Hsing A, Faller D V, Vaziri C
Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 2000 Aug 25;275(34):26024-31. doi: 10.1074/jbc.M002455200.
During previous studies, we found that mdm2 mRNA levels were elevated in benzo[a]pyrene (BaP, a polycyclic aryl hydrocarbon)-treated cells under conditions of DNA damage-induced cell cycle arrest (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). We have identified potential aryl-hydrocarbon receptor-binding sites in the mdm2 promoter. However, we show that induction of mdm2 mRNA by BaP is entirely dependent upon aryl-hydrocarbon-induced genotoxicity and does not involve direct aryl-hydrocarbon receptor-mediated transcriptional activation of the mdm2 gene. Heterologous mdm2 promoter-reporter constructs containing p53-response elements were not responsive to BaP treatment. Therefore the p53-response elements in the mdm2 promoter are insufficient to confer DNA damage-dependent expression of mdm2. Furthermore, mdm2 transcripts were induced by BaP in p53 null cells from transgenic mice (although both basal and BaP-induced mdm2 expression levels were reduced in these cells relative to p53(+/+) cultures). These data show that p53-mediated mechanisms cannot account for BaP/DNA damage-induced mdm2 expression. Mdm2 promoter-reporter gene assays and nuclear run-off analyses of nascent mdm2 transcripts showed that transcriptional induction was unable to account for the large changes in mdm2 transcript levels following BaP treatment. However, mdm2 mRNA half-life measurements showed stabilization of the mdm2 transcript (from approximately 1 h to >4 h) in response to BaP. To our knowledge, this is the first report of control of mdm2 at the post-transcriptional level and in a p53-independent manner. Transient ectopic expression of mdm2 strongly augmented aryl-hydrocarbon-induced apoptosis, demonstrating that mdm2 levels can have a profound effect on the cellular response to DNA damage. Overall, our results suggest a potentially important link between DNA damage signaling and RNA stability that may be relevant to cell cycle regulation, tumor suppression, and environmental carcinogenesis.
在先前的研究中，我们发现，在DNA损伤诱导的细胞周期停滞条件下，苯并[a]芘（BaP，一种多环芳烃）处理的细胞中mdm2 mRNA水平升高（瓦齐里，C.，和法勒，D. V.（1997年）《生物化学杂志》272卷，2762 - 2769页）。我们已在mdm2启动子中鉴定出潜在的芳烃受体结合位点。然而，我们发现BaP对mdm2 mRNA的诱导完全依赖于芳烃诱导的基因毒性，并不涉及芳烃受体直接介导的mdm2基因转录激活。含有p53反应元件的异源mdm2启动子 - 报告基因构建体对BaP处理无反应。因此，mdm2启动子中的p53反应元件不足以赋予mdm2依赖于DNA损伤的表达。此外，BaP在转基因小鼠的p53缺失细胞中诱导了mdm2转录本（尽管相对于p53(+/+)培养物，这些细胞中基础和BaP诱导的mdm2表达水平均降低）。这些数据表明，p53介导的机制无法解释BaP/DNA损伤诱导的mdm2表达。mdm2启动子 - 报告基因测定和新生mdm2转录本的核延伸分析表明，转录诱导无法解释BaP处理后mdm2转录本水平的大幅变化。然而，mdm2 mRNA半衰期测量显示，响应BaP时mdm2转录本稳定（从约1小时延长至>4小时）。据我们所知，这是关于在转录后水平以p53非依赖方式调控mdm2的首次报道。mdm2的瞬时异位表达强烈增强了芳烃诱导的细胞凋亡，表明mdm2水平可对细胞对DNA损伤的反应产生深远影响。总体而言，我们的结果表明DNA损伤信号与RNA稳定性之间可能存在重要联系，这可能与细胞周期调控、肿瘤抑制和环境致癌作用相关。
