Effects of LTEE on Ah-responsiveness and gene expression in MCF-7 cells. (A) Ah-responsiveness decreases in control cells with increasing time in culture. At the passages indicated, control and LTEE cultures were exposed to 0.1% DMSO vehicle or 10 nM TCDD for 48 h, and CYP1 activity was determined using the EROD assay with normalization to total protein content. Significant differences between TCDD-induced EROD activities in control and LTEE cells are shown. Data are the means ± SE; n = 5; ***, P < 0.001. (B) AhR expression remains up-regulated in LTEE cells after withdrawal of E2 and is unaffected by short-term re-exposure to 1 nM E2. RNA from control (white bars) and LTEE cells (black bars) cultured for 49 passages, and from LTEE cultures from which E2 had been withdrawn for 35 days (LTEE (-E2), gray bars) was quantified by real-time PCR. LTEE (-E2) cells were re-exposed to E2 for 6 h as indicated. Primers specific for AhR, CYP1B1, ERα, PGR, and AIP cDNA were used in PCR reactions. Significant differences between treatment groups are shown. Data are the means ± SE; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001.