Survival and death strategies in cells exposed to genotoxins

Survival and death strategies in cells exposed to genotoxins

S8 Abstracts / Toxicology Letters 211S (2012) S4–S23 leads to the appearance of cells that carry mutations, or causes a shift towards induction of t...

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S8

Abstracts / Toxicology Letters 211S (2012) S4–S23

leads to the appearance of cells that carry mutations, or causes a shift towards induction of the senescence or cell death programme. Although for many years it was argued that DNA damage kills cells via apoptosis or necrosis, technical and methodological progress during the last few years has helped reveal that DNA damage might also activate death by autophagy or mitotic catastrophe, which may then be followed by apoptosis or necrosis. The molecular basis underlying the decision-making process is currently the subject of intense investigation. Current knowledge about the response to DNA damage and subsequent signaling, with particular attention to cell death induction and the molecular switches between different cell death modalities following damage will be discussed. doi:10.1016/j.toxlet.2012.03.040

S04-2 Survival and death strategies in cells exposed to genotoxins Bernd Kaina Institute of Toxicology, University Medical Center, Germany

cycle, stimulate DNA repair and to recover mRNA synthesis. If cells do not recover RNA synthesis in a timely fashion, cells will undergo apoptosis. In this manner, the transcription machinery acts as a cellular dosimeter for DNA damage and triggers the elimination of cells that have sustained sufficient damage to prohibit transcription recovery. To study the effect of DNA damage on genome-wide transcription we have developed two new techniques, Bru-Seq and BruChase-Seq, based on bromouridine (Bru) pulse-chase labeling coupled to Illumina deep sequencing. Using these techniques we show that ionizing radiation rapidly alters both the synthesis and the transcript stability of specific sets of genes in human fibroblasts. Furthermore, we show that UV light effectively blocks elongation, but not initiation, of transcription resulting in a strong enrichment of sequencing signals at the beginning of genes. Unexpectedly, we found that UV-irradiation also causes an enrichment of unstable short transcripts generated at enhancer elements. We believe that Bru-Seq, BruChase-Seq and the combination of UV light with the Bru-Seq technique (UVBru-Seq) will allow us to explore the effects of DNA damage on the “transcriptome” and “RNA stabilome” as well as help us map transcription start sites and enhancer elements genome-wide. doi:10.1016/j.toxlet.2012.03.042

Genotoxins induce a plethora of DNA adducts that are usually considered to be harmful for the cell. For methylating agents, which are powerful environmental mutagens and carcinogens and are being used in cancer therapy, it has been shown that both survival and death functions can be triggered by the specific adduct O6-methylguanine (O6MeG), and it is currently a matter of intensive research which of the functions are utilized by the cell. Survival strategies include activation of DNA repair, autophagy, senescence and antiapoptotic functions, whereas death strategies rest on the activation of death receptor and/or mitochondrial apoptotic factors and mitotic catastrophy. We have shown that in some cells the efficiency of O6MeG to trigger the p53 dependent death receptor (Fas) pathway is much higher than the p53 independent endogenous mitochondrial pathway, which has strong impact on the sensitivity of p53 mutated tumor cells to anticancer drugs such as temozolomide. An opposite effect of p53 was found for agents inducing bulky lesions, such as chloroethylnitrosourea and UV light, thus protecting against apoptosis and necrosis. This is due to p53-triggered upregulation of repair genes, notably ddb2 and xpc. Data will be shown to indicate homologous recombination as a key player in the cell’s survival strategy towards monofunctional alkylating agents. For genotoxins inducing bulky lesions and interstrand crosslinks transcriptional inhibition comes into play as well, which may lead to sustained activation of the MAP kinase pathway and other functions that triggers cell death. Acknowledgments: Supported by DFG Ka724 and German Cancer Foundation. doi:10.1016/j.toxlet.2012.03.041

S04-3 Transcriptional inhibition by DNA damage as a trigger for cell death Mats Ljungman, Michelle Paulsen, Artur Veloso, Jayendra Prasad, Tom Wilson

S04-4 Mitochondrial involvement in cell death Sten Orrenius, Erik Norberg, Vladimir Gogvadze, Boris Zhivotovsky Karolinska Institutet, Sweden In addition to the role of the mitochondria in energy metabolism, regulation of cell death has emerged as an important function of these organelles. This seems to be linked to their role as the predominant intracellular source of reactive oxygen species (ROS), which are generated at complexes I and III of the respiratory chain. Excessive ROS production has been implicated in DNA mutations, ageing, and cell death. Although mitochondrial dysfunction can result in ATP depletion and necrosis, these organelles are also involved in the regulation of apoptotic cell death by mechanisms that have been conserved through evolution. Hence, many toxic agents target the mitochondria and cause their release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and other key events in apoptosis. Cytochrome c release is initiated by the dissociation of the hemoprotein from its binding to cardiolipin in the inner mitochondrial membrane (IMM). Similarly, apoptosis inducing factor (AIF) must also be detached from the IMM, before it can be exported from the mitochondria into the nucleus. This occurs by cleavage of the peptide chain that anchors it to the IMM and is preceded by carbonylation of AIF triggered by mitochondrial ROS production. Mitochondrial release of cytochrome c and AIF occurs via pores in the outer mitochondrial membrane formed by pro-apoptotic Bcl-2 family proteins or by Ca2+ /ROS-induced mitochondrial permeability transition. Hence, it is apparent that mitochondrial ROS generation is critically involved in the control of cell death and an important mediator of mitochondrial toxicity. doi:10.1016/j.toxlet.2012.03.043

University of Michigan, United States By transcribing the genetic information stored in DNA, RNA polymerase II generates mRNA blueprints for protein synthesis. Transcription stalling at DNA lesions triggers a cellular stress response that is aimed at arresting proliferating cells in the cell