Dual immunofluorescence labeling with cell-specific markers localizes BRCA1 in both basal and luminal epithelial cells in primary outgrowth from noncancerous mammary ductal and alveolar preparations

Abstract.

The role played by either of the two differentiated mammary epithelial cell types in human breast cancer progression is currently not defined. This work addresses the question of whether the mammary tumor suppressor gene product BRCA1 is localized in basal and/or luminal epithelial cells in noncancerous outgrowth cultured from breast organoids. Primary epithelial cell outgrowths from ductal and alveolar preparations were directly employed to facilitate small-scale analysis under conditions closely approximating intact tissue. BRCA1 immunofluorescence was detected for the most part in cell nuclei of the epithelial outgrowth when using confocal microscopy. Nuclear staining was punctate in the cells with higher labeling intensity. Only minimal nonspecific staining was observed with mouse IgG as a negative primary antibody control or with primary antibody against the cell membrane receptor ErbB2, reported to be expressed in breast cancer, but was either not detectable or weakly expressed in normal breast tissue. Dual labeling was used to distinguish which epithelial cell type(s) stains for BRCA1. Primary monoclonal antibody against vimentin was used to identify basal cells, while antibody against cytokeratin 19 was used to identify luminal cells. Monoclonal antibody against BRCA1 was used for colabeling with each of these markers. Epifluorescence microscopy revealed BRCA1 immunoreactivity in both basal and luminal interphase cells. BRCA1 immunofluorescence was diffusely located about the chromosome mass during mitosis.