Characterization of a Receptor-negative, Hormone-nonresponsive Clone Derived from a T47D Human Breast Cancer Cell Line Kept under Estrogen-free Conditions

We have established an estrogen receptor- and progesterone receptor-negative, hormone-nonresponsive breast cancer cell line from a receptor-positive, hormone-responsive line grown under estrogen-free conditions. T47D breast cancer cells were cultured under estrogenized conditions (in phenol red-containing medium supplemented with whole fetal bovine serum) and cloned to produce line T47D:A18. The parental T47D line was also estrogen deprived (in phenol red-free medium supplemented with dextran-coated charcoal-treated fetal bovine serum) for more than 1 year and subsequently clone T47D:C4 was established. T47D:A18 was estrogen receptor and progesterone receptor positive as determined by both ligand binding assay analysis and enzyme immunoassay analysis. T47D:C4 cells were estrogen receptor and progesterone receptor negative and mRNA for these receptors was not detected. Incubation of hormone-responsive T47D:A18 cells with 17β-estradiol caused a 3-fold increase in cell growth over 8 days when compared to control. This stimulation of growth was completely inhibited by the anti-estrogens 4-hydroxytamox-ifen (0.1 µm) and ICI 164,384 (1.0 µm). Receptor-negative T47D:C4 cells were refractory to the effects of both 17β-estradiol and the antiestrogens. T47D:A18 cells grown under both estrogen-containing and estrogen-free conditions expressed low levels of transforming growth factor (TGF)-α and epidermal growth factor receptor mRNA. In the presence of estrogen, high levels of TGF-β1, mRNA were detected in T47D:A18 cells. These levels decreased when T47D:A18 cells were grown in estrogen-free media. Conversely, TGF-β2 mRNA was not detected in T47D:A18 cells cultured under estrogenic conditions; however, message was detected after the cells were cultured under estrogen-free conditions. T47D:C4 cells expressed low levels of TGF-α, epidermal growth factor receptor, TGF-β1, and TGF-β mRNA. These studies characterize a novel hormone-nonresponsive cell line which has been established from a hormone-responsive cell line grown under estrogen-free and drug-free conditions. Further analysis of these lines should provide valuable information concerning the development of antiestrogen-resistant breast cancer.