The Role of Aryl Hydrocarbon Receptor and Crosstalk with Estrogen Receptor in Response of Breast Cancer Cells to the Novel Antitumor Agents Benzothiazoles and Aminoflavone
Figure 4
(a) 5F 203 induces binding to the XRE sequence of CYP1A1. MCF-7 and AHR100 cells were transfected with XRE-luciferase (pTX.Dir.) or pT81. A schematic of the respective construct is shown below the panel. Transfected cells were treated with DMSO, TCDD (10 nM), or 5F 203 (1 μM) for 9 h. XRE-luciferase activity was determined normalizing to the amount of Renilla reniformis luciferase. The values are expressed as luciferase levels relative to control. (b) 5F 203 induces protein/DNA complexes on the XRE sequence of the CYP1A1 promoter. Nuclear extracts (20 mg) prepared from MCF-7 cells treated with 0.1% DMSO control (lane 1), TCDD (10 nM, 1 h) (lane 2), or 5F 203 (1 μM, 1 h) (lane 3) were incubated with labeled XRE sequence derived from the CYP1A1 promoter for 10 min at room temperature. Free DNA and bound DNA were separated as described. In competition experiments, nuclear extracts from MCF-7 cells treated with 5F 203 (1 μM, 1 h) were incubated with 4 μg of anti-AhR antibody (lane 6), 100-fold excess of unlabeled XRE oligonucleotide (lane 7), 100-fold excess of unlabeled Sp1 oligonucleotide (lane 8), or 4 μg of IgG antiserum (lane 9). Protein/DNA complexes from AHR100 cells were resolved in the same gel. Nuclear extracts from these cells (20 mg) treated with DMSO (lane 4) or 5F 203 (1 μM, 1 h) (lane 5) were incubated with radioactive XRE and resolved by the same procedure [17].
