice-cold RIPA buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell suspensions were sonicated and cellular debris was pelleted by centrifugation. Supernatants were aliquoted and stored at -80°C.
Protein concentration of these lysates was determined by bicinchoninic acid assay (Pierce, Rockford, IL) according to the manufacturer's instructions. Equal amounts of protein were separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat dry milk in TBS with 0.1% Tween 20 v/v (TBST), then probed with antibody against SV40 large T antigen (1
2000, Santa Cruz Biotechnology, Santa Cruz, CA); total p53 (1
40,000), phospho-p53, serine 15 (1
10,000), cleaved caspase 3 (1
1000), and PARP (1
2000) (Cell Signaling Technology, Danvers, MA); p21cip1
1000, BD Pharmingen, San Jose, CA), total ATM (1
1000, Novus Biologicals, Littleton, CO), phospho-ATM, serine 1981 (1
1000, Rockland Immunologicals, Gilbertsville, PA), and γH2AX (1
2000, Trevigen, Gaithersburg, MD). Equal protein loading was confirmed by blotting for G3PDH (1
40,000; Trevigen). Membranes were then probed with horseradish peroxidase-linked secondary antibody (GE Healthcare Biosciences, Piscataway, NJ), and protein bands detected by chemiluminescence with the Amersham ECL Plus kit (GE Healthcare Biosciences) and developed by autoradiography.
Phase contrast and scanning electron microscopy of ULTI cells
ULTI cells subcultured onto plastic dishes were examined with Köhler illumination by phase contrast microscopy on a Zeiss Axiovert 200M inverted microscope using the 20x objective. Cells were also subcultured onto Cyclopore 0.45 µm membrane supports (Becton Dickinson), then once the cells reached confluence fixed with 2% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 hours at 4°C. After rinsing in 0.1 M cacodylate buffer and postfixation with 1% (w/v) OsO4, specimens were chemically desiccated with 1,1,1,3,3,3-hexamethyldisilazane (Sigma). Subsequently, the cells were sputter-coated with a gold/palladium 40% mixture in a Denton Vacuum Desk IV, and examined in a Hitachi S-3000N scanning electron microscope.
Immunofluorescence of cytokeratin 18, ZO-1, and occludin
ULTI cells were seeded onto glass coverslips, and maintained at 37°C in the absence of IFN-γ for four days. Cells were then rinsed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.5% Triton X-100 (Sigma) in PBS. Cells were blocked with 15% FBS in PBS, then incubated either with mouse monoclonal anti-cytokeratin 18 antibody (1
200, Chemicon/Millipore), or a combination of rabbit polyclonal anti-ZO-1 antibody (1
400, Zymed Laboratories, Carlsbad, CA) and mouse monoclonal anti-occludin antibody (1
500, Zymed) in PBS with 1% bovine serum albumin (BSA, Sigma) overnight at 4°C. The cells were rinsed with PBS, then incubated with anti-rabbit or anti-mouse secondary antibodies conjugated to Alexa-Fluor 488 and Alexa Fluor 633 (Molecular Probes/Invitrogen) at 1
500 dilution made in PBS with 1% BSA. Cells were then mounted with ProLong Gold with DAPI (Invitrogen) and coverslips applied to glass slides. Slides were analyzed for cytokeratin 18 with a Zeiss Axiovert 200M inverted fluorescent microscope using 40x and 100x oil-immersion objectives. ZO-1 and occludin expression was assessed with a Zeiss LSM510 confocal microscope equipped with argon 488 nm and HeNe 633 nm laser light sources. Optical sections were taken at 0.5 µm intervals. Orthogonal and z-stack reconstructions were performed with NIH Image J 1.37a software.
Reverse transcriptase PCR of cytokeratin 18, cytokeratin 20, and uroplakin II
ULTI cells as well as YAMC, MSIE, and ImSt gastrointestinal cells and NIH 3T3 fibroblasts were seeded into type I collagen-coated plastic dishes with media as described above without IFN-γ, and maintained at 37°C for four days. Cells were scraped into ice-cold sterile PBS, pelleted, and total cellular RNA was extracted from pelleted cells using TRI reagent (Sigma) according to the manufacturer's instructions and quantitated by spectrophotometry.
In experiments designed to mimic the hyperosmolal bladder microenvironment, ULTI cells were gradually adapted to hyperosmolality in culture by increasing the media osmolality by 50 mOsm/kg every 24 hours from a basal media osmolality (~300 mOsm/kg) to target osmolalities of 450 and 600 mOsm/kg. Cells were seeded into plastic dishes with media lacking IFN-γ and containing 0.5% FBS and maintained at 37°C. Every 24 hours, culture media was aspirated and replaced with fresh media adjusted to the corresponding osmolality by the addition of sterile-filtered 5M NaCl (Sigma) or 5M urea (Fluka). To remove isocyanates, a degradation product of urea, 20 mL of 5M urea stock solution was exchanged with 1 g of AG-501-X8 resin (Bio-Rad) in the method published by Zhang et al 
immediately prior to use. Cells were scraped into PBS and total cellular RNA extracted with TRI reagent as above.
To provide tissue RNA for comparison, total cellular RNA was extracted with TRI reagent from homogenized stomach, intestine, heart, and bladder tissue from euthanized wild-type mice.
Following treatment of 0.3–1 µg of extracted RNA with 2 Units of RNAse-free DNAse I (New England Biolabs, Ipswich, MA) and heat deactivation of the DNAse, cDNA was generated by reverse transcription using oligo-dT20 primers and the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. Aliquots of cDNA were then used to detect cytokeratin 18, cytokeratin 20, uroplakin II, and G3PDH by PCR with AccuPower PCR PreMix tubes (Bioneer, Alameda, CA). Forward and reverse primer sets, PCR reaction annealing temperature, and predicted product sizes are listed in . The PCR program template for all reactions was as follows: 94°C×5 minutes, followed by forty cycles of (94°C×1 minute, annealing temperature×30 seconds, 72°C×30 sec), and final extension of 72°C×10 minutes. PCR products were separated by agarose gel electrophoresis, stained with SYBR® Safe (Invitrogen) and imaged.
Reverse transcriptase PCR primer pairs, annealing temperature (Tannealing), and predicted PCR product size for cytokeratin 18, cytokeratin 20, uroplakin II, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH).