Signaling and damage by localized reactive oxygen species production

The work in this thesis aimed to shed light on how compartmentalization of the subcellular source of reactive oxygen species (ROS) is involved in differential signaling and damage in the context of tumor biology. In the introductory chapter 1, the basics of reactive oxygen species (ROS) in the context of redox biology are discussed. ROS is positioned at a crossroads of signaling and damage. Loss of ROS homeostasis can contribute to the development of disease by dysregulating signaling pathways or enhancing oxidative damage. The biological outcome is dependent on many factors, including the type of ROS, the concentration and its subcellular site of origin. The last of these factors is the least understood, as the tools to study compartmentalized redox biology have been limited. In chapter 2, we review the role of ROS in cancer development and therapy. The ambivalent nature of ROS, being involved in both physiology and pathology, becomes especially apparent in the context of cancer. Over the course of cancer development, ROS can act as a tumor suppressive component by inducing cell cycle arrest and oxidative cell death. However, at each stage, ROS can also have tumor promoting effects. The role of ROS in cancer therapy is just as two-faced. We started investigating the role of the subcellular site of H2O2 production on its biological effects with the work described in chapter 3. The ectopic expression of R. gracilis D-amino acid oxidase (DAAO) has been an upcoming tool to induce localized H2O2 production within cells, but the interpretation of obtained results is problematic when there is no sense of the amount of H2O2 being produced. We solved this problem by developing a method that measures the consumption of O2 by DAAO as a proxy for the equimolar production of H2O2, thus allowing the measurement of DAAO activity in situ. Armed with this newly developed method, we challenge a longstanding dogma regarding subcellular ROS production, namely that the ROS produced by mitochondrial respiration directly damages chromosomal DNA, and this work is described in chapter 4. We observed that supraphysiological H2O2 production at the outer mitochondrial membrane does not cause DNA damage, even at levels orders of magnitude higher than what mitochondrial normally produce. We conclude that for chromosomal DNA oxidation, H2O2 needs to be generated in its close vicinity and that mitochondrial respiration is not a direct source of oxidative damage to nuclear DNA. Finally, in chapter 5, we investigated the type of DNA mutations that occur when H2O2 can reach the DNA. We discover that the mutational consequences of intracellular H2O2 production is not restricted to the archetypical COSMIC SBS18 signature but contains several other mutational signatures. We also investigated the role of intramitochondrial H2O2 in mtDNA mutagenesis. Surprisingly, although mtDNA oxidation led to an increase in various types of single base substitutions, an increase of the expected C>A mutations was largely lacking. We demonstrate that the mutational consequences of intracellular H2O2 is more complex than initially assumed and that they differ between nuclear and mitochondrial DNA.