Figure 1.

HER4 expression regulates tamoxifen induced apoptosis of breast tumor cells. A, Suppression of HER4 expression by RNAi abolishes tamoxifen (5 μM) induced apoptosis of T47D and MCF-7 ERα(+) breast tumor cell lines. Cell lines were cultured in phenol red-free media containing 5% charcoal-stripped FBS 24 hrs prior to treatment with RNAi. Twelve hrs after RNAi treatment cells were left untreated or treated with 100 pM 17-β-estradiol (E2) alone or in combination with 5 μM 4-hydroxytamoxifen (TAM) for 24 and 48 hrs. Apoptosis was determined visually in a Nikon fluorescent microscope by staining the cells with DAPI and determining the percentage of cells with condensed nuclei (mean +/- SE of at least three experiments). Asterisks indicate significant differences in each data set as determined by student t-test. B, HER4 expression is suppressed in tamoxifen resistant MCF-7 cell variants. Western blot analysis of HER4 expression in cell lysates prepared from the parental MCF-7 cell line and three tamoxifen resistant variants TamR, LCC2, and HER2Δ16 each developed in an independent laboratory. Analysis of α-tubulin expression is included as a loading control. C, MCF-7 variants lacking HER4 expression are resistant to tamoxifen induced apoptosis. Each indicated cell line was cultured for 48 hrs in phenol red-free MEM containing 5% charcoal-stripped FBS and then treated for 24 hrs with 100 pM 17-β-estradiol (Estrogen) alone or in combination with 5 μM 4-hydroxytamoxifen (Tamoxifen). Apoptosis was determined visually in a Nikon fluorescent microscope by staining the cells with DAPI and determining the percentage of cells with condensed nuclei.