Senescence in Breast Cancer Figure 1. Classification of breast cancer cell lines as senescent cell progenitor and immortal cell progenitor subtypes. (A) Examples of SABG staining for senescence of breast cancer cell line colonies obtained after plating at low-density clonogenic conditions. Breast cancer cell lines (Table S1) were plated to obtain a few hundred colonies with 1–2 weeks of cell culturing and were subjected to SABG assay, followed by counterstaining with nuclear fast red. T47D, ZR-75-1, MCF-7, CAMA-1 and BT-474 generated heterogeneous colonies composed of with SABG+ and SABG- cells (shown here), but also fully negative and/or fully positive colonies. All other cell lines produced only SABG- colonies (,5% SABG+ cells). Scale bar: 50 mm. (B) Classification of breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes by quantification of the ability to generate senescent progeny. Cell lines with a mean of SABG+ cells higher than 5% were termed SCP, and the other cell lines as ICP. Colonies that were generated and stained as described in (A) were counted manually to calculate % SABG+ cells. At least 10 colonies were counted for each cell line. Error bars represent mean 6 SD. (C) SABG+ senescent cells displayed terminal growth arrest. CAMA-1, T47D and T47D-iso23 colonies were generated as described in (A), labeled with BrdU for 24 h in the presence of freshly added culture medium, and subjected to SABG/BrdU double-staining. SABG+ cells are BrdU-, and vice versa. T47D-iso23 is a clone derived from T47D. Note that parental T47D and T47D- iso23 clones displayed similar staining features. doi:10.1371/journal.pone.0011288.g001 conditions, as senescent cells in permanent cell cycle arrest cannot incorporate BrdU under these conditions [16]. Co-staining of cells for SABG and BrdU from CAMA-1, T47D, and T47D-iso23 colonies provided clear indication that the great majority of SABG+ senescent cells were BrdU-, whereas non-senescent BrdU+ cells were usually SABG- (Fig. 1C). These findings indicated that SABG+ senescent cells were at the terminal differentiation stage with an irreversible loss of DNA synthesis ability. Our observations also indicated that SABG and BrdU tests could be used alternatively to identify senescent (SABG+/BrdU-) and immortal (SABG-/BrdU+) cells under our experimental conditions. Senescent cell progenitor phenotype association with p21Cip1 expression p16Ink4a and p21Cip1 (in a p53-dependent manner or indepen- dently) have been shown to be mediators of senescence arrest in different cells, including mammary epithelial cells [6,15,17,18,19]. We therefore analyzed the expression of p16Ink4a and p21Cip1 in the cell line panel. Heterogeneously positive nuclear p21Cip1 immunoreactivity was observed in four of the five SCP cell lines, but not in any of the seven ICP cell lines (Fig. 2A). The association of p21Cip1 expression with the SCP subtype was statistically significant (P = 0.01). We also compared the expression of p16Ink4a. Three of five SCP cell lines displayed heterogeneously positive immunostaining, whereas three of seven ICP cell lines displayed homogenously positive staining (Fig. S1). The difference of p16Ink4a expression between the two groups was not significant (P = 1). These observations indicated that the SCP phenotype was associated with p21Cip expression in breast cancer cell lines. To test whether p21Cip1 was directly involved in the senescence observed in SCP cells, we first performed p21Cip1/SABG staining in T47D-iso23 cells (hereafter termed T47D). p21Cip1, but not p16Ink4a staining, was associated with SABG staining (Fig. S2). Next, we generated two derivative cell lines following infection of T47D with lentiviral vectors encoding p21Cip1 shRNA (T47D- p21sh) or a scrambled control (T47D-scr). Following the PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11288