title: Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans.
abstract: KillerRed, a red fluorescent protein, is a photosensitizer that efficiently generates reactive oxygen species (ROS) when irradiated with green light. Because KillerRed is genetically encoded, it can be expressed in a spatially and temporally regulated manner under control of a chosen promoter and thus is a powerful tool for studying the downstream cellular effects of ROS. However, information is still limited about the effects of KillerRed-mediated production of ROS inside the mitochondria (mtROS). Therefore, we investigated whether mtROS generated by KillerRed could trigger mitochondrial damage and cell death by generating human cell lines (HEK293T and HeLa cells) that stably expressed mitochondria-targeting KillerRed (mtKillerRed). We found that mtROS generated by mtKillerRed caused depolarization of the mitochondrial membrane and morphological changes, which were partly due to the mitochondrial permeability transition (MPT), as well as inducing both caspase-dependent cell death (apoptosis) and caspase-independent cell death. In order to study the pathological processes initiated by mtROS in animals, transgenic Caenorhabditis elegans expressing mtKillerRed in muscle tissue were generated. Transgenic larvae showed developmental delay following light irradiation, suggesting that mtROS influenced the development of C. elegans larvae. In conclusion, our studies demonstrated that the photosensitizer KillerRed is effective at inducing oxidative damage in the mitochondria, and indicated that our experimental systems may be useful for studying the downstream cellular effects of mtROS.
authors.affiliation: Laboratory of Molecular Genetics, Department of Medical Genetics, Osaka University Medical School, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Shibuya
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Tsujimoto
mesh_terms: D051700:Activating Transcription Factor 3 / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D016923:Cell Death / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000528:radiation effects; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000528:radiation effects; D008928:Mitochondria / Q000378:metabolism* / Q000528:radiation effects*; D009687:Nuclear Proteins / Q000235:genetics; D017319:Photosensitizing Agents / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051742:Transcription Factor CHOP / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000097573:Red Fluorescent Protein; D000097642:GADD45 Proteins
chemical_list: D051700:Activating Transcription Factor 3; D018797:Cell Cycle Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; D051742:Transcription Factor CHOP; D044767:Ubiquitin-Protein Ligases; D000097573:Red Fluorescent Protein; C490449:ATF3 protein, human; C497249:DDIT3 protein, human; C439070:GADD45A protein, human; D000097642:GADD45 Proteins; C111567:parkin protein