The <i>Drosophila white-mottled</i> inversion: a classical system to study long-range regulation of gene expression.

<p>(A) Schematic representation of the <i>Drosophila</i> X chromosome and of the <i>white-mottled</i> (<i>w^m4h</i>) inversion that juxtaposes the <i>white</i> gene to a heterochromatin context, in this case within the 4-Mbp rDNA locus adjacent to the SAT III array. Different features of the proximal end of the chromosome, most of which remains unsequenced, are discussed in the text. Stochastic silencing of <i>white</i> leads to a mottled eye color (bottom left corner) compared to the wild-type red adult eye (top right corner). This phenomenon corresponds to the classical definition of position-effect variegation (PEV). The nearby <i>Notch</i> gene is similarly affected under certain growth conditions (see text for details). (B) Intrinsic loss of symmetry resulting from <i>white</i> and <i>Notch</i> PEV in <i>white-mottled</i> flies. The fly shown is a representative example of an adult phenotype observed at 1–2% frequency in the <i>w^m4h</i> line when grown at 18°C. The facing view illustrates a characteristic mottled color of the left eye, but a wild-type red color on the right. This is more clearly seen in the right and left close-ups shown to the right. This intrinsic propensity for asymmetry in <i>w^m4h</i> PEV manifestations most likely results from <i>Notch</i> misregulation and is important to understand the one-sided phenomena illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone-0023597-g008" target="_blank">Figure 8</a>.</p

Suppression of <i>white-mottled</i> PEV by Topo II poisons.

<p>Photographs show representative eyes of 5-day old <i>w^m4h</i> females hatched from late L3 larvae fed etoposide (VP16), F14512 or teniposide (VM26) at the indicated concentrations. A comparison of the no-drug controls shown in the left column highlights the intrinsic heterogeneity of the <i>white-mottled</i> phenotype. Etoposide and F14512 partly suppress <i>white</i> PEV at higher concentrations and display an apparent threshold effect at 25 µM or 50 µM, respectively. Teniposide feeding leads to full dose-dependent suppression of PEV. Controls (<i>w^m4h</i> or wild-type Oregon R larvae fed 0 or 1% DMSO used to dilute the drugs, a concentration that corresponds to that used in 100-µM drug treatments) are shown in the bottom row. The graph at the bottom shows the corresponding quantifications of extracted eye pigment and represents mean values obtained from three independent experiments performed in duplicate (30 5-day old females scored for each condition). Results are represented as % pigment relative to Oregon R wild-type (WT) flies fed 1% DMSO. Error bars represent the experimental error. Note that the toxicity of F14512 feeding at 25 and 50 µM only permitted analysis of 10 females for each experiment, most likely accounting for the substantial variations observed (p<0.2 in contrast to p<0.01 for other drug treatments).</p

Cellular profiles of drug-induced Topo II-mediated cleavage in SAT III repeats of the <i>Drosophila</i> X chromosome.

<p>(A) Structure of SAT III repeats. 359-bp SAT III repeats accommodate 2 nucleosomes per repeat. One of the two nucleosomal linkers located in each repeat contains a short GC-rich sequence that serves as a highly specific target for Topo II cleavage <i>in vivo</i>, as denoted by scissors. (B) Schneider S2 cells were treated for 30 minutes with teniposide (VM26), etoposide (VP16) or TOP-53 at the concentrations shown, in the absence (-) or presence (+) of distamycin (DIST, 25 µM). Purified DNA was electrophoresed without prior restriction enzyme digestion, transferred to a nylon membrane and hybridized to a labeled SAT III probe. 359-bp: the SAT III monomer (SAT) used as a probe; M: molecular weight standards. (<b>C</b>) S2 cells were treated for 30 minutes with no drug (0, lane 13), F14512 (5, 10, 25 or 50 µM, lanes 14–17) or 50 µM TOP-53 (lane 18), in the presence of 25 µM distamycin (DIST). Omission of the latter gave identical results (not shown). DNA samples were processed as described above. SAT: the 359-bp cloned fragment used as a probe; M: molecular weight standards.</p

<i>Eye wide shut</i>: specific loss- and gain-of-function phenotypes after F14512 oral administration to <i>white-mottled</i> larvae.

<p>(A) Different views of a representative <i>Eye wide shut</i> adult male recovered from feeding 25 µM F14512 to <i>w^m4h</i> 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 <i>Antp</i> gain-of-function and/or <i>ey</i> 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 <i>Eye wide shut</i> phenotype is consistent with the propensity for loss of right/left symmetry in the <i>w^m4h</i> line (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone-0023597-g006" target="_blank">Figure 6B</a>). (B) Close-up views of the left side of F14512-induced <i>Eye wide shut</i> 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 <i>Sex combs reduced</i> (<i>Scr</i>). Note that <i>Scr</i> is closely linked to <i>Antp</i> and is also subject to Polycomb-mediated regulation via mechanisms that implicate Topo II. (C) Asymmetric <i>ey</i> loss-of-function in an F14512-fed <i>w^m4h</i> adult male. Feeding lower concentrations (10 µM) of F14512 to <i>w^m4h</i> 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 <i>ey</i> loss-of-function.</p

<i>In vitro</i> drug-induced cleavage in the <i>Drosophila</i> histone-gene repeat.

