Senescence in Breast Cancer Figure 4. Overexpression of estrogen receptor-a inhibited the production of terminally arrested progeny. (A) ER-overexpressing (ER-5, ER-7, ER-26) and control (C-8, C-10, C-11) clones were established from T47D cells and tested for ER expression by western blotting using decreasing amounts of total proteins. Calnexin was used as loading control. (B–C). Colonies were generated, labeled with BrdU for 24 h and immunostained for BrdU (shown in Fig. S3). Individual colonies were manually counted for quantification of % BrdU cells (B), and mean % BrdU+ cells values were calculated (C). Error bars represent mean 6 SD. ER overexpression caused a significant increase in % ratio of BrdU+ cells (*three ER clones versus three controls; P = 0.034). doi:10.1371/journal.pone.0011288.g004 assay. The mean age of the patients was 58612 yrs, with a mixed menopause status (Table S2). Two tumors (17%) displayed SABG+ cells that were scattered within the tumor area (Fig. S4). Thus, ER+ breast tumors also produced senescent progeny in vivo, but at a lower rate. Senescent cell progenitor and immortal cell progenitor subtypes’ abilities to differentiate into luminal and myoepithelial cell types The cellular specificity of ER expression in the mammary epithelial cell hierarchy is poorly understood. Previous data suggests that normal ER+ cells may represent either relatively differentiated luminal cells with limited progenitor capacity or primitive progenitors with stem cell properties in the luminal cell compartment [4,24,25]. Based on the close association between senescence (which can be considered a manifestation of terminal differentiation) and loss of ER positivity, we hypothesized that ER+ SCP cells may differ from ER- ICP cells by their differentiation potential. We surveyed a few hundred single-cell- derived colonies from each of the 12 cell lines for production of stem/progenitor-like, luminal-like, and myoepithelial-like cells. We used CD44 as a positive stem/progenitor cell marker [26,27], CD24, ER, and CK19 as luminal lineage markers [27,28,29], and ASMA as a myoepithelial lineage marker [29]. Representative examples of marker studies by immunoperox- idase staining in SCP and ICP cell lines are shown in Fig. 5. All five SCP cell lines displayed a heterogeneous pattern of positivity for CD44; some colonies were fully positive, some fully negative, and others were composed of both positive and negative cells. CD44/CD24 double immunofluorescence studies with the T47D cell line indicated that SCP cells produce also CD44+/CD24- stem/progenitor cells, as expected (data not shown). In sharp contrast, five of the seven ICP cell lines generated only fully positive CD44 colonies, indicating they do not produce CD44- cells. One cell line was totally CD44-. Only one cell line displayed PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11288