Characterization of UROtsa Cell Line

We used the UROtsa cell line derived from the ureter epithelium of a 12-year-old female donor, immortalized with SV40 large T-antigen [18 (link)]. It is a non-tumorigenic cell line that displays the phenotypic and morphologic characteristics resembling primary transitional epithelial cells of the bladder [19 (link)]. UROtsa cells were cultured in 25 cm² tissue culture flasks in Dulbeco’s modified Eagle’s medium supplemented with 5% vol/vol fetal bovine serum [20 (link),21 (link)]. Cells were maintained at 37 °C in humidified incubators with 5% CO₂/95% atmospheric air. Cells were fed fresh growth medium every three days. At confluence, cells were subcultured at a 1:4 ratio using trypsin-EDTA (0.05%, 0.02%). For experimentation, cells were grown in 6-well plates containing 2 mL growth media per well (35 mm in diameter). Treatment of cultures with Cd [26 (link)], Zn, BSO [27 (link)], and aza-dC [28 ] were undertaken when cultures reached 80% confluence. Cd, Zn, BSO, and aza-dC were of highest purity grade (Sigma, St. Louis, MO, USA). For an initial dose-response analysis, five cell cultures were used for each Cd concentration. Expression of ZIP and ZnT genes from the initial experiment agreed with microarray data using the Affymetrix 133 Plus 2.0 [29 (link)]. Thus, in later experiments, at least two different batches of cell cultures were used per treatment.