Y this dose of DMBA exposure in vivo; instead the response

September 1, 2017

Y this dose of DMBA exposure in vivo; instead the response to this agent includes a durable (though minor) hyperproliferation of MECs, focalized in “burst centers”. These centers may correlate with compensatory regenerative response centers, at sites of damage repair [39]. Growth stimuli activated by DNA damage repair mechanisms include hypomorphic function for Atm. Thus, surprisingly (given that Atm is essential to growth for most cell lineages), hypomorphic Atm function in mammary epithelial cell models generates a proliferative signal [40]. The phenotype of mammary glands after genotoxic exposure (reduced basal cell number and stem cell activity) was a remarkable phenocopy of glands with loss of function for Wnt signaling. This prompted our evaluation of Wnt signaling function in these glands. Interestingly, activation of Wnt signaling receptors (phospho-S1490 Lrp) was reduced in treated mammary glands, and this loss was durable for weeks, long after the proximal responses to genotoxic exposure were complete. Loss of Lrp activation clearly corresponded with the loss of mammary stem cell activity. In fact, measuring Lrp phosphorylation could act more generally as a proxy measure for the number of stem cells in a larger population, given that luminal cells do not typically express either Lrp receptor [17,41], and Axin2-positive (Wnt signal reporter) are limited to rare basal cells in the adult ductal tree in vivo [42]. Note that Zeng and Nusse have shown that 1313429 Wnt3a (200 ng/ml) supplementation maintains mammary stem cell activity in vitro [42]. Our study of mammary epithelial cell cultures shows that Wnt signaling supports the expansion of the basal epithelial lineage in general. When the basal cell subpopulation is expanded by adding Wnt ligands, these cells became highly sensitized to genotoxic exposure, implying that a specific chemosensitivity is induced by the basal cell response to Wnt signaling.FSC-A) and to analyze the remaining epithelial cells for their expression of CD24 and CD49f (d,h). (B) 1948-33-0 custom synthesis Fractions of cells going forward through each gate are shown. (TIF)Figure S2 DMBA induces double BI 78D3 custom synthesis strand DNA breaks, shown by the appearance of cH2AX, and DDR response activation. (A) Western blotting data; cH2AX activation. As a cross-check of the specificity of immunolocalization results for cH2AX (Fig. 4A, S3), mammary epithelial cells (HC11, NMuMG or BALB/c MECs) were treated with various genotoxins, lysed and transferred to Western blots, probing for the appearance of cH2AX. The genotoxins administered were 10 Gy irradiation (1 hour), 2 mg/ml DMBA 1407003 (24 hours; this is a pro-genotoxin requiring activation by metabolism, or vehicle, DMSO), N-ethylN-nitrosourea (1 hour after addition of 500 mg/ml ENU, a direct acting alkylating agent, or control PCB), or 20 mg/ml camptothecin (1 hour, camptothecin is a topoisomerase I inhibitor, or DMSO). Non-treated cells (NT) are also shown for comparison. (B) pS15-p53 is specifically induced by DNA damage in mammary epithelial cells, whereas pS20-p53 is constitutively present. HC11 mammary epithelial cells and primary mammary epithelial cells (MECs) were treated with ionizing radiation (IR, 10 Gy) and immunostained 1 hour later for the presence of pS15-p53 and pS20-p53. (C) Constitutive nuclear staining of BRCA1. BALB/c MECs were treated with DMBA or IR (as above), fixed and stained for BRCA1 (with a nuclear TOPRO counterstain). BRCA1 staining was ubiquitous and nuclear in all cells, irrespective of D.Y this dose of DMBA exposure in vivo; instead the response to this agent includes a durable (though minor) hyperproliferation of MECs, focalized in “burst centers”. These centers may correlate with compensatory regenerative response centers, at sites of damage repair [39]. Growth stimuli activated by DNA damage repair mechanisms include hypomorphic function for Atm. Thus, surprisingly (given that Atm is essential to growth for most cell lineages), hypomorphic Atm function in mammary epithelial cell models generates a proliferative signal [40]. The phenotype of mammary glands after genotoxic exposure (reduced basal cell number and stem cell activity) was a remarkable phenocopy of glands with loss of function for Wnt signaling. This prompted our evaluation of Wnt signaling function in these glands. Interestingly, activation of Wnt signaling receptors (phospho-S1490 Lrp) was reduced in treated mammary glands, and this loss was durable for weeks, long after the proximal responses to genotoxic exposure were complete. Loss of Lrp activation clearly corresponded with the loss of mammary stem cell activity. In fact, measuring Lrp phosphorylation could act more generally as a proxy measure for the number of stem cells in a larger population, given that luminal cells do not typically express either Lrp receptor [17,41], and Axin2-positive (Wnt signal reporter) are limited to rare basal cells in the adult ductal tree in vivo [42]. Note that Zeng and Nusse have shown that 1313429 Wnt3a (200 ng/ml) supplementation maintains mammary stem cell activity in vitro [42]. Our study of mammary epithelial cell cultures shows that Wnt signaling supports the expansion of the basal epithelial lineage in general. When the basal cell subpopulation is expanded by adding Wnt ligands, these cells became highly sensitized to genotoxic exposure, implying that a specific chemosensitivity is induced by the basal cell response to Wnt signaling.FSC-A) and to analyze the remaining epithelial cells for their expression of CD24 and CD49f (d,h). (B) Fractions of cells going forward through each gate are shown. (TIF)Figure S2 DMBA induces double strand DNA breaks, shown by the appearance of cH2AX, and DDR response activation. (A) Western blotting data; cH2AX activation. As a cross-check of the specificity of immunolocalization results for cH2AX (Fig. 4A, S3), mammary epithelial cells (HC11, NMuMG or BALB/c MECs) were treated with various genotoxins, lysed and transferred to Western blots, probing for the appearance of cH2AX. The genotoxins administered were 10 Gy irradiation (1 hour), 2 mg/ml DMBA 1407003 (24 hours; this is a pro-genotoxin requiring activation by metabolism, or vehicle, DMSO), N-ethylN-nitrosourea (1 hour after addition of 500 mg/ml ENU, a direct acting alkylating agent, or control PCB), or 20 mg/ml camptothecin (1 hour, camptothecin is a topoisomerase I inhibitor, or DMSO). Non-treated cells (NT) are also shown for comparison. (B) pS15-p53 is specifically induced by DNA damage in mammary epithelial cells, whereas pS20-p53 is constitutively present. HC11 mammary epithelial cells and primary mammary epithelial cells (MECs) were treated with ionizing radiation (IR, 10 Gy) and immunostained 1 hour later for the presence of pS15-p53 and pS20-p53. (C) Constitutive nuclear staining of BRCA1. BALB/c MECs were treated with DMBA or IR (as above), fixed and stained for BRCA1 (with a nuclear TOPRO counterstain). BRCA1 staining was ubiquitous and nuclear in all cells, irrespective of D.