2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells.

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.

Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors.

A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.

Origin of cells cultured in vitro from human breast carcinomas traced by cyclin D1 and HER2/neu FISH signal numbers.

BACKGROUND: We have developed and optimized a feeder layer method for cultivation of normal human mammary luminal cells and found it suitable for establishing more than 150 primary cultures from individual human breast carcinomas. Here, we investigated if the malignant cells that are in situ additionally characterized by increased numbers of proto-oncogenes can be traced by the FISH method in the ex vivo-derived cultures. MATERIALS AND METHODS: Paraffin sections from 9 tumors with derived cell cultures kept frozen in our cell bank were screened by FISH for cyclin D1 (CCND1) and c-erb-B2 (HER2/neu) proto-oncogene signal numbers. RESULTS: In 6 tumors (5 primary tumors, 1 cutaneous metastasis), increased numbers of FISH signals were found in 55-99% of cells. Then, the relevant cell cultures were FISH screened; in cell populations maintained for up to 2-6 passages in vitro the incidence of cells with increased FISH signals was found to be low (2-16%). Moreover, the cells with multiplied signals that survived more than one passage in vitro were evidently unable to divide further. However, in all 6 tumors at least a small fraction of cells displaying only two signals of CCND1 or HER2/neu genes was identified directly in invasive tumor structures in the vicinity of cells with multiple signals. CONCLUSION: Our findings suggest that these invasive tumor cells displaying only two proto-oncogene signals were most probably involved in the initiation and propagation of ex vivo tumor-derived primary cell cultures.