<p>(A) Schematic representation of the 5-kbp major histone-gene locus, which contains the 5 genes encoding core and linker histones. The bar with a hook represents the scaffold-associated region (SAR) located in the intergenic spacer between the H1 and H3 genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Mirkovitch1" target="_blank">[74]</a>. It is cleaved by Topo II (denoted by scissors) in the linker regions of 5 positioned nucleosomes (black circles). Other sites (1, 2 and 8–10) map to DNAse I-hypersensitive sites at the 5′ and 3′ ends of the histone genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Ks1" target="_blank">[21]</a> for details). Sites 3-5 mapped at nucleotide resolution by direct genomic sequencing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Ks1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Borgnetto1" target="_blank">[22]</a>. (B) <i>In vitro</i> cleavage induced by teniposide (VM26) and F14512. A uniquely end-labeled DNA clone spanning the histone-gene repeat was incubated with Topo II as described in Materials and Methods in the presence of no drug (lane 1), 50 µM teniposide with 0, 5, 10 or 25 µM distamycin (lanes 5, 4, 3 and 2, respectively) or 5 µM F14512 with 0, 5, 10 or 25 µM distamycin (lanes 6, 7, 8 and 9, respectively). The 4 lanes at the right of the gel are Maxam-Gilbert sequencing reactions of the labeled fragment. Cleavage is induced at numerous identical sites by teniposide and F14512 in the absence of distamycin (compare lanes 5 and 6). Increasing concentrations of distamycin redirect cleavage induced by both drugs to sites 3–5 marked by arrows to the left of the gel and which correspond exactly to the sites cleaved <i>in cellulo</i>.</p

Structure of podophyllotoxin derivatives used in this study.

<p>Structure of F14512. Epipodophyllopoxin is shown to the left, with the substitution of different R groups leading to teniposide (VM26), TOP-53 and etoposide (VP16).</p

ABCB5 expression is increased in melanoma tumors obtained from treated patients.

<p>Skin metastases specimens from respectively 8 untreated and 7 treated patients were analyzed by immunohistochemistry for their ABCB5 protein expression. The ABCB5 staining intensity was ranked in four arbitrary classes according to the intensity and the extent of the labelling. Representative staining of two levels of intensity (left panel: isotypic control) (A). Repartition of the specimens in the different classes (B). The two groups of specimens (untreated versus treated) have been compared with the non parametric Kruskall Wallis test (p<0.30).</p

Quantification of ABCB5- and ABCB1-expressing cells after cytotoxic treatments.

<p>WM-266-4 cells were treated for 72 h with the indicated concentrations of doxorubicin, dacarbazine, vemurafenib, gemcitabine or with vehicle (NT) and ABCB5 expression was analyzed by Western blot. Band intensities were quantified and variations are indicated as fold increases in treated versus untreated samples (<b>A</b>). WM-266-4 cells were treated with various drugs at their EC50 for 72 h. The percentages of positive cells among surviving cells were measured by cell surface labelling and flow cytometry analysis for ABCB5 (<b>B</b>) or ABCB1 (<b>C</b>). The relative mRNA expression of ABCB5, ABCB1, ABCC1, ABCG2 and HMBS as the house-keeping gene was measured by Q-PCR (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036762#pone.0036762.s002" target="_blank">table S1</a>) and the amplified products were run on agarose gel after 29 cycles except for ABCB1 (32 cycles) (<b>D</b>).</p

ABCB5 is expressed on the surface of a subpopulation of melanoma cells.

<p>WM-266-4 cells were surface-labelled with the ABCB5-Ab^Rock antibody and analyzed by flow cytometry. ABCB5⁺ cells (right contour plot) were gated on viable cells (DAPI-negative) according to the isotype control (left contour plot). Inserts (dot plots) display the gating of the positive cells (<b>A</b>). WM-266-4 cells were treated with a siRNA designed to target ABCB5 (si-ABCB5). After 72 h, the cells were analyzed for their ABCB5 mRNA content and ABCB5 surface expression. The left and right histograms show respectively the relative expression of ABCB5 mRNA normalized to the ABCB5 mRNA in cells treated with a control siRNA (si-ctrl), and the percentage of ABCB5⁺ cells among total cells (n = 3). The corresponding contour plots are shown (<b>B</b>). Different melanoma cell lines were analyzed for their ABCB5 surface expression (<b>C</b>) or their ABCB5 mRNA content (<b>D</b>) (n = 3).</p

Effects of fluorescent CD45 mAb intravenously administered over three weeks on the bioluminescence signal of HL60-Luc tumor-bearing mice (A) and on life span (B).

<p>Effects of fluorescent CD45 mAb intravenously administered over three weeks on the bioluminescence signal of HL60-Luc tumor-bearing mice (A) and on life span (B).</p