Senescence in Breast Cancer Figure 2. Growth arrest observed in senescent cell progenitors was inhibited by p21Cip1 silencing. (A) Four of five senescent SCP cell lines (top four from left) generated colonies with heterogeneous expression of p21Cip1; in contrast none of seven ICP cell lines generated p21Cip1 cells. Colonies were immunostained for p21Cip1, with hematoxylin used as counterstain. Scale bar: 50 mm. (B–E) p21Cip1 silencing inhibited the production of the terminally arrested progeny of SCP cells. (B) shRNA-mediated inhibition of p21Cip1 expression. T47D cells were infected with lentiviral vectors encoding p21Cip1 shRNA or scrambled shRNA to generate T47D-p21sh and T47D-scr stable cell lines, and tested for p21Cip1 knockdown by western blotting. (C–E) The ability to generate growth-arrested cells was inhibited by p21Cip1 knockdown. Colonies were generated from respective cell lines, labeled with BrdU for 24 h, immunostained for BrdU, and slightly counterstained with hematoxylin to visualize BrdU+ and negative cells. Scale bar: 50 mm. (C). Individual colonies were manually counted for quantification of % BrdU cells. Each bar represents one colony (D). The silencing of p21Cip1 caused a significant increase (P = 0.0043) in % ratios of BrdU+ cells (E). Mean % BrdU+ cells (6 SD) values were calculated from data presented in (D). Error bars represent mean 6 SD. Tub.; a-tubulin. doi:10.1371/journal.pone.0011288.g002 demonstration of p21Cip1 knockdown in T47D-p21sh cells by western blot assay (Fig. 2B), both cell lines were plated under low- density plating conditions, colonies were grown for 10 days, and subjected to SABG and BrdU staining. It was not possible to quantify SABG+ cells in T47D-p21sh cells because they formed tight clusters in culture (data not shown). We therefore used BrdU staining as an alternative method for senescent cell quantification (Fig. 2C). Randomly selected colonies were counted for the number of BrdU+ and BrdU- cells (Fig. 2D). The T47D-scr cell line generated BrdU+ progeny at a rate of 48620% per colony (n = 18). Under the same conditions, T47D-p21sh cells displayed BrdU+ progeny at a rate of 65612% per colony (n = 18), with a significant (P = 0.0043) increase in the number of cells escaping terminal arrest (Fig. 2E). These results indicated that p21Cip1 was responsible, at least partly, for inducing the senescence observed in the progeny of T47D cells. The control of senescent cell progeny generation by an estrogen receptor As stated above, p21Cip1 is a downstream target of p53 for senescence, but T47D cells do not express wild-type p53 (Table S1). Estrogen inhibits p21Cip1 expression [20] by c-Myc-mediated repression [21], MYC gene being a direct target of ER complex [22]. We therefore tested whether ER could be involved in the senescence observed in T47D cells. The data shown in Fig. 3A indicates that T47D cells displayed nuclear ER immunoreactivity in their great majority, but some progeny was ER-. More interestingly, these ER- cells tended to be SABG+, suggesting that senescence occurred in T47D cells as a result of ER loss. Next, we tested whether experimentally modifying ER activity in T47D cells had any effect on senescence response. After plating at low-density clonogenic conditions, cells were grown in a regular cell culture medium that contained weakly estrogenic phenol red [23] for seven days in order to obtain visible colonies. The culture medium was then changed with phenol-free DMEM complemented with charcoal-treated FCS, grown for two more days, and then cultivated for four more days in the presence of E2 (1029 M), OHT (1029 M to 1026 M), or an ethanol vehicle as control. Colonies were subjected to SABG staining (Fig. 3B). Total and SABG+ cells were counted from 20 randomly selected colonies for each treatment (Fig. 3C). Colonies grown in a phenol-free charcoal-treated control medium complemented with an ethanol vehicle only displayed 31613% SABG+ cells. Complementing this medium with 1029 M PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11288