Like the steroid and thyroid hormone-receptor superfamily, the aryl hydrocarbon receptor (AhR) acts as a nuclear ligand-responsive transcription factor (LTF). The signal transduction mechanism associated with the liganded AhR is a complicated multistep process which occurs in both the cytosolic and nuclear cellular compartments. Based on previous studies and preliminary results, 4 different structural classes of antagonists will be synthesized and investigated and these include alpha- naphthoflavone (alphaNF), 6-substituted-1,3,8-trichlorodibenzofurans (triCDFs), 4'-substituted flavones (F), and 6- and 7-substituted benzocoumarins (Experiment l). The effects of the 4 different structural classes of antagonists on the TCDD-mediated transformation of the AhR will be investigated by determining the kinetics of binding to a synthetic 32P- labeled dioxin responsive element (DRE, 26-mer-duplex) using the gel mobility shift assay. The stoichiometry of the formation of nuclear AhR complexes will be measured in the 4 model cells lines using [3H]TCDD as a ligand and representative model antagonists. This approach will allow the quantitative determination of (i) the total nuclear DRE-binding components (gel shift assay), (ii) the levels of the [3H]TCDD-AhR complex (velocity sedimentation analysis), and (iii) the levels of nuclear AhR complex liganded with the antagonists [(i) - (iii)]. This study, coupled with the parallel structure-activity investigations with substituted analogs, will delineate the role of the nuclear AhR-antagonist complexes as inhibitors of TCDD-induced gene transcription (note: this will include Northern analysis of induced CYP1A1, aldehyde-3-dehydrogenase, plasminogen activator inhibitor-1 -and -2 mRNAs). The final component of Experiment 2 will characterize possible structural differences between AhR complexes liganded with antagonists or agonists. It has been shown that nuclear AhR complexes liganded with some model antagonists are formed in the model cell lines. Experiment 3 will utilize plasmids containing variable length DNA sequences from the 5'-flanking region of the CYP1A1 gene as probes for understanding the molecular mechanisms of AhR antagonists in the 4 model cell lines. Experiment 4 will test the hypothesis that the effects on chromatin structure by the nuclear AhR complex liganded with antagonists are different from those caused by the nuclear TCDD-AhR complex. TCDD- induced gene transcription may also be inhibited at the gene level by trans-acting repressor proteins which bind to cis-acting genomic elements. Preliminary studies in this laboratory have identified 3 cell lines which express the nuclear AhR complex but are not Ah-responsive. The proposed studies in Experiment 5 will utilize a series of plasmid constructs from the 5'-region of the human. CYP1A1 gene to probe the molecular biology of the negative regulation of AhR-mediated gene transcription in the MDA MB 2331, PEO1 and PEO6 human cancer cell lines.
Showing the most recent 10 out of 39 publications
