However, the presence of the polyamine moiety did not perturb the previously characterized sensitivity of Topo II-mediated cleavage to distamycin [38], a DNA minor-groove binder specific for stretches of 4 or more dANdT base pairs [37]. As shown in lanes 2–4 and 7–9, addition of increasing concentrations led, after treatment with either teniposide or F14512, to a striking restriction of Topo II cleavage down to three major sites (marked 3–5 to the left of the gel) that correspond, to the nucleotide, to those cleaved after ex vivo treatment of Kc cells with teniposide, and where distamycin potentiates Topo II-mediated drug-induced cleavage [37]. Hence, the spermine moiety does not perturb drug/Topo II interactions in the presence of distamycin, which can redirect the sequence-specific localization of the protein [3]. In accordance with the observation that these identical patterns require 10-fold less F14512 than teniposide, we conclude from these results that the polyamine moiety might serve to stabilize ternary drug/DNA/ protein complexes, a hypothesis next tested using a well characterized ex vivo system, where polyamine interactions with chromatin might affect sites of drug-induced Topo II cleavage. Differential in cellulo induction of Topo II-mediated cleavage in the highly repeated SAT III region of the X chromosome In cellulo, our experimental system is based on the AT-rich 1.688 g/cm3 satellite III (SAT III) repeat, a 359-bp sequence repeated approximately 30,000 times in tandem near the proximal tip of the Drosophila melanogaster X chromosome (see Figure 6A below). SAT III repeats are highly enriched for Topo II [39] and constitute a hotspot for DNA cleavable-complex formation induced by teniposide, but not by amsacrine or anthracyclines [21,23]. These observations are consistent with the selective association of Topo II with A+T-rich sequences in vitro [20] and are accompanied by a similarly preferential interaction with these sequences ex vivo, as shown for teniposide-stimulated cleavage of SAT III repeats [37]. This huge 11-Mbp target, whose structure is schematized in Figure 3A for 6 adjacent 359-bp monomers can be used as a convenient model system to analyze Topo II cleavage in or ex vivo. The high abundance of the corresponding sequence (almost 5% of the whole genome in diploid tissues) facilitates analysis of rare Topo II cleavage products. As each 359-bp repeat contains a unique Topo II cleavage site located in one of two nucleosomal spacers (Figure 3A), partial cleavage results in a ladder of products of (359-bp)n size. As shown in Figure 3B, a ladder of SAT III cleavage products readily appeared following a short 30-minute treatment of cultured Kc cells with Topo II poisons such as teniposide or TOP-53, but was largely absent in DNA isolated from cells treated with etoposide. In all cases, cells were treated for 30 minutes with each drug at the indicated concentrations, in the absence or presence of distamycin, which renders SAT III sequences more accessible to Topo II. This treatment led to an expected increase [37] in the intensity of the 359-bp cleavage ladder (compare lanes 3, 6 and 12 to lanes 2, 5 and 11). In contrast and similar to the lack of activity of etoposide, we could not detect F14512-induced Topo II cleavage in the SAT III locus, irrespective of the presence of distamycin (Figure 3C, lanes 13-17). This outcome implies that the cellular Topo II-directed activity — if any — of F14512 is comparable to that of etoposide (see Figure 3B), but differs from that of its closest parent, TOP-53. However, we cannot exclude the possibility that either drug is active at this locus, but yields extremely short-lived cleavable complexes whose instability makes it impossible to observe detectable cleavage products. Alternative- ly, the presence of the spermine moiety in F14512 might direct it Studying Anticancer Drug Properties in Drosophila to a discrete subset of chromosomal sites, which in this case might differ from those induced by etoposide, teniposide or TOP-53. Shotgun search for F14512-stimulated Topo II in Drosophila cells Looking for evidence of F14512-induced Topo II-mediated cleavage in a .200-Mbp genome is a daunting task. A low frequency of cleavage sites located on average every 200 kbp (close to 10-fold less than estimates in vertebrates, in line with the overall rarer cleavage induced by F14512 in mammalian cells) still corresponds to a range of 1000 potential targets. We chose instead to use a genome-wide approach similar to that used almost 20 years ago for the discovery of Topo II cleavage of SAT III sequences [21]. In this case, we had used a radio-labeled probe prepared from total Drosophila genomic DNA and hybridized it to undigested genomic DNA extracted from teniposide-treated cells. The kinetics of hybridization of repeated sequences permitted in this case the detection of cleavage products that were eventually shown to originate from SAT III repeats. However, those, as shown above, are not cleaved following treatment with F14512. To detect potential F14512-induced cleavage sites in the Drosophila genome, we introduced modifications to our original strategy. As hybridization to a whole genomic probe did not reveal any F14512-induced cleavage products, as in the case of SAT III repeats, we chose to digest genomic DNA isolated from untreated or treated cells with a restriction enzyme (HindIII in this case) before electrophoresis and hybridization to the whole genomic probe. Another modification was a pre-hybridization step for the denatured whole-genome probe which, under controlled condi- tions, serves to mask hybridization to highly repeated (snap-back simple repeats and Cot1) sequences that might otherwise obscure any specific signal (see Methods for details). Schneider S2 tissue culture cells were used in this case instead of Kc cells as their polyploidy increases copy number per cell and their SAT III repeats are cleaved similarly to those of Kc cells following treatment with teniposide (data not shown). After treatment with TOP-53 or F14512 in the absence or presence of distamycin (see below), genomic DNA was purified, digested with HindIII and hybridized as described in Methods to the pre- hybridized genomic probe. Results of such an experiment are shown in Figure 4. Note that the numerous bands detected in controls (lanes 1 and 7 of Figure 4A) correspond to moderately repeated genomic HindIII fragments and not to Topo II cleavage products. Unique sequences only give rise to a low-level random background smear. This approach yielded encouraging results as it readily identified bands whose detection was sensitive to F14512 treatment, demonstrating the usefulness of such a method when seeking putative —and unknown— drug-induced Topo II cleavage sites in cellulo. Compared to no-drug (lanes 1 and 7) or TOP-53 (lanes 2 and 8) controls, F14512 treatment resulted in the disappearance of two repeated HindIII fragments (labeled ‘‘a’’ and ‘‘b’’) and to the appearance of a new fragment (labeled ‘‘c’’). We surmise that this new fragment might arise from cleavage of the ‘‘a’’ or ‘‘b’’ fragments, or both. A densitometric analysis of lanes 7, 8 and 11 is shown in Figure 4B. While fragment ‘‘a’’ falls in an overexposed and poorly resolved portion of the gel, a significant decrease in the radioactive signal can be observed in this region, while the disappearance of fragment ‘‘b’’ and the appearance of fragment ‘‘c’’ can be clearly discerned. In addition, this pattern was clearly dependent on the concentration of F14512 used. A separate experiment is shown in Figure 4C, using a lower- percentage agarose gel and DNA from cells treated with F14512 and etoposide in the presence of distamycin. Compared to no-drug (lane 1) and etoposide samples (lane 4), F14512-treated cells PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23597