medium for seven to eight days. Then, cells were fed with phenol red-free DMEM (Gibco) supplemented with 5% charcoal-stripped FCS for 48 h, followed by two successive 48 h treatments with 1029 M estrogen (E2; 17b-estradiol; Sigma), 1026 to 1029 M 4- hydroxytamoxifen (4OHT; Sigma) or an ethanol vehicle, under the same conditions. Colonies were then subjected to a SABG assay. Each experimental condition was conducted in triplicate and experiments were repeated three times. Generation of estrogen receptor-overexpressing clones T47D-iso23 cells were transfected with the expression vector pCMV- ERa [12] or an empty vector, using FuGENE-6 (Roche). ER overexpressing and control clones were selected with 500 mg/ ml G418 for three weeks. Isolated single cell-derived colonies were picked and expanded in the presence of G418. Lentiviral infection and generation of p21Cip1 knockdown clones We used mission shRNA plasmid pLKO.1,-puro-p21 (NM_000389.2-640s1c1, Sigma) for p21cip1 knockdown experi- ments. The Control vector shRNA-pGIPz-SCR-puro and a helper packaging mix (Invitrogen) were also used. HEK293T was co- transfected with the appropriate vector and packaging mix, using the CalPhos Mammalian Transfection Kit (Clontech) and following the manufacturer’s instructions. After 48 h of culture, virus-containing culture media were collected, filtered, and used to infect T47D-iso23 cells. After 4 h of infection, stable cells were selected with 1 mg/ml puromycin for seven days. Nude mice tumorigenicity and in vivo senescence assays T47D and MDA-MB-231 cells (56106) were injected subcuta- neously into CD-1 nude mice (Charles River). Females (n = 5 for each cell line) and males (n = 4 for each cell line) were used. Tumor sizes were measured up to 47 days post-injection. In addition, four tumors from each cell line were analyzed for the presence of senescent cells by SABG staining, as described previously [10]. Cluster analysis The two-channel microarray data containing 8102 cDNA genes/clones generated by Sorlie et al. [2] were downloaded from the Stanford Microarray Database (SMD) (http://genome-www. stanford.edu/MicroArray/). In the downloading process, the ‘‘log (base 2) of R/G Normalized Ratio (median)’’ parameter was used for data filtering. We have median-centered expression values for each array. We selected arrays and genes with greater than 75% good data (representing the amount of data passing the spot criteria). Sixty-eight tissue samples were obtained according to this criterion and annotated with the subtypes described by the authors, found in the ‘‘Supplementary Information’’ of the data set in SMD. The expression values of ‘‘500 gene signature,’’ defined by the authors, were extracted from the data. Gene expression profiles of 31 breast cancer cell lines performed by Charafe- Jauffret et al. [13], using the whole-genome cDNA microarray Affymetrix HGU-133 plus 2, was obtained from the ‘‘Supplemen- tary Table’’ of the article. The authors filtered genes with low and poorly measured expression, and with low expression variation, retaining 15, 293 genes. After log transformation of the data, we median normalized the data arrays in R language, using the Bioconductor biostatistical package (www.r-project.org/ and www.bioconductor.org/). The ‘‘500 gene signature’’ tumor data [2] and the normalized breast-cancer cell line data [13] were combined with respect to probe IDs using a set of customized perl Senescence in Breast Cancer routines (source codes are available upon request). A set of 175 genes was common. ‘‘Median center’’ normalization of genes was done for the merged data set for the total samples. We performed unsupervised hierarchical clustering with the 99 samples (the 31 breast cell line [13] and 68 breast tumor [2] samples) by the pair- wise complete-linkage hierarchical clustering parameter, using the Gene-Pattern program. The Pearson correlation method was used for distance measurements. Clustering was visualized by java treeview, again using Gene-Pattern (http://www.broad.mit.edu/ cancer/software/genepattern/). Statistical analyses Significant differences were evaluated using unpaired Student’s t test for compared samples sizes of 10 or higher. Otherwise, one- tailed Fisher’s exact test was used with 262 tables; P,0.05 was considered statistically significant. On the graphical representation of the data, y-axis error bars indicate the standard deviation for each point on the graph. Results Classification of breast cancer cell lines as senescent-cell progenitor and immortal-cell progenitor subtypes Clonogenic assays have been successfully used to test the generation and self-renewal abilities of phenotypically distinct progeny of mammary stem/progenitor cells [14]. We previously applied this technique to test the ability of cancer cells to produce progeny with replication-dependent senescence arrest [10]. Cells were plated under low-density clonogenic conditions and cultivat- ed for one to two weeks until individual cells performed eight to ten PDs and generated isolated colonies composed of several hundred cells. This method permits tracing progeny generated by a few hundred cells under the same experimental conditions. We explored a panel of 12 breast cancer cell lines, composed of luminal (n = 7) and basal (n = 5) subtypes (Table S1). Cell lines formed two groups, according to the presence of senescent cells in isolated colonies. One group of cell lines generated colonies with high rates of senescence, while others did not produce appreciable amounts of senescent cells. Representative pictures of colonies subjected to the SABG assay are shown in Fig. 1A. The percent of SABG+ progeny was calculated by manual counting of at least 10 different colonies for each cell line. Colonies derived from five cell lines generated SABG+ cells at high rates (means: 5-40%) Senescence rates were negligible (means ,5%) in the progeny of the remaining seven cell lines. The first group, the senescent cell progenitor subtype, included T47D, BT-474, ZR-75-1, MCF-7, and CAMA-1 cell lines. The second group, the immortal cell progenitor subtype, included MDA-MB-453, BT-20, SK-BR-3, MDA-MB-468, HCC1937, MDA-MB-231 and MDA-MB-157 (Fig. 1B). In order to verify whether the occurrence of senescent cells in the SCP group was intrinsic to each cell line or due to the presence of a side population, we generated clones from the T47D (n = 20) cell line, and subjected them to the SABG assay at different intervals. All clones acted similarly to the parental T47D cell line with similar rates of SABG+ progeny. No clone gained the ICP phenotype. More importantly, none of the clones tested over a long period of time (.60 PDs) entered full senescence (data not shown), unlike normal mammary epithelial cells that undergo two stages of senescence arrest over a period of ,20 PDs [7]. The SABG assay can provide false-positive responses, especially when cells remain under conflu- ence for a long period [15]. Although all our tests used low-density clonogenic conditions, we wanted to confirm the senescence arrest by a long-term (24 h) BrdU labeling assay under mitogenic PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11288