Senescence in Breast Cancer Figure 3. Generation of senescent cell progeny was controlled by the estrogen receptor-a. (A) SCP cells (T47D) expressed nuclear ER. Colonies were co-stained for senescence by SABG and for ER expression by immunoperoxidase. The MDA-MB-231 cell line was used as a negative control. (B–D) The production of senescent progeny in SCP cells was inhibited by estrogen (E2), but enhanced by tamoxifen (4OHT) treatment. After plating in low-density clonogenic conditions, T47D cells were grown in standard cell culture medium for seven days, followed by phenol-free DMEM complemented with charcoal-treated fetal calf serum for two days, and then cultivated for four days in the presence of E2, OHT, or an ethanol vehicle (control). Colonies were subjected to SABG staining (B). Total and SABG+ and SABG- cells were counted from 20 randomly selected colonies (C), and mean % SABG+ cells (6 SD) values were calculated (D). Error bars represent mean 6 SD. The inhibition of senescence by E2 and its activation by OHT was statistically significant when compared to ethanol-complemented control cells (P values 0.0093, 0.0002 and ,0.0001 for 1029 M E2, 1027 M OHT and 1026 M OHT, respectively). doi:10.1371/journal.pone.0011288.g003 E2 generated colonies with 17618% SABG+ cells. Senescence inhibition by E2 was nearly 50% and statistically significant when compared to the ethanol-complemented control cells (P = 0.0093). In contrast to E2, OHT provoked a dose-dependent increase in the proportion of SABG+ cells. At the maximum dose used (1026 M OHT), 90613% of colony-forming cells displayed a SABG+ signal (Fig. 3D), indicating that tamoxifen-mediated inactivation of ER can induce almost a complete senescence response in these cells (P,0.0001). The increase in senescence rate was also significant with 1027 M OHT (P = 0.0002). Our findings strongly suggested that the senescence observed in the SCP T47D cell line was due to a loss of expression and/or function of ER in a subpopulation of the progeny of these cells. For confirmation, we constructed ER-overexpressing stable clones from T47D cells. The thhree clones with the highest ER expression were selected. In addition, three clones with endoge- nous expressions of ER were selected from stable clones obtained with an empty vector (Fig. 4A). Progeny obtained from these six clones were tested by BrdU assay (Fig. S3). Randomly selected colonies (n = 10) from each clone were evaluated for total and BrdU+ number of cells (Fig. 4B). Consistently higher levels of BrdU+ cells were observed with clones ectopically expressing the ER protein (Fig. 4C). Overexpression of ER resulted in a significant increase in the BrdU+ progeny (P = 0.034). The protective effect of ER overexpression was not as important as the senescence-promoting effects of ER inhibition. This was not unexpected, since the parental cells used for the ER overexpres- sion studies were already expressing high levels of endogenous ER (Fig. 4A), displaying a baseline anti-senescence activity due to the serum estrogen and phenol red found in the cell culture medium. The close relationship between ER and senescence in the ER+ T47D cell line, and the highly effective treatment of ER+ breast tumors with tamoxifen, which induced senescence in our experimental model, suggested that senescence induction might be a relevant mechanism involved in anti-estrogen treatments. As fresh tumor tissues cannot be obtained from tamoxifen-treated patients for obvious ethical reasons, we analyzed untreated ER+ breast tumor samples for evidence of spontaneously occurring in vivo senescence. We screened a panel of 12 snap-frozen ER+ breast tumor tissues from 11 patients for senescence by an SABG PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11288