the formation of meta-stable ‘‘cleavable complexes’’ [13] located 30–50 kpb apart that can be converted to DNA double-stranded breaks as a result of collisions with replication or transcription machineries. Collisions with any DNA motor complex can result in the conversion of the readily reversible cleavable complex into an irreversible cytotoxic lesion. Accumulation of these lesions, which cannot be easily handled by the cellular DNA repair machinery, usually induces apoptotic pathways that result in the onset of cell death within approximately 48–72 hours in the case of dividing cells [14,15,16,17,18]. Detailed studies have shown that different classes of Topo II poisons differ in their activities, even though they all act through the same basic mechanism of retarding the religation reaction at the end of the catalytic cycle [13,19]. This has previously been extensively analyzed in a system of Drosophila melanogaster tissue- culture cells, in which we initially characterized sites of Topo II/ chromatin interactions, both ex vivo and in vitro, at nucleotide resolution [12,20,21]. Different classes of poisons (epipodophyllo- toxins, acridines and anthracyclines) typically differ in the cleavage sites they induce, as well as in their stability and reversibility [22,23]. The strict molecular basis for these differences remains poorly understood. In the case of etoposide and teniposide, which exert strong cytotoxic effects and stimulate Topo II-mediated cleavage in cells, the generation of derivatives with more specific activities and/or a restricted subset of genomic targets constitutes an attractive strategy towards the development of new, more effective drugs. F14512 is a novel epipodophyllotoxin derivative which possesses antitumor properties in mice harboring human tumors [24,25]. Using both enzymatic and cellular assays and similar to other previously characterized epipodophyllotoxin-based compounds, F14512 has been shown to inhibit Topo II. F14512 differs from its analogues by the presence, on the epipodophyllotoxin moiety, of a polyamine chain, spermine in this case. Polyamines, such as spermine and spermidine, are natural polycationic molecules required for cell growth [26]. Tumor cells have a higher requirement for polyamines relative to normal quiescent or cycling cells and often overexpress ornithine decarboxylase, the rate- limiting enzyme in polyamine biosynthesis [27,28,29,30]. In addition, to accumulate sufficient polyamine amounts required for their growth, tumor cells also increase their uptake of polyamines from the extracellular compartment via the Polyamine Transport System (PTS) [31,32]. We previously showed that F14512 was preferentially imported by PTS+ human tumors xenografted in mice [24]. Moreover, the polyamine targeting moiety of F14512 endows it with significantly higher antitumor properties than the structurally analogous etoposide, in which a sugar is found instead of the spermine chain [24,25]. In the test tube, in the presence of purified Topo II, F14512 also proved to be markedly more potent than etoposide at inhibiting the enzyme and generating DNA double strand breaks [24]. Surprisingly, this higher potency of F14512 is not reflected by a higher frequency of DNA cleavage induced in treated cells [25]. We attempted to further dissect the possible intricacies of F14512 activity on DNA and chromatin, presumably due to the sole presence of its spermine moiety, and which could explain these apparently contradictory observations. Because of the presence of spermine in the F14512, we also decided to compare this novel compound, in addition to etoposide and teniposide, to the previously described TOP-53, an epipodophyllotoxin derivative that contains a structurally analogous alkyl-amide moiety and possesses anti- tumor activity [33,34]. Because of the inherent difficulty of addressing such a question in classical cellular models, we report here on the use of a fully Studying Anticancer Drug Properties in Drosophila integrated Drosophila melanogaster model system whose evident complexity is offset by the powerful genetic tools it provides. Using assay systems and loci previously characterized in vitro and in cellulo for their response to Topo II inhibitors such as teniposide, we tested F14512 for possible differences with its polyamine-less analogs. In addition, we extended our analysis to in vivo studies in this organism, where the use of a defined genetic background revealed some unique features of F14512 that provide new insights in the properties of this novel drug. Our results also demonstrate that it is possible to use the Drosophila melanogaster model organism as an assay system that provides a low-cost, rapid and effective complement to mammalian models and permits the elucidation of fundamental mechanisms of action of candidate drugs of therapeutic interest in humans. Methods Drosophila melanogaster fly and cell culture Flies were grown at 22uC on standard cornmeal-glucose-yeast medium. An Oregon R laboratory stock was used as a wild-type control strain. A laboratory stock of the white-mottled inversion (ln(I)wm4h), referred to as wm4h here, was maintained at a growth temperature of 18uC. Drosophila Schneider S2 and Kc cells were grown at 24uC in Schneider Drosophila medium (Invitrogen) and Echalier’s D22 medium (Sigma), respectively, supplemented with 5% heat-inactivated newborn calf serum. Under these conditions, S2 and Kc cells have doubling times of approximately 26 and 24 hours, respectively. S2 cells were grown at sub-confluence in 75-cm2 flasks or in the 2-cm diameter wells of multi-well plates. Kc cells were grown in spinner cultures at densities ranging between 26106 to 46106 cells/ml and transferred to multi-well plates (2- cm diameter wells) for drug treatments. When used, distamycin A3 (Sigma) was added to the culture medium for 60 minutes, followed by addition of Topo II poisons at the concentrations indicated in the text. Incubations were continued for an additional 30 minutes (cleavage experiments) or for up to 96 hours (cytotoxicity experiments). The procedure used for cleavage experiments is detailed below. Drugs Etoposide, TOP-53 and F14512 were obtained from Pierre Fabre Medicament and teniposide from Bristol-Myers Squibb. The detailed synthesis of F14512, based on previously reported chemical procedures [35], will be described elsewhere. The drug TOP-53 (initially developed by Taiho, Japan) is an epipodophyl- lotoxin derivative that contains a [[(dimethylamino)ethyl]-N- methylamino]ethyl group reminiscent of the polyamine chain of F14512 (Figure 1) [33,34]. All compounds were prepared as 5 mM stock solutions in DMSO. Water-soluble F14512 was also prepared in DMSO to equalize solvent concentrations in all experiments, except for drug feeding where it was dissolved in water. In vitro Topo II cleavage experiments In vitro cleavage experiments (Figure 2) were performed exactly as described [20], using 2 ng (104 cpm) of a cloned uniquely end- labeled Drosophila 5-kb histone-repeat DNA fragment and 200 ng of sonicated salmon sperm DNA (average size ,2 kb) as competitor. These conditions optimize the specificity of the cleavage reaction. 50-ml reaction mixtures were incubated for 15 minutes with 0, 5, 10 or 25 mM distamycin as shown in Figure 2B, before addition of 0.5 ml of human Topo IIa purified using an expression vector kindly provided by Dr. J. L. Nitiss. Incubation was continued for 5 minutes at 37uC to allow binding to occur PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23597