Molecular mechanism of cytochrome P4501A1 expression.

描述(由申请人提供)：多环芳烃(PAHs)存在于香烟烟雾、颗粒物和木炭烤肉中。细胞色素P450(CYP)1A酶在多环芳烃与DNA共价相互作用的代谢产物的活化中起着关键作用，这是启动癌症发生的关键事件。这一应用的中心假设是，CYP1A1和CYP1A2酶在多环芳烃介导的致癌过程中扮演着相互作用的角色，依赖于CYP1A2的MC代谢物(S)有助于在CYP1A1启动子的调节区[例如，AHR反应元件(AHRE)]上形成序列特异性的DNA加合物，从而产生一种新的机制，MC-DNA加合物(S)将通过该加合物抑制CYP1A1基因的表达。提出了以下具体目标。1.探讨CYP1A1和1A2酶在多环芳烃介导的肺癌中的作用机制。这个目标有两个子目标：(I)检验这样的假设，即缺乏CYP1A2基因的小鼠将更容易受到影响，而那些缺乏Cyp1a1基因的小鼠将比类似暴露的WT小鼠更不容易受到PAH诱导的肺癌和肿瘤的影响。(Ii)测试一项假设，即人源化的CYP1A1小鼠较WT小鼠更易患肺癌和肿瘤，而人源化的CYP1A2小鼠则较不容易患肺癌。2.探讨MC体内持续诱导和抑制肝、肺细胞色素P1A1酶活性的分子机制。这一目标有两个子目标：(I)检验肝脏细胞色素P1A2对MC对持续的肝和肺细胞色素P1A1的抑制有机械作用的假设。(Ii)验证一种假设，即MC通过相应启动子的持续转录激活而诱导持续的人细胞色素P1A1的诱导，以及肝脏细胞色素P1A2将抑制在Cyp1a1-或Cyp1a1/1a2缺失背景上表达人细胞色素P1A1(hCYP1A1-Luc或hCYP1A1-GFP)的转基因人源化小鼠肝和肺细胞色素P1A1表达的持续诱导。3.探讨抑制肺细胞色素P4501A1诱导的分子机制。我们将检验这一假设，即CYP1A2衍生的MC代谢物通过在CYP1A1启动子的AHRE上的序列特异性DNA加合物而有助于抑制肺细胞。这个目标有两个子目标。(I)验证一种假设，即在体外，转染含有CYP1A2衍生DNA加合物的质粒的细胞将在人肺细胞(例如A549、BEAS-2B)中显示出对CYP1A1转录的抑制。(Ii)测试高表达细胞色素P1A2的人肺细胞暴露于MC将显示持续的细胞色素P1A1诱导减弱的假设。本研究的长期目标是：(I)明确多环芳烃调节细胞色素P1A1基因表达的分子机制，(Ii)阐明基因特异性DNA加合物在细胞色素P1A1分子调控中的可能作用，以及(Iii)开发合理的策略来预防和治疗由环境化学物质引起的人类癌症。

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.