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A novel human mammary epithelial cell line, HME348, was established from benign breast tissue from a 44-year-old germ-line BRCA2mutation carrier with a history of stage 1 breast cancer. Mutation analysis showed that the patient had a known 6872del4 BRCA2heterozygous mutation. The human mammary epithelial cells passaged in culture exhibited cellular replicative aging as evidenced by telomere shortening, lack of telomerase activity, and senescence. Ectopic expression of telomerase (hTERT) reconstituted telomerase activity in these cells and led to the immortalization of the cells. When grown on glass, the majority of immortalized HME348 cells expressed ESA and p63 with a small population also expressing EMA. In three-dimensional Matrigel®culture, HME348 cells formed complex branching acini structures that expressed luminal (EMA, CK18) and myoepithelial (p63, CALLA, CK14) markers.Three clones derived from this culture were also p63+/ESA+/EMA+/−on glass but formed similar acinar structures with both luminal and myoepithelial cell differentiation in Matrigel®confirming the mammary progenitor nature of these cells. Additionally, the experimentally immortalized HME348 cells formed acini in cleared mammary fat pads in vivo. As this is the first report establishing and characterizing a benign human mammary epithelial cell line derived from a BRCA2patient without the use of viral oncogenes, these cells may be useful for the study of BRCA2function in breast morphogenesis and carcinogenesis.A novel human mammary epithelial cell line, HME348, was established from benign breast tissue from a 44-year-old germ-line BRCA2mutation carrier with a history of stage 1 breast cancer. Mutation analysis showed that the patient had a known 6872del4 BRCA2heterozygous mutation. The human mammary epithelial cells passaged in culture exhibited cellular replicative aging as evidenced by telomere shortening, lack of telomerase activity, and senescence. Ectopic expression of telomerase (hTERT) reconstituted telomerase activity in these cells and led to the immortalization of the cells. When grown on glass, the majority of immortalized HME348 cells expressed ESA and p63 with a small population also expressing EMA. In three-dimensional Matrigel®culture, HME348 cells formed complex branching acini structures that expressed luminal (EMA, CK18) and myoepithelial (p63, CALLA, CK14) markers.Three clones derived from this culture were also p63+/ESA+/EMA+/−on glass but formed similar acinar structures with both luminal and myoepithelial cell differentiation in Matrigel®confirming the mammary progenitor nature of these cells. Additionally, the experimentally immortalized HME348 cells formed acini in cleared mammary fat pads in vivo. As this is the first report establishing and characterizing a benign human mammary epithelial cell line derived from a BRCA2patient without the use of viral oncogenes, these cells may be useful for the study of BRCA2function in breast morphogenesis and carcinogenesis.

Breast cancer is the most commonly diagnosed cancer in women worldwide and consequently has been extensively investigated in terms of histopathology, immunochemistry and familial history. Advances in genome-wide approaches have contributed to molecular classification with respect to genomic changes and their subsequent effects on gene expression. Cell lines have provided a renewable resource that is readily used as model systems for breast cancer cell biology. A thorough characterization of their genomes to identify regions of segmental DNA loss (potential tumor-suppressor-containing loci) and gain (potential oncogenic loci) would greatly facilitate the interpretation of biological data derived from such cells. In this study we characterized the genomes of seven of the most commonly used breast cancer model cell lines at unprecedented resolution using a newly developed whole-genome tiling path genomic DNA array. Breast cancer model cell lines MCF-7, BT-474, MDA-MB-231, T47D, SK-BR-3, UACC-893 and ZR-75-30 were investigated for genomic alterations with the submegabase-resolution tiling array (SMRT) array comparative genomic hybridization (CGH) platform. SMRT array CGH provides tiling coverage of the human genome permitting break-point detection at about 80 kilobases resolution. Two novel discrete alterations identified by array CGH were verified by fluorescence in situhybridization. Whole-genome tiling path array CGH analysis identified novel high-level alterations and fine-mapped previously reported regions yielding candidate genes. In brief, 75 high-level gains and 48 losses were observed and their respective boundaries were documented. Complex alterations involving multiple levels of change were observed on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Furthermore, alignment of whole-genome profiles enabled simultaneous assessment of copy number status of multiple components of the same biological pathway. Investigation of about 60 loci containing genes associated with the epidermal growth factor family (epidermal growth factor receptor, HER2, HER3 and HER4) revealed that all seven cell lines harbor copy number changes to multiple genes in these pathways. The intrinsic genetic differences between these cell lines will influence their biologic and pharmacologic response as an experimental model. Knowledge of segmental changes in these genomes deduced from our study will facilitate the interpretation of biological data derived from such cells.