Interplay between benzo(a)pyrene-induced senescence and transcriptonal repression of DNA repair

苯并芘诱导的衰老与 DNA 修复转录抑制之间的相互作用

• 批准号: 388225306
• 负责人: Professor Dr. Markus Christmann
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/388225306?language=en
• 关键词: Interplay; between; benzo; pyrene; induced

A coordinated and faithful DNA repair is of central importance for maintaining genomic integrity and survival. A significant influence on the outcome of DNA damage processing is expected if the relative abundance of key repair factors is altered. This could disrupt the balance between individual repair pathways and the effectiveness of the DNA repair. Transcriptional activation of DNA repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress conditions. However, it appears that the inverse strategy, i.e. downregulation of gene expression in response to DNA damage, also plays a role in the fine-tuned regulation of complex DNA repair pathways. In our previous work, we analysed the regulation of DNA repair in response to benzo(a)pyrene 9,10-diol-7,8-epoxide (BPDE), the active metabolite of benzo(a)pyrene (B[a]P), which is the most important carcinogen formed by incomplete combustion during food preparation and smoking. We showed that low (nontoxic) BPDE concentrations cause AP-1 and p53 dependent upregulation of several NER genes, leading to enhanced NER activity and consequently reducing the effectiveness of a challenge dose (adaptive response). To further elucidate alterations in the expression of DNA repair genes following BPDE exposure, qPCR microarrays were performed, revealing a strong repression of the mismatch repair factors MSH2, MSH6 and EXO1, as well as of Rad51, the central component of the homologous recombination. The repression of these genes was mediated by abrogation of the E2F1 pathway. As part of the present application, we will investigate the molecular mechanisms leading to the repression of MSH2, MSH6, EXO1 and RAD51, focussing on mechanisms underlying abrogation of the E2F1 pathway and on the impact of histone modification.Since we could show that repression of these DNA repair mechanisms is a specific trait of senescent cells, the repression may represent a great danger for the organism. In absence of these important DNA repair mechanisms, smoking induced DNA lesions, may lead to accumulation of mutations and chromosomal aberrations in senescent cells. Furthermore, since the nontoxic BPDE concentrations only induce a transient DNA damage response, the cells may exit senescence with unrepaired genomic alterations, which could contribute to the carcinogenic potential of smoking. To test this hypothesis, the mechanisms responsible for induction and maintenance of senescence will be analysed using senescent and non-senescent cells separated by FACS upon BPDE exposure. In detail, we will focus on the impact of the DNA damage response and the SASP phenotype. In addition open approaches using kinome and transcriptome profiling will be performed. Finally we will analyse whether cells can escape from B[a]P/BPDE-induced senescence and whether these cells harbour increased genomic alterations.

协调和忠实的DNA修复对于维持基因组的完整性和生存至关重要。如果关键修复因子的相对丰度发生变化，预计将对DNA损伤处理的结果产生重大影响。这可能会破坏个体修复途径和DNA修复有效性之间的平衡。DNA修复基因的转录激活是细胞适应遗传毒性应激条件的重要调控机制。然而，相反的策略，即下调基因表达以响应DNA损伤，似乎也在复杂的DNA修复途径的微调调节中发挥作用。在我们以前的工作中，我们分析了苯并(A)芘(B[a]P)的活性代谢物苯并(A)芘9，10-二醇-7，8-环氧化物(BPDE)对DNA修复的调节，苯并(A)芘是食物制备和吸烟过程中不完全燃烧形成的最重要的致癌物。我们发现，低浓度(无毒)的BPDE导致AP-1和P53依赖的几个NER基因上调，导致NER活性增强，从而降低了挑战剂量(适应性反应)的有效性。为了进一步阐明暴露于BPDE后DNA修复基因表达的变化，进行了qPCR微阵列，结果显示错配修复因子MSH2、MSH6和EXO1以及同源重组的中心成分RAD51受到强烈抑制。这些基因的抑制是通过取消E2F1途径来实现的。作为本应用的一部分，我们将研究导致MSH2、MSH6、EXO1和RAD51抑制的分子机制，重点是E2F1途径取消的机制和组蛋白修饰的影响。由于我们可以证明抑制这些DNA修复机制是衰老细胞的特有特征，因此这种抑制可能对生物体构成巨大的危险。在缺乏这些重要的DNA修复机制的情况下，吸烟导致DNA损伤，可能导致衰老细胞中突变和染色体异常的积累。此外，由于无毒的BPDE浓度只会诱导短暂的DNA损伤反应，细胞可能会退出衰老，出现未经修复的基因组变化，这可能有助于吸烟的致癌潜力。为了验证这一假说，我们将使用BPDE暴露时FACS分离的衰老和非衰老细胞来分析诱导和维持衰老的机制。具体来说，我们将重点讨论DNA损伤反应和SASP表型的影响。此外，还将使用染色体组和转录组分析的开放式方法。最后，我们将分析细胞是否能够逃脱B[a]P/BPDE诱导的衰老，以及这些细胞是否存在更多的基因组变化。