could be due to a perturbation of signaling pathways and/or genes implicated in structural symmetry. The Notch (N) gene is required for the maintenance of certain aspects of lateral symmetry [51,52] and, perhaps more importantly in this case, has been implicated in the establishment of the axis of mirror symmetry in the Drosophila eye. Notch lies within 338.5 kbp of white and is therefore also relocalized near X-chromosome heterochromatin in the white- mottled inversion (Figure 6A; [47,71]). Accordingly, we were able to document a drug-independent asymmetric variegation of eye color in large white-mottled collections (Figure 6B), most likely reflecting a unique tendency for one-sided effects in this genetic background, where the characteristic eye-color phenotype is highly sensitive to Topo II localization, activity or poisoning thereof [39,53]. We surmise that the white-mottled background might provide a highly sensitized context that permits the expression of phenotypes such as Eye wide shut. In this case, the identification of exquisitely sensitive targets is in line with what appears to be a limited subset of F14512-induced Topo II cleavage sites in tissue-culture cells (Figure 4; [24]) even though this drug behaves similarly to other Topo II poisons in vitro (Figure 2). These complex in vivo results obtained in a whole developing organism —although they can be reconciled with known genetic anomalies— constitute a remarkable instance of specific alter- ations of gene expression by ingestion of a drug. F14512 is unique in this respect: the Eye wide shut phenotype was never recovered after feeding teniposide, m-amsa, etoposide or idarubicin (data not shown) to L3 white-mottled larvae. Similarly, feeding F14512 to wild-type Oregon R larvae gave rise to no observable phenotypes, other than death, at the highest concentrations tested. We consider it particularly significant that F14512 exhibits a range of activities not found in related drugs such as etoposide and teniposide, suggesting singular mechanisms of action consistent with unusual targeting of discrete Topo II cleavage sites. We hypothesize that F14512 effects might be promoted, at least in part, by facilitated uptake, via its polyamine moiety, by larvae and tissues therein [72]. Drosophila-based anticancer pharmacology Taken together, these results establish the validity of using a fully integrated Drosophila melanogaster system to study the mode of action of a new anticancer drug, tentatively identify its targets and to unravel its most specific aspects as obtained phenotypes can be References 1. Adachi Y, Luke M, Laemmli UK (1991) Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell 64: 137–148. 2. Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 9: 327–337. 3. Poljak L, Ka¨s E (1995) Resolving the role of topoisomerase II in chromatin structure and function. Trends Cell Biol 5: 348–354. 4. Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev MolCell Biol 3: 430–440. 5. Heisig P (2009) Type II topoisomerases--inhibitors, repair mechanisms and mutations. Mutagenesis 24: 465–469. 6. Nitiss JL (2009) Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9: 338–350. 7. Pommier Y, Leo E, Zhang H, Marchand C (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol 17: 421–433. 8. Li TK, Liu LF (2001) Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol 41: 53–77. 9. Liu LF (1989) DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 58: 351–375. 10. Larsen AK, Escargueil AE, Skladanowski A (2000) Resistance mechanisms associated with altered intracellular distribution of anticancer agents. Pharma- colTher 85: 217–229. 11. Larsen AK, Skladanowski A (1998) Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death. Biochim Biophys Acta 1400: 257–274. 12. Laemmli UK, Ka¨s E, Poljak L, Adachi Y (1992) Scaffold-associated regions: cis- acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev 2: 275–285. Studying Anticancer Drug Properties in Drosophila easily linked to target genes. While not a substitute for experimentation in the mouse for example, which provides invaluable cancer and tumor xenograft models, Drosophila offers, as shown here, a powerful, rapid and inexpensive complement that could also integrate feeding of multiple drugs that might affect the effects of drugs such as F14512 and their polyamine moieties and could thus serve as adjuvants. We expect that other genetic backgrounds would extend the suitability of this system to the analysis of different classes of drugs. As recently discussed, such a notion is indeed fully consistent with the outcome of recent elegant molecular studies that, due to its ‘‘genetic tractability,’’ firmly establish Drosophila melanogaster as a system of choice for the study of tumorogenesis and cancer therapy [73]. In conclusion, we have documented the usefulness of the Drosophila model system to characterize the properties of F14512, a novel targeted antitumor agent, and to distinguish it from epipodophyllotoxin-containing Topo II inhibitors. In addition to the presence of a spermine moiety which targets F14512 preferential entry into cancer cells —the rational basis for its initial design— our study, using a predisposed genetic background that is hypersensitive to Topo II dysfunction or mislocalization, identifies a unique subset of target genes. Such a property might of course be extremely useful to develop new drugs whose targets belong to a restricted subset, perhaps enhancing cytotoxic effects while limiting, in normal cells, the genome-wide damage that can result from the accumulation of frequent drug-induced DNA lesions. As such, F14512, which is currently being evaluated in a phase I clinical trial in patients with acute myeloid leukemia, is expected to exert promising anticancer activities. Acknowledgments We thank Je´roˆme Filiol (CROE) for expert technical assistance in performing cytotoxicity tests on Kc cells. Author Contributions Conceived and designed the experiments: EK SV NG JMB SC CMW. Performed the experiments: SC CMW SV. Analyzed the data: EK SV CB NG SC CMW. Contributed reagents/materials/analysis tools: CB NG JMB SV. Wrote the paper: EK SV CB NG JMB. 13. Chen GL, Yang L, Rowe TC, Halligan BD, Tewey KM, et al. (1984) Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259: 13560–13566. 14. El-Awady RA, Ali MM, Saleh EM, Ghaleb FM (2008) Apoptosis is the most efficient death-pathway in tumor cells after topoisomerase II inhibition. Saudi Med J 29: 558–564. 15. Kluza J, Mazinghien R, Irwin H, Hartley JA, Bailly C (2006) Relationships between DNA strand breakage and apoptotic progression upon treatment of HL- 60 leukemia cells with tafluposide or etoposide. Anticancer Drugs 17: 155–164. 16. Nam C, Doi K, Nakayama H (2010) Etoposide induces G2/M arrest and apoptosis in neural progenitor cells via DNA damage and an ATM/p53-related pathway. Histol Histopathol 25: 485–493. 17. Solary E, Bertrand R, Pommier Y (1994) Apoptosis induced by DNA topoisomerase I and II inhibitors in human leukemic HL-60 cells. Leuk Lymphoma 15: 21–32. 18. Sordet O, Khan QA, Kohn KW, Pommier Y (2003) Apoptosis induced by topoisomerase inhibitors. Curr Med Chem - Anti-Cancer Agents 3: 271–290. 19. Charcosset JY, Soues S, Laval F (1993) Poisons of DNA topoisomerases I and II. Bull Cancer 80: 923–954. 20. Adachi Y, Ka¨s E, Laemmli UK (1989) Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions. EMBO J 8: 3997–4006. 21. Ka¨s E, Laemmli UK (1992) In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics. EMBO J 11: 705–716. 22. Borgnetto ME, Tinelli S, Carminati L, Capranico G (1999) Genomic sites of topoisomerase II activity determined by comparing DNA breakage enhanced by three distinct poisons. J Mol Biol 285: 545–554. PLoS ONE | www.plosone.org 14 August 2011 | Volume 6 | Issue 8 | e23597