Ionizing radiation is the exogenous agent best proven to induce breast cancer. c-erbB2/neu amplification and overexpression are known to occur in breast cancer and are correlated with aggressive tumor growth and poor prognosis. We have developed simian virus 40-immortalized cell lines from normal human breast epithelial cells (HBECs) with luminal and stem-cell characteristics. In this study, we examined whether x-rays and a mutated neu oncogene are capable of inducing tumorigenicity in these cells. The results indicated that x-rays were effective in converting immortal non-tumorigenic HBECs to weakly tumorigenic cells that then could be transformed to highly tumorigenic cells by the neu oncogene. The in vitro growth of these tumorigenic cells was significantly faster than that of the parental non-tumorigenic cells in growth factor- and hormone-supplemented or -depleted media. The neu oncogene, however, had no tumorigenic effect on immortal non-tumorigenic cells. The expression of p185(c-erb82/neu) was elevated in neu-transduced immortal or weakly tumorigenic cell lines. However, only in the latter was p185(c-erbB2/neu) found to be phosphorylated at tyrosine residues. Thus, x-rays appear to induce a genetic alteration that confers weak tumorigenicity on immortal HBECs and interacts with p185(c-erbB2/neu) directly or indirectly to give rise to fast-growing tumors.

Although approximately two-thirds of breast cancers are estrogen receptor (ER)-positive, only a small proportion of epithelial cells in the mammary gland express the ER. The origin of the ER-positive breast cancers is unknown. Recently, we have developed a culture method to grow two morphologically and antigenically distinguishable types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. In this report, we studied the expression of ER in these two types of cells and their transformed cell lines. The results indicate that Type I HBEC with luminal and stem cell characteristics expressed a variant ER (approximately 48 kd) by Western blot analysis. This variant ER contains a deletion in the DNA binding domain (exon 2) as revealed by RT-PCR analysis. The lack of the DNA-binding domain of the variant ER was also confirmed by the ER-estrogen responsive element binding assay, as well as by the immunofluorescence staining of the ER using anti-ER antibodies which recognize either the C-terminal or N-terminal region. In contrast, Type II HBEC with basal epithelial phenotype are ER-negative. Simian virus 40 (SV40) transformed Type I and Type II HBEC lines also expressed the variant ER. Tumors formed in athymic nude mice by in vitro transformed tumorigenic Type I cell lines, however, expressed a high level of wild type ER which was undetectable in these cells grown in vitro before and after tumor formation. Thus, there appears to be a differential ER mRNA splicing between the in vitro and in vivo mileu.

Ionizing radiation is the exogenous agent best proven to induce breast cancer. c-erbB2/neu amplification and overexpression are known to occur in breast cancer and are correlated with aggressive tumor growth and poor prognosis. We have developed simian virus 40-immortalized cell lines from normal human breast epithelial cells (HBECs) with luminal and stem-cell characteristics. In this study, we examined whether x-rays and a mutated neu oncogene are capable of inducing tumorigenicity in these cells. The results indicated that x-rays were effective in converting immortal non-tumorigenic HBECs to weakly tumorigenic cells that then could be transformed to highly tumorigenic cells by the neu oncogene. The in vitro growth of these tumorigenic cells was significantly faster than that of the parental non-tumorigenic cells in growth factor- and hormone-supplemented or -depleted media. The neu oncogene, however, had no tumorigenic effect on immortal non-tumorigenic cells. The expression of p185(c-erb82/neu) was elevated in neu-transduced immortal or weakly tumorigenic cell lines. However, only in the latter was p185(c-erbB2/neu) found to be phosphorylated at tyrosine residues. Thus, x-rays appear to induce a genetic alteration that confers weak tumorigenicity on immortal HBECs and interacts with p185(c-erbB2/neu) directly or indirectly to give rise to fast-growing tumors.