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

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改变，预计对DNA损伤处理的结果会产生重大影响。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，似乎相反的策略，即响应于DNA损伤下调基因表达，也在复杂DNA修复途径的微调调节中发挥作用。在我们以前的工作中，我们分析了响应于苯并（a）芘9，10-二醇-7，8-环氧化物（BPDE）的DNA修复的调节，苯并（a）芘（B[a]P）的活性代谢物，其是在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（无毒）BPDE浓度导致AP-1和p53依赖性上调几个NER基因，导致增强NER活性，从而降低有效性的挑战剂量（适应性反应）。为了进一步阐明BPDE暴露后DNA修复基因表达的变化，进行了qPCR微阵列，揭示了错配修复因子MSH 2、MSH 6和EXO 1以及Rad 51（同源重组的中心组分）的强烈抑制。这些基因的阻遏是通过E2 F1途径的废除来介导的。作为本申请的一部分，我们将研究导致MSH 2、MSH 6、EXO 1和RAD 51阻遏的分子机制，重点关注E2 F1途径被废除的潜在机制和组蛋白修饰的影响。由于我们可以证明这些DNA修复机制的阻遏是衰老细胞的一个特定特征，因此这种阻遏可能对生物体构成极大的危险。在缺乏这些重要的DNA修复机制的情况下，吸烟诱导的DNA损伤可能导致衰老细胞中突变和染色体畸变的积累。此外，由于无毒的BPDE浓度仅诱导短暂的DNA损伤反应，细胞可能会退出衰老与未修复的基因组改变，这可能有助于吸烟的致癌潜力。为了检验这一假设，将使用在BPDE暴露后通过FACS分离的衰老和非衰老细胞分析负责诱导和维持衰老的机制。详细地说，我们将集中在DNA损伤反应和SASP表型的影响。此外，将进行使用激酶组和转录组谱分析的开放方法。最后，我们将分析细胞是否可以逃避B[a]P/BPDE诱导的衰老，以及这些细胞是否含有增加的基因组改变。

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.