13 research outputs found
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<sup>Rock</sup> antibody and analyzed by flow cytometry. ABCB5<sup>+</sup> 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<sup>+</sup> 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
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
Actinic keratosis modelling in mice: A translational study
<div><p>Background</p><p>Actinic keratoses (AK) are pre-malignant cutaneous lesions caused by prolonged exposure to ultraviolet radiation. As AKs lesions are generally accepted to be the initial lesions in a disease continuum that progresses to squamous cell carcinoma (SCC), AK lesions have to be treated. They are also the second most common reason for visits to the dermatologist. Several treatments are available but their efficacy still needs to be improved. The UV-B-induced KA lesion mouse model is used in preclinical studies to assess the efficacy of novel molecules, even though it is often more representative of advanced AK or SCC.</p><p>Objectives</p><p>Here we report on a translational study, comparing the various stages of AK development in humans and in the UV-B irradiated mouse model, as well as the optimization of photograph acquisition of AK lesions on mouse skin.</p><p>Methods</p><p>Human and mouse skin lesions were analysed by histology and immunohistochemistry. Mouse lesions were also assessed using a digital dermatoscope.</p><p>Results</p><p>An histological and phenotypic analysis, including p53, Ki67 and CD3 expression detection, performed on human and mouse AK lesions, shows that overall AK modelling in mice is relevant in the clinical situation. Some differences are observed, such as disorganization of keratinocytes of the basal layer and a number of atypical nuclei which are more numerous in human AK, whereas much more pronounced acanthosis is observed in skin lesion in mice. Thanks to this translational study, we are able to select appropriate experimental conditions for establishing either early or advanced stage AK or an SCC model. Furthermore, we optimized photograph acquisition of AK lesions on mouse skin by using a digital dermatoscope which is also used in clinics and allows reproducible photograph acquisition for further reliable assessment of mouse lesions. Use of this camera is illustrated through a pharmacological study assessing the activity of CARAC<sup>®</sup>.</p><p>Conclusion</p><p>These data demonstrate that this mouse model of UV-B-induced skin lesions is predictive for the identification of novel therapeutic treatments for both early and advanced stages of the disease.</p></div
ABCB5-expressing cells survive upon dacarbazine treatment.
<p>WM-266-4 (<b>A,D,G</b>), G-361 (<b>B,E,H</b>) and SK-MEL-28 cells (<b>C,F,I</b>) were treated at the indicated concentrations of dacarbazine (<b>A–C</b>), vemurafenib (<b>D–F</b>) and doxorubicin (<b>G–I</b>). After 72 h, the total viable cells were numbered using an automated cell viability analyzer. The percentages of viable ABCB5-expressing cells (among viable cells gated on DAPI-negative cells) were analyzed by flow cytometry. The numbers of total cells (white symbols) are reported as percentages of the number of cells in the untreated control sample. The numbers of viable ABCB5<sup>+</sup> cells (black symbols) were calculated from the total cell numbers and ABCB5<sup>+</sup> cells ratio, and reported as percentages of the viable ABCB5<sup>+</sup> cells number in the control sample. ABCB5<sup>+</sup> cells represent respectively 3%, 3.5% and 5% of the total cells in the WM-266-4, G-361 and SK-MEL-28 cell lines.</p
Ingenol mebutate inhibits UV-B-induced AK lesions.
<p>Mice were exposed to UV-B irradiation for 74 days, and then topically treated for 5h with either 0.015% ingenol mebutate or control vehicle (5% DMSO, 70% Glycerol and 25% H<sub>2</sub>O) on day 78 after irradiation initiation. A) Skin appearance after ingenol mebutate treatment, at days indicated after irradiation initiation. B) Histology of UV-B-irradiated mouse skin topically treated with either control vehicle or 0.015% ingenol mebutate. B1-B2) Hematoxylin and eosin staining of control vehicle-treated skin. B3) Ki67 staining of control vehicle-treated skin. B4) p53 staining of control vehicle-treated skin. B5 and B6) Hematoxylin and eosin staining of 0.015% ingenol mebutate-treated skin. B7) Ki67 staining of 0.015% ingenol mebutate-treated skin. B8) p53 staining of 0.015% ingenol mebutate-treated skin. Experiments involved three mice per experimental group. Scale bars represent 100 μm.</p
0.5% 5-FU inhibits UV-B-induced AK lesions.
<p>Mice were exposed to UV-B irradiation for 74 days, then topically treated for 30h with either 0.05% 5-FU or control vehicle (5% DMSO, 70% Glycerol and 25% H<sub>2</sub>O), starting on day 78 after irradiation initiation. The chamber systems containing the 0.05% 5-FU cream were then removed for 20h, and mice were again treated topically for 30 additional hours. Skin appearance after either control vehicle (A) or 5-FU (B) treatment, at indicated days after treatment initiation. Scale bars represent 500 μm. C) Assessment of the number of skin lesions in control vehicle and 5-FU treated mice, before and after treatment (on day 28 after treatment initiation). Experiments involved five mice per experimental group.</p
ABCB5-expressing cells are enriched in the residual tumors after an anti-melanoma treatment <i>in vivo.</i>
<p>WM-266-4 cells (5×10<sup>6</sup> cells) were injected subcutaneously in Swiss nude mice. Fourteen days later, mice were treated by repeated i.p. injections of either temozolomide (80 mg/kg) or vehicle following the schedule indicated by the black arrows. The tumoral volumes were monitored and the means of measured volumes respectively for temozolomide-treated and vehicle treated tumors are as follows: 180 mm<sup>3</sup> and 175 mm<sup>3</sup> at day 14; 264 mm<sup>3</sup> and 295 mm<sup>3</sup> at day 16; 178 mm<sup>3</sup> and 444 mm<sup>3</sup> at day 18; 84 mm<sup>3</sup> and 699 mm<sup>3</sup> at day 21. (<b>A</b>). 24 h after the injections at days 16 and 21 (d17 and d22), tumors were recovered, dissociated and the cell suspensions were searched for the presence of human ABCB5<sup>+</sup> cells by flow cytometry (<b>B</b>) (medians are represented as black lines). Tumors recovered at day 17 were analyzed by immunohistochemistry for their ABCB5 expression (<b>C</b>).</p
Comparative histology of human and mouse UV-B-induced AK lesions.
<p>Hematoxylin and eosin staining. Representative cases: A1) Human normal skin, case 399. A2) Human early-stage AK, case P14-1532. A3) Human early-stage AK, case P14-1358. A4) Human intermediate-stage, case P14-1363. A5) Human advanced-stage AK, case P14-1521. A6) Human advanced-stage AK, case P14-1828. B1) Mouse normal skin, case D181P14. B2) Mouse early-stage AK, case 203P21. B3) Mouse early-stage AK, case D195P16. B4) Mouse intermediate-stage, case 198P39. B5) Mouse advanced-stage AK, case 488. B6) Mouse advanced-stage AK, case D221-1. Scale bars represent 100 μm.</p
Comparison of macroscopic skin lesions.
<p>Photographs taken with either a standard camera (A1-A3) or a calibrated digital dermatoscope (B1-B3). Mice were exposed to UV-B irradiation for 108 days and photographs were taken on the indicated days after irradiation initiation. Scale bars represent 500 μm.</p