Different sensitivity to tamoxifen in BT and BT/HerR cells. (A) Effect of tamoxifen and ICI on ER-mediated transcription in BT, BT/HerR0.2 and BT/HerR1.0 cells. Cells were transiently transfected with an ERE-containing luciferase reporter construct (ERE) in steroid-deprived medium, followed by a 16h incubation in the presence or absence of 1 nM 17β-estradiol (E2) with or without 1 μM tamoxifen (Tam) or 10nM ICI. Normalized luciferase activity from triplicate wells was expressed relative to the luciferase activity of each cell line in the absence of 17β-estradiol, tamoxifen and ICI. (B) Effects of tamoxifen and ICI on the proliferation in BT, BT/HerR0.2 and BT/HerR1.0 cells. Cells were deprived of steroid for 72 h and then incubated in the presence or absence of 1 nM 17β-estradiol with or without 1 μM tamoxifen or 10 nM ICI for 6 days. Cell proliferation from triplicate wells was expressed relative to optical density at 490 nm of each cell line in the absence of 17β-estradiol, tamoxifen and ICI. Results in A and B are expressed as mean ± SD of at least three separate experiments. *, P<0.05, compared to control in presence of 17β-estradiol. #, P<0.05, compared to control in absence of 17β-estradiol, tamoxifen and ICI. (C) Real time-PCR analysis of the estrogen responsive gene pS2 in BT and BT/HerR cells. BT and BT/HerR1.0 cells were deprived of steroid for 72 h and incubated in the presence or absence of 1 nM 17β-estradiol (E2) with or without 1 μM tamoxifen (Tam) for 24h. The expression of pS2 from triplicate wells was quantified and normalized to that of β-actin. Results are expressed as mean ± SD of at least three separate experiments. *, P<0.05, compared to BT cells. #, P<0.05, compared to control in absence of 17β-estradiol and tamoxifen. (D). Levels of ER and Her2 receptors in BT and BT/HerR cells. BT and BT/HerR1.0 cells were cultured in regular media to 80% confluence. Lysates of cells were prepared and tested for ER and HER2 content by immunoblot analysis. β-actin was used as a control for equal loading and transfer. Blots are representative of three experiments.