Molecular mechanism of cytochrome P4501A1 expression.

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DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs) are present in cigarette smoke, particulate matter, and in charcoal broiled meats. The cytochrome P450 (CYP)1A enzymes play pivotal roles in the activation of PAHs to metabolites that are interact covalently with DNA, a critical event in the initiation of carcinogenesis. The central hypothesis of this application is that CYP1A1 and CYP1A2 enzymes play reciprocal roles in PAH-mediated carcinogenesis, and that a CYP1A2-dependent MC metabolite(s) contributes to the formation of sequence- specific DNA adducts on the regulatory regions of the CYP1A1 promoter [e.g., AHR response elements (AHREs)], leading to a novel mechanism by which MC-DNA adduct(s) will suppress CYP1A1 gene expression. The following Specific Aims are proposed. 1. To determine the mechanistic role of CYP1A1 and 1A2 enzymes in PAH-mediated lung cancers. This aim has two sub-aims: (i) To test the hypothesis that mice lacking the gene for Cyp1a2 will be more susceptible, and those lacking the gene for Cyp1a1 will be less susceptible to PAH-induced lung carcinogenesis and tumorigenesis than similarly exposed WT mice. (ii) To test the hypothesis that humanized CYP1A1 mice will be more susceptible to lung carcinogenesis and tumorigenesis than WT mice, while humanized CYP1A2 mice will be less susceptible. 2. To determine the molecular mechanisms of sustained induction and suppression of hepatic and pulmonary CYP1A1 by MC in vivo. This aim has two sub-aims: (i) To test the hypothesis that hepatic CYP1A2 mechanistically contributes to the suppression of sustained hepatic and pulmonary CYP1A1 by MC. (ii) To test the hypothesis that MC elicits persistent human CYP1A1 induction by sustained transcriptional activation of the corresponding promoter, and that hepatic CYP1A2 will suppress persistent induction of hepatic and pulmonary CYP1A1 expression in transgenic humanized mice expressing the human CYP1A1 (hCYP1A1-luc or hCYP1A1-GFP) on Cyp1a1- or Cyp1a1/1a2-null backgrounds. 3. To determine the molecular mechanisms of suppression of pulmonary CYP1A1 induction. We will test the hypothesis that CYP1A2-derived MC metabolites contribute to suppression of pulmonary cells via sequence-specific DNA adducts on the AHREs of the CYP1A1 promoter. This aim has two sub-aims. (i) To test the hypothesis that cells transfected with plasmids containing CYP1A2-derived DNA adducts will display suppression of CYP1A1 transcription in human pulmonary cells (e.g., A549, BEAS-2B) in vitro. (ii) To test the hypothesis that exposure of human lung cells overexpressing CYP1A2 to MC will display attenuation of sustained CYP1A1 induction. The long-term objectives are to: (i) define the molecular mechanisms of regulation of CYP1A1 gene expression by PAHs, (ii) elucidate the possible role of gene- specific DNA adducts in molecular regulation of CYP1A1, and (iii) develop rational strategies for the prevention/treatment of human cancers caused by environmental chemicals.