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The normal duct-lobular system of the breast is lined by two epithelial cell types, inner luminal secretory cells and outer contractile myoepithelial cells. We have generated comprehensive expression profiles of the two normal cell types, using immunomagnetic cell separation and gene expression microarray analysis. The cell-type specificity was confirmed at the protein level by immunohistochemistry in normal breast tissue. New prognostic markers for survival were identified when the luminal-and myoepithelial-specific molecules were evaluated on breast tumor tissue microarrays. Nuclear expression of luminal epithelial marker galectin 3 correlated with a shorter overall survival in these patients, and the expression of SPARC (osteonectin), a myoepithelial marker, was an independent marker of poor prognosis in breast cancers as a whole. These data provide a framework for the interpretation of breast cancer molecular profiling experiments, the identification of potential new diagnostic markers, and development of novel indicators of prognosis.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other ''immortalizing'' genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Introduction Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells.