Altered phenotypic characteristics of T47D human breast cancer cells after prolonged growth in estrogen-deficient medium

T47D human breast cancer cells were cultured in estrogen-deficient media for up to 32 months and the resulting cell line (L(hE^-)) exhibited unique phenotypic and genotypic characteristics. Compared to low passage (L) cells, the L(hE^-) cells exhibited a significantly higher rate of proliferation, unique morphological features, advanced ploidy status and 5- to 10-fold higher levels of the estrogen receptor (ER) as determined by ligand binding and Western blot analysis. Sequence analysis of the DNA binding domain of the ER revealed a C→A transversion which resulted in a H513N amino acid change. Treatment of L cells with 10 nM 17β-estradiol (E2) resulted in a greater than two-fold increase in cell proliferation which was inhibited by tamoxifen, 4'-hydroxytamoxifen, ICI 164,384 and ICI 182,780. In contrast, 10 nM E2 caused a 70% decrease in growth of L(hE^-) cells and this antimitogenic activity was blocked by ICI 164,384 and ICI 182,780 but not by tamoxifen or 4'-hydroxytamoxifen. L(hE^-) cells were E2-responsive in transient transfection studies using a plasmid containing an estrogen-responsive element derived from the vitellogenin A2 gene promoter. These data show that the phenotypic and genotypic characteristics of L(hE^-) T47D cells resemble those described for ER-negative cell lines stably transfected with the ER.