title: Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology.
abstract: The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: arif.chishti@dlr.de.
authors.forename: Arif A
authors.initials: AA
authors.lastname: Chishti
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: christine.hellweg@dlr.de.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Hellweg
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: thomas.berger@dlr.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Berger
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: christa.baumstark-khan@dlr.de.
authors.forename: Christa
authors.initials: C
authors.lastname: Baumstark-Khan
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: sebastian.feles@dlr.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Feles
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: tkaetzel@uni-bonn.de.
authors.forename: Thorben
authors.initials: T
authors.lastname: Kätzel
authors.affiliation: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, D-51147 Köln, Germany. Electronic address: guenther.reitz@dlr.de.
authors.forename: Günther
authors.initials: G
authors.lastname: Reitz
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation / Q000528:radiation effects*; D002470:Cell Survival / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005118:Extraterrestrial Environment; D057809:HEK293 Cells; D020450:Heavy Ions / Q000009:adverse effects*; D006801:Humans; D018499:Linear Energy Transfer; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D011853:Radiobiology; D014965:X-Rays / Q000009:adverse effects*; D000097573:Red Fluorescent Protein
chemical_list: D015415:Biomarkers; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Cytotoxicity; Fluorescent protein; Mammalian cells; Proliferation; Radiation response; tdTomato