The long-term objective of this proposal is to understand the molecular mechanisms underlying the biological responses to dioxin (2,3,7,8- tetrachlorodibenzo-p-dioxin; TCDD) exposure. TCDD is the prototype of a large group of halogenated aromatic compounds to which humans are environmentally exposed. In animal models, dioxin produces a variety of apparently unrelated toxic effects, including developmental teratogenesis, skin, liver and squamous cell hyperplasia, splenic, thymic and testicular atrophy, immunosuppression, wasting syndrome, and death. Dioxin is poorly, if at all, metabolized; its biological half- life in humans is 7 to 10 years and many of its biological effects are likely to result from long-term, sustained exposure. At the molecular level, dioxin is a ligand for the aromatic hydrocarbon (Ah) receptor (AHR) which, as a heterodimer with the Ah receptor nuclear translocator protein ARNT, mediates the transcriptional activation of genes in the CYP1 family of cytochrome P450 monooxygenases. Our laboratory has shown that dioxin also induces the expression of the immediate-early competence genes fos and jun, and we have characterized the mechanisms responsible for TCDD-dependent upregulation of transcription factor AP-1, a FOS/JUN dimer, and the role of the Ah receptor in this process. AP-1 activation drives quiescent cells into the cell cycle, yet TCDD exposure does not always lead to cell cycle progression. Depending on cell type and lineage, TCDD-exposed cells may differentiate, arrest or die from apoptosis, suggesting that TCDD perturbs the functions of a second cell cycle regulatory component. The proposed research is based on our experimental evidence indicating that the activated Ah receptor binds to the retinoblastoma protein (RB), a key cell cycle regulatory factor. The goal of the proposed experiments is to test the hypothesis that a TCDD-activated AHR sequesters RB, derails expression of cyclin- dependent kinases and their inhibitors, and causes premature entry into S phase. Major objectives of this work are, (1) to identify at the molecular level the AHR domains involved in AHR/RB interactions, (2) to analyze AHR/RB interactions in mammalian cells exposed to TCDD, (3) to determine the effect of TCDD exposure on the function of cyclin- dependent kinase, and cdk inhibitors, and (4) to analyze the consequences of TCDD exposure on cell cycle progression and on the expression of S phase-specific genes. Results from these experiments will be crucial for our understanding of the long-range biological consequences of exposure to dioxin and to other organochlorinated compounds and will help formulate an adequate rationale to deal with health problems arising from an ever-increasing exposure to these environmental agents.
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