yielded an identical a-b-c pattern, establishing the reproducibility and drug specificity of these results, for which the region spanning fragment ‘‘a’’ is better resolved. Quite interestingly —and unusually for treatment of intact cells with Topo II poisons— this behavior was quite quantitative, possibly indicating a highly specific and potent cleavage activity. Because we know of no other sequence-specific nuclease activities that can be induced in Drosophila cells under the conditions used, these results necessarily seem to implicate Topo II in this phenomenon. Simple interpretations of these preliminary results lead us to propose several important conclusions. First, treatment of Drosophila cells with F14512 results in a pattern of DNA cleavage at discrete moderately repeated sites that is not seen following treatment with TOP-53, its closest relative, or with teniposide or etoposide. As teniposide and TOP-53 induce cleavage in SAT III repeats while etoposide and F14512 do not, the a-b-c pattern clearly reflects a property unique to F14512. Importantly, this F14512-specific behavior was amplified in the presence of distamycin (compare lanes 3–6 to 9–12, in Figure 4A, where the disappearance of fragment ‘‘b’’ occurs faster than in the absence of this AT-specific minor-groove binder). Indeed, note that bands a-c are difficult to discern in the absence of distamycin, which opens AT-rich chromatin and makes it more accessible to non-histone chromatin-associated proteins such as Topo II [37]. Another plausible conclusion then is that F14512-induced cleavage in the moderately repeated sequences detected here occurs in generally AT-rich regions, suggesting that the presence of a spermine moiety influences cleavage-site localization in chromatin but not sequence preference [40]. Because Topo II can play a role as a transcriptional regulator [41,42,43,44], F14512-specific targets might give rise to unique changes in patterns of gene expression, which we investigated next by exploring the possible phenotypic effects of this new anticancer drug in vivo. F14512 inhibits the proliferation of Drosophila cells and induces unique morphological changes Altogether, the presented data demonstrate that F14512 is a Topo II poison that induces an atypical cellular cleavage pattern compared to the reference compounds etoposide, teniposide and TOP-53. We then asked whether this originality translates into different antiproliferative properties of F14512 in Drosophila Kc tissue-culture cells, compared to those of related epipodophyllo- toxins lacking the polyamine moiety. Viability measurement clearly indicated that all the molecules tested, F14512 as well as the Topo II inhibitors etoposide, teniposide and TOP-53, inhibited proliferation as early as 24 hours after drug addition (Figure 5A). The number of viable cells was further decreased after 48 and 72 hours. In addition, dose- and time-dependent effects were evident for each compound, suggesting that all four drugs exert comparable effects in Kc cells. F14512 has demonstrated IC50 values for proliferation in the nanomolar range for a 72-hour treatment of a series of mammalian cancer cell lines [24]. Kc cells seem then to appear more resistant to F14512. Since these cells are non cancereous one possibility is that they express less PTS, which would expose them to lower intracellular concentrations of compound. However, given the differences highlighted by the cellular cleavage patterns, we asked whether F14512 might exert effects different from those of its related parent drugs. As a straightforward way of addressing this question, Kc cells exposed to the same compounds were examined by phase contrast microscopy 24 hours post-treatment. As shown in Figure 5B, untreated Kc cells are round and refringent and growing cultures are enriched in doublets of these non-adherent cells. Treatment Studying Anticancer Drug Properties in Drosophila with either TOP-53 or etoposide at 10 or 50 mM concentrations led to the appearance of apoptotic cells and/or debris in a dose- dependent fashion (arrowheads in Figure 5B), a known and expected effect of prolonged exposure to podophyllotoxins-based Topo II poisons [17,45,46]. Concentrations .50 mM resulted in 100% killing over 72 hours of treatment (data not shown). In contrast, treatment with 10 and 50 mM F14512 led a loss of refringence, with doublets becoming rarer in a population of dispersed cells. One characteristic manifestation was the rapid generation of a significant number of giant, multi-nucleated cells (arrowheads, in Figure 5B), which appeared within 2 hours of treatment (data not shown and see Discussion). Treated cells also became adherent and elongated, forming filamentous projections and/or aggregates of seemingly fused cells. These effects became more marked with increasing drug concentrations (compare the 10 and 50 mM treatments). It is significant to note that, even at high F14512 concentrations, we could observe no obvious apoptosis products or cellular debris thereof, in contrast to the effects of etoposide and, significantly, the closely related TOP-53 lacking a spermine moiety. We investigated these effects in more detail, using lower drug concentrations and by comparing phase-contrast images of intact cultures with those of DAPI-stained nuclei of fixed treated cells. As shown in Figure 5C, F14512 treatment led to effects strikingly different from those of etoposide and that could be observed at concentrations as low as 1 mM. Nuclei became enlarged and more heterogeneously stained. Some nuclei were clearly fused and their chromatin intimately linked. In some cases, anaphase bridges could be observed, suggestive of segregation defects. Other nuclei were regrouped in closely associated aggregates that most likely correspond to multinucleated giant cells observed by phase- contrast microscopy. Small hypercondensed structures that correspond to apoptotic bodies prevalent after treatment with etoposide or TOP-53 could only very rarely be observed. These observations clearly suggest that the mode of action of F14512 and its greater cytotoxicity rest on mechanisms different from those of its close relative TOP-53, or of the etoposide podophyllotoxin used in the clinic. The white mottled inversion and PEV We extended our studies to developing Drosophila larvae, using a well defined genetic system characterized by H. J. Muller over 80 years ago [47]. As schematized in Figure 6A, the white-mottled (wm4h) chromosomal inversion of most of the euchromatic arm of the X chromosome places the white gene next to pericentromeric heterochromatin. white encodes a pigment transporter required for the normal red eye color of wild-type flies. Partial inactivation of white in wm4h flies results in a salt-and-pepper (‘‘mottled’’) white/ red eye color, a phenomenon referred to as position-effect variegation (PEV). PEV reflects the stochastic inactivation of genes placed in or near heterochromatin, the resulting silencing or expression pattern, once established, being then stably propagated to daughter cells. This differential effect is classically attributed to varying extents of heterochromatin spreading into euchromatin regions as a result of the loss of insulator or boundary elements that are thought to prevent such propagation in normal chromosomes ([48,49] and references therein). While a mottled eye color is the most obvious and easily scored phenotype of adult wm4h flies, this line also grows more slowly than wild-type and the extent of eye-color variegation strongly depends on growth conditions. Growth delays might reflect a misregulation of rRNA expression as the rDNA locus lies very close to or even within the proximal breakpoint. The nearby Notch gene, 338.5 kbp distant from white, is also subject to PEV in wm4h flies, an effect that PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23597