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

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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.