Figure 4.

Ara-C incorporation produces top1 cleavage complexes in human leukemia CEM cells. Detection of top1 cleavage complexes was performed by using the ICE assay as described in Fig. 3, and top1 immunoblotting of the DNA-containing fractions (numbers at the bottom of each panel) are presented. Dose-response and kinetics of top1 trapping by ara-C in CEM cells are shown in A and B, respectively. (C) Aphidicolin cotreatment inhibits the production of top1-DNA complexes by ara-C. Cells were treated with 1 μM aphidicolin alone (Aph), 10 μM ara-C alone (Ara-C) for 7 h, or with a combination of ara-C and aphidicolin (Ara-C + Aph) (1 h pretreatment with aphidicolin followed by aphidicolin and ara-C for 6 h) at 37°C. (D) Persistence of ara-C-induced top1-DNA complexes. Cells were treated with Ara-C (1 μM, 15 h), and top1-DNA complexes were measured either immediately (Ara-C) or after removal of the drug and incubation in drug-free medium for 1 or 2 h (Rev. 1 h and Rev. 2 h, respectively).