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DNA-damaging aryl hydrocarbons induce Mdm2 expression via p53-independent post-transcriptional mechanisms
A Hsing1, D V Faller, C Vaziri
1Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Abstract:
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.
Insights
Benzo[a]pyrene (BaP) induces mdm2 mRNA by stabilizing its transcript post-transcriptionally, independent of p53. This post-transcriptional regulation of mdm2 impacts cellular responses to DNA damage and apoptosis.
Area of Science:
- Molecular Biology
- Cellular Biology
- Environmental Toxicology
Background:
- Previous studies indicated elevated mdm2 mRNA in benzo[a]pyrene (BaP)-treated cells during DNA damage-induced cell cycle arrest.
- Potential aryl-hydrocarbon receptor-binding sites were identified in the mdm2 promoter.
Purpose of the Study:
- To investigate the mechanism of BaP-induced mdm2 mRNA elevation.
- To determine if aryl-hydrocarbon receptor (AhR) directly activates mdm2 transcription.
- To elucidate the role of p53 in BaP-induced mdm2 expression.
Main Methods:
- Reporter gene assays with mdm2 promoter constructs.
- Analysis of mdm2 expression in p53 null cells.
- Nuclear run-off assays to assess mdm2 transcription.
- Measurement of mdm2 mRNA half-life.
Main Results:
- BaP induction of mdm2 mRNA is dependent on genotoxicity, not direct AhR transcriptional activation.
- p53-response elements in the mdm2 promoter are insufficient for DNA damage-dependent expression.
- BaP induced mdm2 in p53 null cells, indicating p53-independent regulation.
- Transcriptional induction did not account for mdm2 mRNA level changes; instead, mdm2 mRNA half-life increased significantly.
- Ectopic mdm2 expression enhanced BaP-induced apoptosis.
Conclusions:
- BaP induces mdm2 expression primarily through post-transcriptional stabilization of its mRNA, independent of p53.
- This study reveals a novel p53-independent mechanism for mdm2 regulation at the RNA stability level.
- The findings suggest a link between DNA damage signaling, RNA stability, and cellular responses like apoptosis, relevant to cancer and environmental carcinogenesis.