The Use of ProteoTuner Technology to Study Nuclear Factor kappa B Activation by Heavy Ions

Nuclear factor kappa B (NF-kappa B) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-kappa B binding sites (HEK-pNF kappa B-DD-tdTomato-C8). Using this reporter cell line, NF-kappa B activation after exposure to different energetic heavy ions (O-16, 95 MeV/n, linear energy transfer-LET 51 keV/mu m; C-12, 95 MeV/n, LET 73 keV/mu m; Ar-36, 95 MeV/n, LET 272 keV/mu m) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-kappa B-dependent DD-tdTomato expression. Approximately 44 hits of O-16 ions and & AP;45 hits of C-12 ions per cell nucleus were required to double the NF-kappa B-dependent DD-tdTomato expression, whereas only & AP;3 hits of Ar-36 ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of Ar-36 ions doubled NF-kappa B-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-kappa B activation detection by a factor of three, allowing the detection of single particle hits' effects.