FdUMP[10] is a potent inhibitor of TS and traps Top1CCs. (A) TS inhibition assay. HL60 cells were exposed to 10nM FdUMP[10] or 100nM 5-FU for the indicated time, lysed, and assayed for TS activity. Activity is plotted as percentage of control. (B) ICE bioassay for Top1CCs. THP-1, Jurkat, and HL60 cells were incubated with 100nM FdUMP[10] for 48 hours. Cells were lysed and subjected to ICE bioassay (see “ICE bioassay/Top1 cleavage complex detection”), and DNA-containing fractions were blotted for Top1. (C) Murine AML TS competition assays. Murine AML cells were partially infected with TS and GFP-expressing retrovirus and treated with the indicated drug for 72 hours. All experiments were performed in triplicate. The GFP⁺ percentage in the viable cell population is indicated; representative histograms are shown. (D) HL60 TS competition assays. HL60 cells were partially infected with TS and GFP-expressing retrovirus and exposed to the indicated drug for 72 hours. Percentage of GFP⁺ cells was determined as in panel C and normalized to untreated controls. All experiments were done in triplicate. (E) Results of TS and Top1 Western blot analyses. K562 (K), HL60 (H), Kg1a (Kg), MLL-ENL murine AML (M3), or 3 primary AML patient samples (B1-3) were blotted with the indicated Ab. Error bars represent SE. *P < .05.