c Putative protein sequence alignment of the splice variants. all lines. These results demonstrate that expression of the characteristic 35 kDa BRMS1 protein is not sufficient to prevent metastasis. The differential expression of alternative splice variants suggests caution should be taken when evaluating mRNA in clinical samples. was introduced [7-9]. Further mechanistic studies have exhibited that BRMS1 regulates the expression of multiple genes that have been linked to metastasis including osteopontin (into cells that have little to no detectable levels of endogenous BRMS1. Clinically, loss of BRMS1 protein has been correlated with reduced disease-free survival when patient samples were stratified by loss of estrogen or progesterone receptor (ER, PR) or expression of HER2 . Consistent with the protein data, mRNA correlated with PR and Ambroxol HCl loss of HER2 and loss of mRNA correlated with poor prognosis . However, mRNA studies have not been completely consistent as some groups found no correlation with breast cancer markers and mRNA expression was associated with shorter disease-free survival . The current study was undertaken to evaluate the expression level of BRMS1 in isogenic cell lines representing normal breast epithelium, pre-malignancy, ductal carcinoma in situ (DCIS), and metastasis. We demonstrate that although BRMS1 is present and localized to the nucleus, its expression is not sufficient to prevent metastasis, suggesting that other protein interactors are important Mouse monoclonal to TAB2 for BRMS1 mediated metastasis suppression. Materials and methods Cell lines and cell culture The MCF10 breast cancer progression model system was a kind gift from Drs. Fred Miller and Herbert Soule (Karmanos Cancer Institute, Detroit, MI). The immortalized breast epithelial cell line MCF10A, pre-malignant MCF10AT, comedo ductal carcinoma in situ MCF10DCIS.com, and two metastatic clones MCF10CAa.1 and MCF10CAd.1 were cultured in a mixture (1:1, v/v) of Dulbeccos modified Eagles medium and Hams F12 medium supplemented with 5% horse serum, 2 mM l-glutamine (Invitrogen), and 0.02 mM non-essential amino acids (Mediatech, Herndon, VA) without antibiotics or antimycotics. Additional supplements for the MCF10A and MCF10AT cell lines were 10 ng/ml EGF, 500 ng/ml hydrocortisone, 100 ng/ml cholera toxins, and 10 g/ml insulin (Sigma). All cultures were confirmed unfavorable for spp. contamination using a PCR-based test (TaKaRa, Shiga, Japan). Cells were maintained on 100 mm Corning tissue culture dishes at 37C with 5% CO2 in a humidified atmosphere. When cultures reached 80-90% confluence they were passaged using a solution of 0.05% Trypsin/EDTA (1) (Invitrogen). Metastasis assays Spontaneous and experimental metastasis Ambroxol HCl assays with the MCF10CA clones were performed as described previously [3, 4], with the exception that 2 105 cells were injected i.v. into the lateral tail vein. For the MCF10A and MCF10AT cell lines, 2 106 cells were injected into the mammary fat pad. Ten mice per experimental group were used. Orthotopic tumor size was evaluated weekly by taking orthogonal measurements and plotted by the mean tumor diameter (MTD = (diameterx) (diametery)0.5). All animals were maintained under the guidelines of the National Institutes of Health and the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Food and water were provided ad libitum. Antibodies, co-immunoprecipitation, and western blotting Nuclear and cytoplasmic fractions were obtained according to the manufacturers protocol (NE-PER kit, Thermo Fisher Scientific). Co-immunoprecipitation and western blotting was performed as previously described . Monoclonal BRMS1 antibodies 1a5.7 and 3a1.21 were described previously [13, 14, 17]. Other antibodies used in this study were purchased as indicated: anti-ARID4A clone LY11 (Upstate Biotechnology, Lake Placid, NY) and anti-lamin A/C (Cell Signaling Technology, Danvers, MA). PCR amplification of genomic DNA Genomic DNA was collected using Trizol (Invitrogen) according Ambroxol HCl to manufacturers protocol. Primers to the individual exons were used for PCR (see Fig. 3b) with GoTaq (Promega). The products were visualized on a 2% agarose gel and sequenced. Open in a separate window Fig. 3 Genomic sequence of in cell lines reveals no mutations. a Genomic DNA was collected from the genetically related MCF10A-derived cell lines. PCR products were visualized on.