CNE1 < BX1 > cells have been previously described and were cultured in Dulbecco’s Modified Eagle Medium plus 10% fetal bovine serum with 700 ug/ml G418 (Gibco/Life Technologies, Grand Island, NY) [
27]. Animal experiments were performed in accordance with Tulane University Institutional Animal Care and Use Committee approved protocols and standards. Six to eight week old outbred NU/NU mice (strain code 088) weighing 18–20 grams were obtained from Charles River Laboratories (Wilmington, MA) and housed in sterile conditions. CNE1 < BX1 > cells (7.5×10
6 cells/mouse) were trypsinized and mixed with growth factor reduced matrigel (BD Bioscience, Franklin Lakes, New Jersey) and injected subcutaneously on the right flank of each mouse. Tumor growth was assessed for 5 days at which time the average tumor volume was 250 mm
3. Animals were randomly assigned to control (n = 6), GCV (100 mg/kg/d, n = 6), ATO (1 mg/kg/d, n = 7), or CoTx (100 mg/kg/d GCV + 1 mg/kg/d ATO, n = 7). Working solutions were diluted such that injection volumes for each group were equal. Tumor volume was assessed at 3–5 day intervals by a blinded investigator using the L*W
2/2 formula. At 22 days post injection, mice were euthanized and tumors were harvested for paraffin sectioning and mRNA isolation. Isolation of mRNA and quantitative real time reverse transcription PCR detection of BMRF1 mRNA levels was done as previously described with the following exceptions [
27]. At harvest, ½ of the tumor volume was flash frozen in liquid nitrogen and stored at −80 deg C. Samples were homogenized utilizing homogenized utilizing a rotor-stator homogenizer in RLT Plus buffer. Data is representative of repeated experiments.