Characterization of normal breast epithelial cells in primary cultures: differentiation and growth factor receptors studies

Abstract

The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures from breast tissue biopsies from 42 mammoplasty patients. Cells were grown for up to 7 mo. in low calcium medium. HMEC cultures displayed heterogeneous growth patterns, according to the average doubling time of 44 +/- 6 h for 32 generations. Proliferation peaked at Day 30. HMEC maintained a normal karyotype and were organized in ductlike structures when cultured in collagen gel matrix. The cultures retained several phenotype traits of the epithelial lineage, including the expression of cytokeratins 18 and 19, specific mammary gland antigens, as shown by indirect HMEC immunostaining by the monoclonal antibodies DF3, EMA, 7B10, and 1BE12. Estrogen receptors were undetectable, whereas progesterone receptors were present at very low density. High-affinity cell surface receptors for epidermal growth factor (EGF) (Kd = 1.1 x 10(-10) M) were observed at a density of 50,000 to 100,000 sites per cell. Accordingly, [3H]thymidine incorporation in HMEC was optimally stimulated by EGF at concentrations of 10(-11) to 10(-10) M. HMEC were also seen to possess functional VIP receptors linked to the adenylate cyclase system, as we previously observed in seven human breast cancer cell lines. These results show that long-term cultures of HMEC provide useful models for studying the growth and differentiation of the normal human mammary gland, and the role of growth factors and hormones in these functions.