The role of Nuclear Factor kB in the cellular response to different radiation qualities

Kurzfassung

Radiation is currently one of the most important limiting factors for manned space flight. During such missions, there is a constant exposure to low doses of galactic cosmic radiation and in particular high-energy heavy ions. Together this is associated with an increased cancer risk which currently cannot be sufficiently reduced by shielding. As such, cellular radiation response needs to be further studied in order to improve risk estimation and develop appropriate countermeasures. It has been shown that exposure of human cells to accelerated heavy ions, in fluences that can be reached during long-term missions, leads to activation of the Nuclear Factor kB (NF-kB) pathway. Heavy ions with a linear energy transfer (LET) of 90 to 300 keV/μm were most effective in activating NF-kB. NF-kB as an important modulating factor in the cellular radiation response could improve cellular survival after heavy ion exposure, thereby influencing the cancer risk of astronauts. The NF-kB pathway may be a potential pharmacological target in the mitigation of radiation response during space missions; such as the prevention of massive cell death after high dose irradiation (acute effects), in addition to neoplastic cell transformation during chronic low-dose exposure (late effects). The aim of this work was to examine the role of NF-kB in the cellular response to spacerelevant radiation. Firstly, NF-kB activation in human embryonic kidney cells (HEK) after exposure to different radiation qualities and quantities was investigated. Key elements of different NF-kB sub-pathways were chemically inhibited to analyze their role in NF-kB activation induced by low and high LET ionizing radiation. Finally a cell line, stably transfected with a plasmid coding for a short-hairpin RNA (shRNA) for a knockdown of the NF-kB subunit RelA, was established to assess the role of RelA in the cellular response to space-relevant radiation. The knockdown was verified on several levels and the cell line was characterized concerning proliferation, cell cycle progression and gene expression. Additionally, the effects of the RelA knockdown on cell cycle progression, cellular survival and gene expression after exposure to low and high LET radiation were investigated. It was shown that activation of NF-kB depends on radiation quality and quantity. Experiments with chemical inhibitors revealed that NF-kB activation by ionizing radiation is strictly ATM dependent and degradation of the NF-kB inhibitor IkB by the proteasome is essential for both the classical and genotoxic stress-induced NF-kB pathway. Absence of NF-kB dimers containing RelA resulted in a prolonged lag-phase but did not affect cell cycle progression significantly in untreated cells. After irradiation, a dose and radiation quality dependent arrest in the G2 phase of the cell cycle occurred and also upon downregulation of RelA expression. RelA knockdown resulted in higher sensitivity of HEK cells to the killing effect of X-irradiation. In contrast, RelA knockdown did not further reduce the cellular survival after heavy ion exposure. Further, NF-kB target genes were not inducible in the RelA knockdown cell line. NF-kB-dependent gene expression rely on radiation dose and LET. Chemokine expression (e.g. CXCL1, 2, 8 and 10) was induced in a proportional manner to radiation quality and quantity, emphasizing the role of NF-kB in the bystander effect. These NF-kB regulated genes are interesting targets for countermeasure development against the effects of space radiation.