Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer

Sleep and physical activity patterns in adults and children with Bardet–Biedl syndrome

Abstract Background Overweight and obesity are common features of the rare disease Bardet–Biedl syndrome (BBS). Sleep and physical activity are behaviors that might impact overweight and obesity and thus may play a key role in the health and well-being of people with BBS. Objectively-measured sleep and physical activity patterns in people with BBS are not well known. We evaluated objectively-measured sleep and physical activity patterns in the largest cohort to date of people with BBS. Results Short sleep duration, assessed using wrist-worn accelerometers, was common in both children and adults with BBS. Only 7 (10%) of adults and 6 (8%) of children met age-specific sleep duration recommendations. Most adults 64 (90%) achieved recommended sleep efficiency. The majority of children 26 (67%) age 6–12 years achieved recommended sleep efficiency, but among children age 13–18, only 18 (47%). In both adults and children, sleep duration was significantly negatively correlated with duration of prolonged sedentary time. In children age 6–12 sleep duration was also significantly related to total activity score, children with lower sleep duration had lower total activity scores. Conclusions Insufficient sleep duration is very common in people with BBS. Prolonged sedentary time and short sleep duration are both potentially important health-related behaviors to target for intervention in people with BBS

Features of the actively dividing cells in CD45–CD31– RMECs.

<p>(<b>A</b>) Flow cytometric histogram showing gating for actively dividing cells (cells in S/G2+M phase of the cell cycle) by having >2n cellular DNA content (left panel); representative dot plot showing the actively dividing cells overlaid on CD24 and CD29 expression in the RMECs (right panel). (<b>B</b>) Representative dot plot showing gating for RMECs expressing high levels of both CD24 and CD29 (CD24hiCD29hi gate; left panel); bar graph (right panel) showing mean ± sem percentage of cells in S/G2+M phase of the cell cycle in total RMECs or CD24hiCD29hi-gated cells (n = 24 each). A significant enrichment of actively dividing cells was detected in the CD24hiCD29hi-gated cells (p<0.05; indicated with an asterisk). (<b>C</b>) Overlaid histogram showing CD49f expression in the total RMECs and actively dividing cells (left panel); bar graph (right panel) showing mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD49f in the total RMECs and actively dividing cells (n = 14 each). Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative dot plot showing CD49f expressing cells (CD49f+) overlaid on CD24 and CD29 expression of the RMECs (left panel); bar graph showing mean ± sem percentage of CD24hiCD29hi-gated cells in total RMECs or the CD49f expressing population (right panel). A significant enrichment of CD24hiCD29hi-gated cells was detected in the CD49f expressing population (p<0.05; indicated with an asterisk). For all panels, rats of 12 weeks of age were used.</p

Epithelial cell differentiation in mammary glands and carcinomas from 7,12-dimethylbenz(a)anthracene (DMBA)-or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-exposed rats.

<p>(<b>A</b>) Schematic representation of the modulation of RMEC differentiation 1 week after exposure of rats to the mammary carcinogens DMBA or MNU. DMBA exposure increases CD49f expression and proliferation (not shown here). MNU exposure disrupts the luminal and basal homeostasis. (<b>B</b>) Schematic representation of the changes of RMEC differentiation in carcinomas as compared to mammary gland from untreated age-matched (22 weeks of age) control rats. Note that the RMECs from animals of 22 weeks of age have a higher percentage of luminal cells as compared to younger animals (comparing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g001" target="_blank">Fig. 1A</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">Fig. 4A</a>). In both panels cell surface expression of CD24, CD29, CD49f and CD61 and intracellular expression of focal adhesion kinase (FAK) and Y397-phosphorylated FAK (pFAK) in basal, luminal and CD24hiCD29hi-gated cells are shown.</p

Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers.

<p>(<b>A</b>) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). (<b>B</b>) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. (<b>C</b>) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.</p

Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU).

