reverted exactly to that observed in the presence of distamycin (data not shown), a result identical to that obtained with the single Topo II isoform purified from the yeast S. cerevisiae or S. pombe [21,37]. Hence, the cleavage activity and specificity of type II topoisomerases isolated from different organisms may differ but revert to a unique pattern upon addition of distamycin, reflecting a competitive redistribution of the enzyme to drug-free sites that correspond to those cleaved in cellulo [3]. In this case, one hallmark of F14512 activity —induction of Topo II-mediated cleavage— is comparable in both Drosophila and human cells in that drug treatment results in a cleavage frequency that is markedly lower than that induced by etoposide or teniposide [25]. This suggests comparable modes of action in both organisms, in spite of the absence in one and the presence in the other, of multiple isoforms. These observations also raise the question of which Topo II isoform —a, b or both— is the target of F14512 in human cells. Given the distinct roles of human Topo IIa and b, for example the specific function of the latter in transcriptional regulation [59], identification of the isoform(s) and associated processes targeted by F14512 might help explain the unique potency of the drug. Given that the single Drosophila Topo II enzyme shares similarities with both a and b human isoforms and that both Topo IIa and Topo IIb are efficient targets for F14512 in vitro [57], our data do not allow us to answer this question at present. This important point might be addressed in the future by examining Topo IIa and Topo IIb expression and activity levels in cells selected for resistance to F14512. Surprisingly, the similar effects of F14512 and teniposide on Topo II-mediated cleavage observed in vitro did not translate into cleavage of the SAT III region of the X chromosome of growing Drosophila cultured cells (Figure 3C) and F14512 behaved more similarly to etoposide in this case (Figure 3B). This negative result is quite unexpected, as SAT III repeats constitute a massive storage site for Topo II in vivo [39] and most likely reflects an intrinsic inability of F14512 to form sufficiently stable ternary complexes with Topo II and DNA at this locus, in accordance with the demonstrated differential activity of different classes of drugs at specific Drosophila loci [22,23]. While we cannot exclude the possibility that this essential enzyme is not a major target of F14512 in cultured cells, it is important to note that such a hypothesis would not be consistent with previous data indicating that cells deficient for Topo II are less sensitive to F14512 than Topo II-proficient cells [24,25], hence strengthening the conclu- sion that Topo II plays a direct role in the cytotoxic action of the drug. A global approach to a search for chromosomal cleavage F14512-induced Topo II cleavage sites reveals that TopoII is a pivotal component of drug activity. Our results show that F14512 is indeed capable of inducing cleavage at discrete sites in the Drosophila genome (Figure 4). In this case, we were able to document the dose-dependent F14512-specific disappearance of 2 HindIII fragments, concomitant with the appearance of a third fragment that most likely corresponds to a cleavage product. Regardless of the exact identity of fragments a, b and c, their behavior is unique to F14512-treated cells and is not seen in control or etoposide-treated samples. We consider this last point to be particularly important as it serves to establish a significant difference between these compounds. Again, with the caveat that more work will be needed before firmer conclusions can be drawn, we propose that the spermine moiety grafted onto F14512, and absent from etoposide or other Topo II poisons, endows this new molecule with unique properties that target it to discrete sites in chromatin, a conclusion supported by the remarkable effects of F14512 in whole developing animals. Studying Anticancer Drug Properties in Drosophila Drug-induced chromatin remodeling and alteration of gene expression in vivo A particularly detailed understanding of Drosophila melanogaster genetics, which extends over one century of research, renders this organism eminently suitable for classical biological studies but also, as demonstrated here, to search for insights into the mechanism of action of new anti-cancer drugs and to identify their molecular targets. Feeding Drosophila larvae different classes of drugs ranging from Topo II poisons to minor-groove binders as well as histone- deacetylase inhibitors was previously shown to result in gain-of- function phenotypes [39,53,60,61,62,63]. Here, we have extended this approach to the study of the effects of F14512 treatment in developing larvae and of the ensuing phenotypes in surviving adults. Results of our experiments demonstrate effects that, in addition to being largely chromatin-mediated (Figure 7), also denote gains or losses of functions that affect specific and readily identifiable genes. Feeding F14512 to mutant white-mottled Drosophila larvae leads to the striking Eye wide shut phenotype recovered at a high frequency of surviving adults (Figure 8). From 5 independent experiments starting with 30 L3 larvae in duplicate, we obtained 14 Eye wide shut individuals, all males that had undergone a homeotic transformation of the left eye into a first thoracic segment. Remarkably, the Eye wide shut phenotype corresponds exactly to that obtained following ectopic expression of the Antennapedia (Antp) gene in the eye imaginal disks of larvae [64]. Antp is a HOX-family gene involved in the differentiation of the thoracic and head segments of the fly. It is normally repressed in segments where its expression is stably extinguished by a Polycomb-group repression complex that, significantly, has been shown to require Topo II for its activity [43]. Hence, the transformation we obtain suggests that F14512 may promote aberrant Topo II activity at least at this locus and, in flies that survive treatment, is manifested in one eye in an exquisitely specific fashion. The accompanying unilateral disappearance of male sex combs on the same side as the transformed eye can similarly be correlated to a loss-of-function of the Sex combs reduced (Scr) gene. Scr, which also encodes a homeobox-containing transcription factor, lies within the Antp complex and is similarly subject to Polycomb-mediated regulation [65]. However, it is differentially affected by F14512 feeding, relative to the closely linked Antp gene. The residual shrunken eye portion found within the anterior side of the transformed eye or the multiple small eyes seen after treatment with lower concentrations of F14512 (Figure 8C) also suggest a partial loss-of-function of the eyeless (ey) gene [66]. ey encodes the Drosophila ortholog of the PAX-6 transcription factor and is a ‘‘master control’’ gene for eye development [67]. Antp antagonizes its activity [64] and it is plausible that the Eye wide shut and shrunken-eye or small double-eye phenotypes both reflect Antp gain-of-function and concomitant ey loss-of-function. It should also be noted that ey is located on chromosome 4 [68], the smallest and most highly condensed of the 4 chromosome pairs present in Drosophila melanogaster. Chromosome 4 consists largely of hetero- chromatin, in which euchromatin regions are interspersed [69]. Alternatively, ey loss-of-function induced by F14512 might reflect a perturbation of the normal distribution of heterochromatin and associated proteins in the white-mottled line. In this respect, it is perhaps significant that we only recovered Eye wide shut males: the large Y chromosome of Drosophila consists almost entirely of heterochromatin and can perturb heterochromatin-mediated effects by titrating heterochromatin-associated proteins (reviewed in [70]). The striking loss of symmetry that characterizes eye transfor- mation and loss of sex combs is more difficult to explain. This PLoS ONE | www.plosone.org 13 August 2011 | Volume 6 | Issue 8 | e23597