Didanosine

Assays to monitor the potential cytotoxicity of RT inhibitors were performed as previously described (68 (link)). RT inhibitors were first made as 50 mM stock solutions in DMSO and then diluted in tissue culture media to arrive at the concentrations used in the respective experiments. Briefly, untransfected HT-29 cells were plated on 10 cm plates (500 cells/plate) with and without the RT inhibitors Didanosine and Zidovudine (Sigma Aldrich). Ten days later, cells were fixed to the plate and stained with 0.1% crystal violet using the same procedure described above for retrotransposition assays. The number of foci was counted to calculate colony formation ability and compared to cells treated with equal concentrations of DMSO alone.

Mitomycin C, zinc pyrithione, methy-umbelliferyl-glucuronate (MUG), and the reagents used for the Miller assays for recA were purchased from Sigma-Aldrich (St. Louis, MO). Zidovudine, 5-fluorouracil, 5-azacytidine, paraquat, arsenic trioxide, and didanosine were also from Sigma-Aldrich. Ciprofloxacin was obtained from Bayer Pharmaceuticals. E-test strips were from Biomerieux (Durham, NC).

The cell-based retrotransposition assay was conducted as described above. Seventy-two hours after transfection, the cells were trypsinized and plated onto three 10cm plates in RPMI medium containing 600 μg/ml G418 and 50 μM RT inhibitors Didanosine and Zidovudine (Sigma Aldrich). RPMI medium containing 600 μg/ml G418 and 50 μM DMSO was added to the mock treated plates. Two weeks post-transfection, G418 resistant cells were fixed to the plate and stained with 0.1% crystal violet, using the same procedure described above for retrotransposition assays, to calculate the retrotransposition frequency. Three independent assays were performed for each experiment.