Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology. (January 2015)

• Record Type: Journal Article
• Authors: Chishti, Arif A.; Hellweg, Christine E.; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther
• Is Part Of: Life sciences in space research. Volume 4(2015)
• Page Start: 35
• Page End: 45
• Publication Date: 2015-01
• Subjects: Mammalian cells -- Radiation response -- Fluorescent protein -- Cytotoxicity -- Proliferation -- tdTomato; A260 absorbance at 260 nm -- A280 absorbance at 280 nm -- BP bandpass -- CellRad Cellular Responses to Radiation in Space -- CMV Cytomegalovirus -- Cu copper -- DMSO dimethyl sulfoxide -- DsRed red fluorescent protein from Discosoma sp. -- EGFP Enhanced Green Fluorescent Protein -- ESA European Space Agency -- F fluence -- FA formaldehyde -- FBS fetal bovine serum -- FT "Farbteiler" – dichroic mirror -- GANIL Grand Accélérateur National d'Ions Lourds -- GCR galactic cosmic rays -- Gy Gray -- GSI GSI Helmholtzzentrum für Schwerionenforschung GmbH -- HEK/293 Human embryonic kidney -- IBER Investigations into Biological Effects of Radiation Using the GSI Accelerator Facility -- ISS International Space Station -- LEO low Earth orbit -- LET linear energy transfer -- LP longpass -- MDR multidrug resistance -- MTT 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide -- NADH reduced nicotinamide adenine dinucleotide -- PBS phosphate buffered saline -- RBE relative biological effectiveness -- RBE50%PR RBE for 50% proliferation reduction -- SCR solar cosmic rays -- SpaceLife Helmholtz Space Life Sciences Research School -- Sv Sievert -- tdTomato tandem dimer Tomato -- μ×g microgravity; Space biology -- Periodicals; 571.0919
• DOI: 10.1016/j.lssr.2014.12.005
• Languages: English
• ISSNs: 2214-5524

Abstract

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. Highlights: HEK cells were stably transfected with a constitutive tdTomato vector. Fast proliferation monitoring by fluorescence measurement. Radiation-induced proliferation reduction successfully monitored with this cell line. Relative biological effectiveness of heavy ions reached a maximum at 90 keV/μm. Reporter cell line for assessment of shielding, countermeasures, combined effects etc.