ALTERED PHENOTYPIC CHARACTERISTICS OF T47D HUMAN BREAST CANCER CELLS AFTER PROLONGED GROWTH IN ESTROGEN‐DEFICIENT MEDIUM

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Abstract
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 10nm17β‐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, 10nmE2 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.

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