Epithelial cells isolated from mammary gland cells undergo two successive senescence states in cell culture, termed ‘‘stasis’’ and ‘‘agonescence’’ [6,7]. In contrast to normal mammary epithelial cells, established breast cancer cells are immortal by definition. They may owe this phenotype of immortalization to genetic and epigenetic inactivation of senescence checkpoints and reactivation of telomerase reverse transcriptase expression [7]. Either in relation to these changes or independently, breast cancer cells may also present a stem/progenitor phenotype that is less subjected or resistant to senescence barriers. However, the abundance of non-tumorigenic and differentiated cells both in breast tumors and cell lines strongly suggests that replicative immortality cannot be assigned to all cells within a tumor or a cancer cell line, and that spontaneous senescence after a limited number of population doublings (PD) is likely to occur. If this hypothesis is correct, then the rate of generation of senescent progeny may reflect the potential of a cancer stem/progenitor cell to produce terminally differentiated progeny. We tested this hypothesis using a panel of luminal and basal-like breast cancer cell lines (Table S1). Although a single cell line is not representative of breast tumor heterogeneity, a panel of cell lines might reproduce the heterogeneity that is observed in primary breast tumors, albeit with some limitations [5,8]. Therefore, we hoped that in vitro studies with a panel of cell lines might help to better understand breast tumor heterogeneity. Our senescence tests allowed us to classify breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. We also show that senescent progeny are observed exclusively in ER-positive cells, as a result of ER inactivation, partly mediated with p21Cip1 protein. The ability to produce senescent progeny was associated with the ability to produce luminal-like and myoepithelial-like progeny from stem/ progenitor-like cells. In contrast, most of the cell lines lacking senescent progeny were also unable to generate differentiated progeny. Finally, we show that SCP-subtype cells cluster with luminal A and ‘‘normal-like’’ breast tumor types and are less tumorigenic, whereas ICP-subtype cell lines cluster with luminal B and basal-like tumor types and are more tumorigenic. Materials and Methods Ethics Statement We used archival tumor samples remaining from a previous study by BB and IGY described in Gur-Dedeoglu et al. [9], for which the use of the tissue material was approved by the Research Ethics Committee of Ankara Numune Research and Teaching Hospital (decision date: 04/07/2007). The tumor samples in this study were used anonymously. All animals received care according to the Guide for the Care and Use of Laboratory Animals. All animal experiments have been pre-approved by the Bilkent University Animal Ethics Committee (Decision No: 2006/1; Decision date: 10/5/2006). Clinical samples and cell lines Freshly frozen tumor specimens were collected at Ankara Numune Hospital. Breast cancer cell lines used in this study were obtained from ATCC (http://www.atcc.org) and listed in Table S1. Cell line authenticity was verified by short tandem repeat profiling, as recommended by ATCC (Dataset S1). Isogenic clones from the T47D (n = 20) cell line were obtained from single cell- derived colonies. Briefly, cells were plated in 96-well plates to obtain single colonies in fewer than 70% of the wells. Isolated colonies were then transferred to progressively larger wells, and to T25 flasks. Clones were subcultivated weekly at 1:4 dilution ratios, Senescence in Breast Cancer and maintained in culture for 25–30 passages to reach .60 PD before testing. Primary antibodies The following antibodies were used: anti-CD44 (559046; BD Pharmingen), anti-CD24 (sc53660; Santa Cruz,), anti-ASMA (ab7817; Abcam), anti-CK19 (sc6278; Santa Cruz,), anti-p21Cip1 (OP64; Calbiochem), anti-p16Ink4a (NA29, Calbiochem), anti-ERa (sc8002; Santa Cruz). Low-density clonogenic assays Cells were seeded as low-density on coverslips in six-well plates (500–2000 cells, according to plating efficiency) and allowed to grow in DMEM supplemented with 10% fetal calf serum (FCS), with medium change every three days, until they formed colonies of a few hundred cells. Depending on the cell line, this took one to two weeks. For bromodeoxyuridine (BrdU) incorporation assays, cells were labeled for 24 h prior to immunocytochemistry, as described previously [10]. Immunocytochemistry For simple immunoperoxidase assays, cells were fixed with cold methanol for five minutes, then blocked with 10% FCS in phosphate-buffered saline (PBS) for 1 hour. This was followed by incubation with a primary antibody for 1 h. Cells were then washed with PBS three times and subjected to immunostaining using the Dako-Envision-dual-link system and the liquid diaminobenzidine (DAB) substrate chromogen system (Dako, CA, USA), according to the manufacturer’s instructions. Hematoxylin was used as a counter-stain when the visualization of cells was necessary. For SABG-immunoperoxidase co-staining studies, unfixed cells were first subjected to SABG assay, and then fixed prior to immuno- staining assays. Hematoxylin counter-staining was omitted for co- staining experiments, unless cells were negative for SABG staining. Immunoblot analyses Cell pellets were incubated in an NP-40 lysis buffer containing 50 mM Tris–HCl, pH 8.0, 250 mM NaCl, 0.1% Nonidet P-40, and a protease inhibitor cocktail (Roche) for 30 minutes in a cold room. Cell lysates were then cleared by centrifugation, and a Bradford assay was performed to quantify their protein concen- tration. 30 mg of protein was denatured and resolved by SDS- PAGE using 10% or 12% gels. The proteins were then transferred to the PVDF or nitrocellulose membranes. Membranes were treated for 1 h with a blocking solution of TRIS-buffered saline containing 0.1% Tween-20 and 5% non-fat milk powder (TBS-T) and probed with a primary antibody for 1 h. Next, membranes were washed three times with TBS-T and incubated with an HRP- conjugated secondary antibody for 1 h. Immunocomplexes were then detected by an ECL-plus (Amersham) kit on the membrane. a-tubulin was used as an internal control. SABG assay and BrdU/SABG co-staining SABG activity was detected as described [11], except that cells were counterstained with eosin or nuclear fast red following SABG staining. For BrdU/SABG co-staining, cells were first labeled with BrdU (10 mg/ml) for 24 h in a freshly added culture medium as described [10]. Next, cells were subjected to a SABG assay, fixed in 70% methanol, and subjected to BrdU immunostaining. Estrogen and tamoxifen treatment Cells were seeded under low-density clonogenic conditions onto coverslips in six-well plates, and cultivated in a standard culture PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11288