Molecular mechanism of cytochrome P4501A1 expression.

描述（由申请人提供）：多环芳烃（PAH）存在于香烟烟雾、颗粒物和木炭烤肉中。细胞色素P450（P450）1A酶在多环芳烃活化为与DNA共价结合的代谢产物中起着关键作用，这是启动致癌作用的关键事件。本申请的中心假设是CYP 1A 1和CYP 1A 2酶在PAH介导的致癌作用中起相互作用，并且CYP 1A 2依赖性MC代谢物有助于在CYP 1A 1启动子的调节区上形成序列特异性DNA加合物[例如，AHR反应元件（AHRE）]，导致MC-DNA加合物抑制CYP 1A 1基因表达的新机制。提出了以下具体目标。1.确定CYP 1A 1和1A 2酶在PAH介导的肺癌中的机制作用。这一目标有两个次级目标：（i）检验缺乏Cyp 1a 2基因的小鼠对PAH诱导的肺癌发生和肿瘤发生的易感性高于类似暴露的WT小鼠，而缺乏Cyp 1a 1基因的小鼠对PAH诱导的肺癌发生和肿瘤发生的易感性低于类似暴露的WT小鼠的假设。(ii)检验人源化CYP 1A 1小鼠比WT小鼠更易发生肺癌和肿瘤，而人源化CYP 1A 2小鼠不太易发生肺癌和肿瘤的假设。2.探讨MC对肝、肺细胞色素P450 1A 1（CYP 1A 1）的持续诱导和抑制作用的分子机制。这一目标有两个次级目标：（i）检验肝CYP 1A 2机制有助于MC持续抑制肝和肺CYP 1A 1的假设。(ii)在Cyp 1a 1-或Cyp 1a 1/1a 2-null背景下表达人CYP 1A 1（hCYP 1A 1-luc或hCYP 1A 1-GFP）的转基因人源化小鼠中，检验MC通过相应启动子的持续转录激活来增强人CYP 1A 1的持续诱导，以及肝CYP 1A 2将抑制肝和肺CYP 1A 1表达的持续诱导的假设。3.探讨抑制肺细胞色素P4501 A1诱导的分子机制。我们将检验CYP 1A 2衍生的MC代谢物通过CYP 1A 1启动子AHRE上的序列特异性DNA加合物抑制肺细胞的假设。这一目标有两个次级目标。(i)为了检验用含有CYP 1A 2衍生的DNA加合物的质粒转染的细胞将在人肺细胞中显示CYP 1A 1转录抑制的假设（例如，A549，BEAS-2B）。(ii)检验过表达CYP 1A 2的人肺细胞暴露于MC将显示持续CYP 1A 1诱导减弱的假设。长期目标是：（i）确定多环芳烃调控CYP 1A 1基因表达的分子机制，（ii）阐明基因特异性DNA加合物在CYP 1A 1分子调控中的可能作用，以及（iii）制定预防/治疗环境化学品引起的人类癌症的合理策略。 公共卫生相关性：多环芳烃（PAH）存在于香烟烟雾、颗粒物和木炭烤肉中。拟议的研究将导致发展的合理战略，化学预防和/或化疗的肺癌引起的环境化学品在人类。长期目标是：（i）确定多环芳烃调控细胞色素P450（P450）1A 1基因表达的分子机制，（ii）阐明基因特异性DNA加合物在药物代谢酶分子调控中的可能作用，以及（iii）制定合理的策略预防/治疗环境化学品引起的人类癌症。

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. PUBLIC HEALTH RELEVANCE: Polycyclic aromatic hydrocarbons (PAHs) are present in cigarette smoke, particulate matter, and in charcoal broiled meats. The proposed studies would lead to development of rational strategies for the chemoprevention and/or chemotherapy of lung cancers caused by environmental chemicals in humans. The long-term objectives are to: (i) define the molecular mechanisms of regulation of cytochrome P450 (CYP)1A1 gene expression by PAHs, (ii) elucidate the possible role of gene-specific DNA adducts in molecular regulation of drug metabolizing enzymes, and (iii) develop rational strategies for the prevention/treatment of human cancers caused by environmental chemicals.