<p>(<b>A</b>) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p<0.05). (<b>B</b>) Bar graphs showing the mean ± sem percentage of RMECs containing >2n cellular DNA (actively dividing cells in S/G2+M phase of cell cycle). Significantly different percentage comparing RMECs from carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>C</b>) Representative overlaid histograms showing upregulation of CD29 expression (upper left panel), upregulation of CD49f expression (upper middle panel) and downregulation of CD61 expression (upper right panel) in RMECs of a DMBA-induced or MNU-induced carcinoma as compared to a control mammary gland; bar graphs quantifying the mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD29 (lower left panel), CD49f (lower middle panel) and CD61 (lower right panel) on RMECs from control mammary glands and carcinomas. Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative pseudo-color dot plot showing gating for CD29 and focal adhesion kinase (FAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hiFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>E</b>) Representative pseudo-color dot plot showing gating for CD29 and Y397-phosphorylated focal adhesion kinase (pFAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hi pFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). In the entire <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">figure 4</a>, age-matched untreated control mammary glands: n = 16, DMBA-induced mammary carcinomas: n = 10 and MNU-induced mammary carcinomas: n = 10.</p

The gene desert mammary carcinoma susceptibility locus Mcs1a regulates Nr2f1 modifying mammary epithelial cell differentiation and proliferation.

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in 'triple-negative' (ER-PR-HER2-) breast cancers as compared with 'receptor-positive' breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk

The rat mammary carcinoma susceptibility locus <i>1a</i> (<i>Mcs1a</i>) is located in a gene desert and confers resistance to three distinctly acting carcinogenic treatments.

<p>A) Genetic map of the congenic lines contributing to the positional identification of the <i>Mcs1a</i> locus on rat chromosome <i>2</i>. Each congenic line, as defined by genotyping the genetic markers indicated along the vertical scale bar (also listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s005" target="_blank">Table S1</a>), represents a segment from the resistant Copenhagen (Cop) inbred strain introgressed into the susceptible Wistar-Furth (WF) genetic background. The critical interval for the <i>Mcs1a</i> resistance allele is defined by resistant congenic lines (filled bars) showing a <i>7,12</i>-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma multiplicity that is lower than that of the susceptible congenic control line (WF.Cop), and susceptible congenic lines (open bars) showing a DMBA-induced mammary carcinoma multiplicity not different than that of the susceptible congenic control line. The grey boxes illustrate the areas of recombination. The coordinates (in bp) along the vertical axis are from the 2004 version of the rat genome (UCSC Genome Browser, rn4). B) DMBA-induced mammary carcinoma multiplicity phenotype for <i>Mcs1a</i> resistant congenic lines Q (n = 83), R3 (n = 24), V4 (n = 24), W4 (n = 28), Y4 (n = 45), and W5 (n = 41) and susceptible congenic lines P5 (n = 16), V5 (n = 56), R5 (n = 30), A4 (n = 24), Y3 (n = 38) and WF.Cop (n = 44). Congenic line Q originally defined the <i>Mcs1a</i> interval in our previous publication and is used as a reference for resistance <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549-Haag1" target="_blank">[33]</a>. C) <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-induced mammary carcinoma multiplicity phenotype of resistant congenic lines W4 (n = 20) and W5 (n = 23), susceptible congenic line R5 (n = 14) and the susceptible congenic control line WF.Cop (n = 28). D) Mammary carcinoma multiplicity phenotype induced by mammary ductal infusion of retrovirus expressing the activated <i>HER2/neu</i> oncogene (<i>HER2/neu</i>) for resistant congenic line R3 (n = 15) and susceptible congenic line A4 (n = 14). In all graphs, resistant congenic lines are displayed as filled bars, susceptible congenic lines are displayed as open bars. Significant difference (P<0.05) from the susceptible congenic control line (panels B and C) or from susceptible congenic line A4 (panel D) is indicated by an asterisk.</p