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

Nuclear factor κB (NF-κB) 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-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

The role of linear energy transfer in modulating radiation-induced NF-κB activation and its down-stream target genes

To enable long-term human spaceflight, the cellular radiation response to heavy ions with a high linear energy transfer (LET) needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions in provoking DNA damage response pathways as a gateway to cell death or survival is of major concern not only for spaceflight but also for new regimes of tumor radiotherapy. It has been shown that NF-κB, the main actuator in inflammation and immune response, can be activated by DNA double strand breaks (DNA damage dependent subpathway) and is discussed to contribute to anti-apoptosis and survival improvement of cells containing residual DNA damage after ionizing irradiation. The biological relevance of the recently discovered LET depencency of NF-κB activation is unknown, especially the resulting profile of NF-κB target gene expression. Therefore, the effect of heavy ions of a broad LET range (0.3-10,000 keV/μm) on cellular survival, regulation of the cell cycle and activation of NF-κB and the induction of its target genes were investigated. Furthermore, the role of NF-κB activation in cellular survival, cell cycle progression and DNA damage response after X-irradiation was assessed using HEK and RelA knock-down (HEK shRNA RelA) cells. Use of a NF-κB reporter cell line revealed that exposure to heavy ions resulted in maximal NF-κB activation, cell killing and G2/M arrest in the LET range of 90-200 keV/μm. Here, a doubling of NF-κB was reached with radiation doses compliant to induce as less as 1 DNA double strand break per cell. The cellular survival after X-irradiation decreases in HEK RelA knock-down cells whereas activation of NF-κB by tumor necrosis factor alpha (TNF-α) 6 h in advance to X-irradiation slightly improves cellular survival. The results show that NF-κB down regulation leads cells not only towards high radiosensitivity and lower survival but also, for the first time, to a delayed DNA damage response and cell cycle progression. In this research work it has been shown for the first time that heavy ions up-regulate NF-κB-dependent chemokines (CXCL1, CXCL2, CXCL10 and IL-8) and CD83 expression with maximal potency in the LET range of 50-300 keV/μm. These up-regulated chemokines are important for cell-cell communication with hit as well as unhit cells (bystander effect). The latter are believed to contribute to the tissue radiation response especially after exposure to low doses of high LET radiation relevant for spaceflight and surrounding tissue in tumor therapy. Taken together, this study clearly demonstrates that the cellular radiation response modulation of NF-κB activation and NF-κB-dependent gene expression is highest in the LET range of ~50-300 keV/μm. The results obtained suggest the NF-κB pathway to be a promising target for pharmacological modulation of cellular radiation response either to improve tumor cell killing during radiotherapy with heavy ions or to mitigate radiation late effects such as carcinogenesis in astronauts

NF-κB ACTIVATION AFTER HEAVY ION EXPOSURE: INCREASING THE SENSITIVITY OF THE REPORTER SYSTEM

Biological effects of ionizing radiation are strongly influenced by the radiation quality. The biological effectiveness of accelerated heavy ions (which constitute the biologically most important radiation type in space) with medium to high linear energy transfer (LET), for affecting DNA damage response pathways as a gateway to cell death or survival, is of major concern for space missions

Molecular Signaling in Response to Charged Particle Exposures and its Importance in Particle Therapy

Energetic, charged particles elicit an orchestrated DNA damage response (DDR) during their traversal through healthy tissues and tumors. Complex DNA damage formation, after exposure to high linear energy transfer (LET) charged particles, results in DNA repair foci formation, which begins within seconds. More protein modifications occur after high-LET, compared with low-LET, irradiation. Charged-particle exposure activates several transcription factors that are cytoprotective or cytodestructive, or that upregulate cytokine and chemokine expression, and are involved in bystander signaling. Molecular signaling for a survival or death decision in different tumor types and healthy tissues should be studied as prerequisite for shaping sensitizing and protective strategies. Long-term signaling and gene expression changes were found in various tissues of animals exposed to charged particles, and elucidation of their role in chronic and late effects of charged-particle therapy will help to develop effective preventive measures

Radiation-induced NF-κB activation and expression of its down-stream target genes as biomarker of radiation quality

Activation of Nuclear Factor κB (NF-κB) and the resulting gene expression profile after exposure to different radiation qualities have been evaluated to a very limited extent. Therefore, the activation of NF-κB after exposure to low and high linear energy transfer (LET) radiation and the expression of its target genes was analyzed in human embryonic kidney (HEK) cells

Linear Energy Transfer Modulates Radiation-Induced NF-kappa B Activation and Expression of its Downstream Target Genes

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3–9,674 keV/μm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/μm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/μm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50–200 keV/μm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell–cell communication among hit as well as nonhit cells (bystander effect)

Imaging of nuclear factor κB activation induced by ionizing radiation in human embryonic kidney (HEK) cells

Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. Nuclear factor kappa B (NF-κB) is one of the most important transcription factors that respond to changes in the environment of a mammalian cell. NF-κB plays a key role not only in inflammation and immune regulation but also in cellular radiation response. In response to DNA damage, NF-κB might inhibit apoptosis and promote carcinogenesis. Our previous studies showed that ionizing radiation is very effective in inducing biological damages. Therefore, it is important to understand the radiation-induced NF-κB signaling cascade. The current study aims to improve existing mammalian cell-based reporter assays for NF-κB activation by the use of DD-tdTomato which is a destabilized variant of red fluorescent protein tdTomato. It is demonstrated that exposure of recombinant human embryonic kidney cells (HEK/293 transfected with a reporter constructs containing NF-κB binding sites in its promoter) to ionizing radiation induces NF-κB-dependent DD-tdTomato expression. Using this reporter assays, NF-κB signaling in mammalian cells was monitored by flow cytometry and fluorescence microscopy. Activation of NF-κB by the canonical pathway was found to be quicker than by the genotoxin- and stress-induced pathway. X-rays activate NF-κB in HEK cells in a dose-dependent manner, and the extent of NF-κB activation is higher as compared to camptothecin