These cells are characterized by deficiency in gap junctional intercellular communication, the abil ity to form budding and ductal organoids on Matrigel, the expression of luminal epithelial cell markers, estrogen receptor alpha and the stem cell pluri potency gene Oct 4, similar to the phenotypes of breast carcinoma meanwhile cells such as the MCF 7 cell line. Furthermore, Type 1 HBECs were highly susceptible to telomerase activation and immortalization following SV40 large T antigen transfection which is known to inacti vate p53 and Rb as well as to transactivate a CCAAT box binding factor. Both changes have been reported for human breast cancer. These immortal cells can be further transformed to weakly tumorigenic and highly tumorigenic cells by successive X ray irradiation and ecto pic expression of C erbB2/neu.

Recently, we found that M13SV1R2 cells became non tumorigenic after grow ing in a growth factor/hormone deprived medium for 10 passages. Unlike M13SV1R2 cells, these R2d cells contain CD44 CD24 cells pre viously identified as breast cancer stem cells and were responsive to estrogen for cell Inhibitors,Modulators,Libraries growth and tumor develop ment. In this study, we developed M13SV1R2N1 under the same growth factor/hormone deprived condi tion. This provides an oppor tunity to analyze unambiguously the effects of HER2 on tumor development and gene expressions underlying tumorigenic mechanisms by comparative study of R2d and R2N1d cells with homogeneous genetic background under same cell culture condition.

Results Development of R2N1d cells In order to investigate HER2 effect on Inhibitors,Modulators,Libraries tumor develop ment and gene expression, M13SV1R2N1 cells were cul tured under similar condition as R2d cells, i. e. in MSU Inhibitors,Modulators,Libraries 1 medium without growth factors/hormones except 5% FBS for more than 10 passages. Morphologically, R2N1d cells were more heterogeneous, i. e. increased intercellular separa tion and scattering of cells and the formation Inhibitors,Modulators,Libraries of pseudo podia. Similar to parental M13SV1R2N1 cells, R2N1d cells expressed ERa and HER2 by immu nocytochemical study. R2N1d cells expressed stem cell related genes The Notch signaling pathway is implicated in the regula tion of cell differentiation and self renewal of mammary stem cells. Over expression of the active form of Notch 4 inhibits differentiation of breast epithelial cells.

Musashi 1 is a positive regulator of Notch signal ing, and both Msi 1 and Notch 1 are key regulators of asymmetrical cell division in human breast epithelial stem cells. We have examined whether R2N1d cells express genes involved in stem cell function and self renewal, Inhibitors,Modulators,Libraries i. e. Oct 4 and Notch pathway selleck chem inhibitor by flow cytometric analysis. The results show that R2N1d cells expressed the stem cell pluripotency gene, Oct 4, and the Notch pathway related genes, Notch1, Notch4 and Msi1. The expression of these genes in R2N1d was similar to R2d cells.