In the presence of DMBA (Fig. 5 C,D). Only basal epithelial cells express Lrp proteins, and are predicted to respond to Wnt ligands with a canonical Wnt signal in vivo. In order to test whether Wnt signaling could explain lineage specific responses to genotoxic exposure, MECs were cultured with or without Wnt3a and DMBA, and the activation of theFigure 2. Genotoxin exposure during juvenile development affects differentiation and stem cell frequency in adult ductal trees. (A) Stem cell assay. Mammary epithelial cells populations were prepared from adults (9?0 weeks old), either exposed to DMBA at 5 weeks or not (administered tricaprylin vehicle), and injected into cleared fat pads at various limiting cell numbers. Four to six weeks later, glands were assessed for colonization and scored as a take (more than 25 colonization), or no take. The data fit the limiting dilution model (see Methods section; likelihood ratio test of single-hit model: P,0.000001) and stem cell frequencies were estimated on the basis of the PD-1/PD-L1 inhibitor 1 chemical information LimDil statistical program (Docosahexaenoyl ethanolamide site difference between groups: P,0.0001). (B) Flow cytometric analysis of MEC populations from adults exposed to DMBA as juvenile mice. Mammary epithelial cells were dissociated from 3 mice each (DMBA-treated and control), and stained according to [9,17], to resolve basal and luminal epithelial cell populations (see Fig. S1 for gating details). Representative flow cytograms are shown. The two principal cell types, luminal and basal, were quantified, and the ratio of luminal/basal cell is shown. doi:10.1371/journal.pone.0049902.gGenotoxins Inhibit Wnt-Dependent Mammary Stem CellFigure 3. Development of genotoxin-exposed glands during pregnancy. (A, B) Analysis of lobulo-alveolar development during pregnancy. Whole mount preparations from timed pregnant mice (6d p.c, 10 week old mice), exposed as juveniles to DMBA (or not). To examine the pattern of growth in more detail, paraffin sections from mammary glands were immunostained with Ki67 (to show cells in cycle; green) and counterstained with a luminal cell-specific stain, CK8 (K8; red; scale bar = 25 mm). Quantitation of the Ki67 index showed no significant difference between genotoxinexposed mice and the control cohort. (C) Lobulo-alveolar development during pregnancy in Lrp52/2 glands. Samples were processed according to (B), and were similarly stained with Ki67 and CK8, and also with CK5 (K5; blue) to visualize basal epithelial cells. doi:10.1371/journal.pone.0049902.gDDR was measured in basal and luminal cells (triple stains at 24 hours after DMBA exposure; Fig. 6A), 1407003 together with the effect of this on the mitotic index of each lineage. Fig. 6B illustrates theoverlaid dual-stained images analyzed to generate the quantitative information 15857111 shown in Fig. 6C. Approximately 12 of luminal or basal cells showed activation of the DDR, 24 and 48 hours afterGenotoxins Inhibit Wnt-Dependent Mammary Stem CellFigure 4. Evaluation of the DNA damage response (DDR) in basal and luminal epithelial cells after genotoxin administration in vivo. (A) DNA damage focus assembly. Mice were treated with either 10 Gy c-irradiation and harvested 30 minutes later (positive control), or administered DMBA (2 mg/ml) or vehicle (tricaprylin), and their mammary glands were harvested 2 days or 7 weeks later (as shown). Paraffin sections were tested for the formation of nuclear-associated cH2AX foci (green) in basal and luminal epithelial cells (K5, blue and K8, red respectively, counte.In the presence of DMBA (Fig. 5 C,D). Only basal epithelial cells express Lrp proteins, and are predicted to respond to Wnt ligands with a canonical Wnt signal in vivo. In order to test whether Wnt signaling could explain lineage specific responses to genotoxic exposure, MECs were cultured with or without Wnt3a and DMBA, and the activation of theFigure 2. Genotoxin exposure during juvenile development affects differentiation and stem cell frequency in adult ductal trees. (A) Stem cell assay. Mammary epithelial cells populations were prepared from adults (9?0 weeks old), either exposed to DMBA at 5 weeks or not (administered tricaprylin vehicle), and injected into cleared fat pads at various limiting cell numbers. Four to six weeks later, glands were assessed for colonization and scored as a take (more than 25 colonization), or no take. The data fit the limiting dilution model (see Methods section; likelihood ratio test of single-hit model: P,0.000001) and stem cell frequencies were estimated on the basis of the LimDil statistical program (difference between groups: P,0.0001). (B) Flow cytometric analysis of MEC populations from adults exposed to DMBA as juvenile mice. Mammary epithelial cells were dissociated from 3 mice each (DMBA-treated and control), and stained according to [9,17], to resolve basal and luminal epithelial cell populations (see Fig. S1 for gating details). Representative flow cytograms are shown. The two principal cell types, luminal and basal, were quantified, and the ratio of luminal/basal cell is shown. doi:10.1371/journal.pone.0049902.gGenotoxins Inhibit Wnt-Dependent Mammary Stem CellFigure 3. Development of genotoxin-exposed glands during pregnancy. (A, B) Analysis of lobulo-alveolar development during pregnancy. Whole mount preparations from timed pregnant mice (6d p.c, 10 week old mice), exposed as juveniles to DMBA (or not). To examine the pattern of growth in more detail, paraffin sections from mammary glands were immunostained with Ki67 (to show cells in cycle; green) and counterstained with a luminal cell-specific stain, CK8 (K8; red; scale bar = 25 mm). Quantitation of the Ki67 index showed no significant difference between genotoxinexposed mice and the control cohort. (C) Lobulo-alveolar development during pregnancy in Lrp52/2 glands. Samples were processed according to (B), and were similarly stained with Ki67 and CK8, and also with CK5 (K5; blue) to visualize basal epithelial cells. doi:10.1371/journal.pone.0049902.gDDR was measured in basal and luminal cells (triple stains at 24 hours after DMBA exposure; Fig. 6A), 1407003 together with the effect of this on the mitotic index of each lineage. Fig. 6B illustrates theoverlaid dual-stained images analyzed to generate the quantitative information 15857111 shown in Fig. 6C. Approximately 12 of luminal or basal cells showed activation of the DDR, 24 and 48 hours afterGenotoxins Inhibit Wnt-Dependent Mammary Stem CellFigure 4. Evaluation of the DNA damage response (DDR) in basal and luminal epithelial cells after genotoxin administration in vivo. (A) DNA damage focus assembly. Mice were treated with either 10 Gy c-irradiation and harvested 30 minutes later (positive control), or administered DMBA (2 mg/ml) or vehicle (tricaprylin), and their mammary glands were harvested 2 days or 7 weeks later (as shown). Paraffin sections were tested for the formation of nuclear-associated cH2AX foci (green) in basal and luminal epithelial cells (K5, blue and K8, red respectively, counte.
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