Use of Camptothecin-resistant Mammalian Cell Lines to Evaluate the Role of Topoisomerase I in the Antiproliferative Activity of the Indolocarbazole, NB-506, and Its Topoisomerase I Binding Site

Fig. 1.

Schematic representation of the top1 mutations in the CPT-resistant cell lines used and cross-resistance of CPT-resistant top1 mutants to NB-506. A, the COOH-terminal domain, the linker region, the core domain, and the NH₂ terminus domain are indicated from right to left as black, gray, dotted, and white rectangles, respectively. Mutations (and corresponding cell lines) used in the present study are indicated above the top1 schematic representation. ∗, position of other CPT resistance mutations (for details, see Ref. 2 ). B, nuclear extracts from parental and top1-mutant cells were used for DNA cleavage assay. ³²P-end-labeled PvuII-HindIII 161-bp fragment from pBluescript was incubated with purified top1 or nuclear extracts (from left to right, CEM and CEM/C2, DC3F and DC3F/C10, DU-145 and DU-145/RC1, respectively) in the presence or absence of CPT or NB-506 at 25°C for 30 min. CPT was used at 1μ m, and the numbers above the lanes represent the concentrations of NB-506 inμ m (from 1 to 100 μm). C, control without drug. Reactions were stopped with SDS (final concentration, 0.5%) and resolved in 7% sequencing gels. Imaging and quantification were performed with a PhosphorImager.