Figure 1. Structure of podophyllotoxin derivatives used in this study. Structure of F14512. Epipodophyllopoxin is shown to the left, with the substitution of different R groups leading to teniposide (VM26), TOP-53 and etoposide (VP16). doi:10.1371/journal.pone.0023597.g001 before addition of teniposide (50 mM) or F14512 (5 mM) and ATP (1 mM). A stop mix was added (1% SDS, 5 mM EDTA, 100 mg/ ml proteinase K in a 300 ml final volume brought up with TE) after a 5-minute additional incubation. Following incubation for 3 hours at 55uC, DNA samples were purified by organic extractions and ethanol purification in the presence of 0.2 M NaCl and electrophoresed on 5% acrylamide/6M urea sequenc- ing gels in TBE at 2000 volts. Gels were fixed in 10% acetic acid, dried and exposed to Kodak BioMax MS-1 film with BioMax MS intensifying screens. Maxam-Gilbert chemical sequencing reac- tions performed on the same labeled histone-repeat fragment were loaded on the gels. Because the 59 end of the fragment (as shown on the map in Figure 2A) was labeled, sequencing gels and sequencing reactions allow the precise assignment of cleavage sites 3–5. Similar experiments not shown here were performed with a cloned uniquely end-labeled SAT III monomer fragment (see below), yielding comparable results. Ex vivo SAT III cleavage experiments Approximately 56106 exponentially growing S2 and Kc cells, cultured as described above, were incubated with Topo II poisons for 30 minutes. In some experiments, cells were first treated with distamycin (25 mM) before addition of teniposide, etoposide, TOP- 53 or F14512 added at the concentrations shown in the Figure 3. After a 5-minute centrifugation (3000 x g), cell pellets were resuspended in PBS supplemented with drugs, MgCl2 (5 mM) and ATP (1 mM) before sequential addition of SDS (1% final concentration), EDTA (5 mM final concentration) and proteinase K (Roche) to 200 mg/ml. Samples were gently mixed following addition of each component to the lysis mix, which was incubated for 3 hours to overnight at 55uC. DNA samples were purified by organic extractions and ethanol precipitation. Washed and dried DNA pellets were resolubilized in 20 ml TE. 10 ml aliquots were ran on 1.2% agarose gels in TBE (35 volts for 14 hours). After staining with ethidium bromide and photography, gels were transferred to charged nylon membranes and hybridized to a radioactively labeled cloned SAT III 359-bp monomer exactly as described [37], except that GeneScreen Plus membranes (Perkin- Elmer) were used instead of Hybond-N+ (GE Healthcare). Filters were washed at high stringency (0.1x SSPE, 1% SDS at 65uC) and Studying Anticancer Drug Properties in Drosophila Figure 2. In vitro drug-induced cleavage in the Drosophila histone-gene repeat. (A) Schematic representation of the 5-kbp major histone-gene locus, which contains the 5 genes encoding core and linker histones. The bar with a hook represents the scaffold- associated region (SAR) located in the intergenic spacer between the H1 and H3 genes [74]. It is cleaved by Topo II (denoted by scissors) in the linker regions of 5 positioned nucleosomes (black circles). Other sites (1, 2 and 8–10) map to DNAse I-hypersensitive sites at the 59 and 39 ends of the histone genes (see [21] for details). Sites 3-5 mapped at nucleotide resolution by direct genomic sequencing [21,22]. (B) In vitro cleavage induced by teniposide (VM26) and F14512. A uniquely end- labeled DNA clone spanning the histone-gene repeat was incubated with Topo II as described in Materials and Methods in the presence of no drug (lane 1), 50 mM teniposide with 0, 5, 10 or 25 mM distamycin (lanes 5, 4, 3 and 2, respectively) or 5 mM F14512 with 0, 5, 10 or 25 mM distamycin (lanes 6, 7, 8 and 9, respectively). The 4 lanes at the right of the gel are Maxam-Gilbert sequencing reactions of the labeled fragment. Cleavage is induced at numerous identical sites by teniposide and F14512 in the absence of distamycin (compare lanes 5 and 6). Increasing concentrations of distamycin redirect cleavage induced by both drugs to sites 3–5 marked by arrows to the left of the gel and which correspond exactly to the sites cleaved in cellulo. doi:10.1371/journal.pone.0023597.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23597