is not as strong as on white expression and is also sensitive to changes in growth conditions [50]. PEV of Notch might account for some abnormal growth phenotypes as its gene product is implicated in a broad array of developmental processes (see http://flybase.org/reports/FBgn0004647.html), including estab- lishment of lateral symmetry [51,52]. This probably accounts for a striking loss of symmetry of white PEV observed in about 1% of adult wm4h flies grown at 18uC (Figure 6B) and which extends to the unique phenotypes described below. F14512, Topoisomerase II and suppression of white- mottled PEV The white-mottled system lends itself to the identification of factors that can perturb Topo II activity in a developing whole organism and of their effects on a readily measurable phenotype. We previously showed that Topo II is a structural and functional component of heterochromatin and can modulate the white-mottled phenotype [39]. Accordingly, the eye color of wm4h flies is uniquely sensitive to the chromosomal localization of Topo II [53] and feeding teniposide to developing wm4h larvae suppresses white- mottled PEV, as shown by the recovery of adults with wild-type eye color [39]. We compared this effect to that of F14512 and etoposide. For the experiments shown in Figure 7, wm4h late-stage third-instar (L3) larvae were grown overnight with Topo II poisons added to the food at the sub-lethal concentrations shown. Adults were photographed 5 days after hatching, before pigment extraction from the heads of 30 or 10 females (see Methods) and quantification. Feeding teniposide (10–50 mM, see Figure legend for details) to wm4h late L3 larvae led to a dose-dependent suppression of the PEV phenotype, with a wild-type eye-color obtained at 50 mM teniposide (see graph at the bottom of Figure 7). Drug concentrations above 100 mM resulted in .90% lethality (data not shown). While etoposide feeding led to a similar lethality, it resulted in only moderate suppression of white-mottled PEV. F14512 was found to be significantly more toxic (about 30% hatched adults at 50 mM), but behaved similarly to etoposide in moderately suppressing PEV in the 10–50 mM range, albeit with a relatively wide spread between experiments. We attribute these differences between teniposide and etoposide and F14512 to their differential effects on the stimulation of Topo II cleavage, as in the case of SAT III repeats (see Figure 3 and Discussion). DMSO alone (the solvent for all drugs used here) had no effect on white-mottled PEV or on wild-type Oregon R flies. However, we did detect strikingly specific properties of F14512 in this system, consistent with its plausible induction of cleavage at a select subset of genomic sites (see Figure 4), and which we describe next. Eye wide shut: a unique side effect of F14512 ‘‘oral administration’’ Feeding F14512 at concentrations above 50 mM led to 100% lethality (no adults hatched). At concentrations of 50 mM, approximately 30% of treated larvae hatched into adults, of which about 10%, which all died within 2 days, showed the remarkable effects illustrated in Figure 8. These dying young adults possess one wild-type eye with normal or near-normal eye color. Quite amazingly, their other eye has been replaced by a first thoracic segment, which also retains a shrunken remnant of a white-colored eye (Fig. 8A, white arrowhead in the inset showing a bottom view of a head). This phenotype affected exclusively the left side of the fly and was only scored in males, denoting a sex- dependent loss-of-symmetry which untreated wm4h flies appear to be prone to at some frequency (Figure 6B). Studying Anticancer Drug Properties in Drosophila Closer views are shown in Figure 8B and reveal additional differences. A disappearance of the male-specific sex combs present on the front legs could also be observed and always occurred on the same side as the transformed eye. Because the most impressive manifestation of oral administration of F14512 to developing wm4h larvae is the asymmetric homeotic transformation of one eye of affected flies, we refer to this phenotype as ‘‘Eye wide shut’’. The Eye wide shut phenotype was reproducible: we carried out 5 series of independent experiments starting with 30 wm4h L3 larvae in duplicates, each yielding 1 to 2 adults showing these striking abnormalities. Feeding etoposide, teniposide or DMSO never produced these changes, while feeding F14512 to wild-type Oregon R larvae led to no effects other than lethality at high concentrations. Intermediate asymmetric effects were also ob- served at a lower frequency, as shown in Figure 8C, where a dying adult retains a mottled right eye, while its left eye has been duplicated into smaller structures. As discussed below, these striking phenotypes can be correlated to precise genetic gains- or losses-of-function and all point to an implication of site-specific Topo II dysfunction in the effects we detect using the white-mottled model system following treatment with F14512, but not etoposide. Discussion F14512 is a novel targeted Topo II inhibitor possessing strong anticancer activity in mouse models of human tumors [24,25]. The results reported here demonstrate that, in addition to halting Drosophila cell growth in culture, F14512 induces unique features including rapid adherence, flattening and multi-nucleation but, at the concentrations used here, without the signs of apoptosis that are the hallmarks of etoposide and TOP-53, its closest relative. Although we cannot exclude more complex explanations, is appears reasonable to propose that the spermine moiety of F14512 might be responsible for these specific effects. In spite of these profound differences, F14512 remains highly cytotoxic and other possible pathways of growth arrest and cell death are currently under investigation. Recently, F14512-induced senescence has been evidenced with different human tumor cell lines (unpublished data). We note that the rapid appearance of multinucleated cells induced by F14512 treatment is reminiscent of the effects of dexrazoxane (ICRF-187), a potent Topo II catalytic inhibitor [54], suggesting the possibility of similar mechanisms of action. However, unlike dexrazoxane [55,56], F14512 does not induce apoptosis, even after prolonged treatment (Figure 5). Most importantly, similar to etoposide and teniposide, F14512 has been shown to act as a poison that stabilizes the Topo II cleavable complex [57], albeit with a greater potency that can be attributed to the presence of the spermine moiety. These observations rule out the possibility that, similar to dexrazoxane, F14512 acts as a Topo II catalytic inhibitor. Podophyllotoxin derivatives differ in their Topo II poison activities As expected for an epipodophyllotoxin derivative, F14512 exhibits a potent poisoning activity directed against Topo II and efficiently stabilizes Topo II cleavable complexes when used on a purified DNA target [57]. Using the Drosophila histone-gene repeat as a well understood model, F14512 behaved identically to teniposide and showed a similar sensitivity to the effects of distamycin (Figure 2), which affects Topo II localization in vitro [20,38] and in cellulo [37]. It is important to note that purified human Topo IIa was used in these experiments. Experiments carried out with purified Drosophila Topo II, of which only one isoform exists [58], yielded a slightly different cleavage pattern that PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e23597