Studying Anticancer Drug Properties in Drosophila Figure 8. Eye wide shut: specific loss- and gain-of-function phenotypes after F14512 oral administration to white-mottled larvae. (A) Different views of a representative Eye wide shut adult male recovered from feeding 25 mM F14512 to wm4h larvae. The right side shows suppression of PEV by the drug while, on the left side, the eye has been replaced by a first thoracic treatment, best explained by a left side-only Antp gain-of- function and/or ey loss-of-function. The latter is also suggested by the presence of a small remnant of an eye (arrowhead in bottom view; see also panel C). The striking asymmetry of the F14512-induced Eye wide shut phenotype is consistent with the propensity for loss of right/left symmetry in the wm4h line (Figure 6B). (B) Close-up views of the left side of F14512-induced Eye wide shut male adults. Homeotic transformation of the left eye into a first thoracic segment can be clearly seen, white arrowheads indicate a small remnant of an eye. The inset shows an enlarged view of a normal right side, with male-specific sex combs on the first tarsal segment of the fore leg indicated by the shaded arrowhead. Sex combs are absent on the left side where the eye is also transformed in a first thoracic segment, suggesting a loss-of-function of the homeotic gene Sex combs reduced (Scr). Note that Scr is closely linked to Antp and is also subject to Polycomb-mediated regulation via mechanisms that implicate Topo II. (C) Asymmetric ey loss- of-function in an F14512-fed wm4h adult male. Feeding lower concentrations (10 mM) of F14512 to wm4h larvae occasionally leads to the recovery of other phenotypes at a frequency of about 5%. The right side of the fly again shows a normal eye with a characteristic mottled color. The left eye has been diminished to two distinct smaller eye structures characteristic of partial ey loss-of-function. doi:10.1371/journal.pone.0023597.g008 Drosophila (Figure 2A). Teniposide-induced cleavage sites have been previously characterized in this 5-kbp sequence at nucleotide resolution ex vivo [21,22] and in vitro. DNA incubation with human Topo IIa before addition of teniposide or F14512 yielded essentially identical cleavage patterns (Figure 2B, compare lanes 5 and 6). Note, however, that F14512 was used at a 10-fold lower concentration (5 mM) than teniposide (50 mM) to obtain compa- rable extents of cleavage of the 5-kbp fragment. Hence, addition of a spermine moiety to a podophyllotoxin-based molecule does not, in this system, alter the basic properties of the Topo II poison and F14512 retains an identical selectivity for cleavage-site selection. It is however likely, given the 10-fold differences in drug concentra- tions used to yield comparable extents of cleavage products that the presence of spermine promotes the formation of a more stable cleavable complex. Such a possibility is supported by the previously characterized interaction of F14512 with DNA [24]. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23597