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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.

Space radiation contains a complex mixture of particles comprised primarily of protons and high-energy heavy ions. Radiation risk is considered one of the major health risks for astronauts who embark on both orbital and interplanetary space missions. Ionizing radiation dose-dependently kills cells, damages genetic material, and disturbs cell differentiation and function. The immediate response to ionizing radiation-induced DNA damage is stimulation of DNA repair machinery and activation of cell cycle regulatory checkpoints. To date, little is known about cell cycle regulation after exposure to space-relevant radiation, especially regarding bone-forming osteoblasts. Here, we assessed cell cycle regulation in the osteoblastic cell line OCT-1 after exposure to various types of space-relevant radiation. The relative biological effectiveness (RBE) of ionizing radiation was investigated regarding the biological endpoint of cellular survival ability. Cell cycle progression was examined following radiation exposure resulting in different RBE values calculated for a cellular survival level of 1 %. Our findings indicate that radiation with a linear energy transfer (LET) of 150 keV/μm was most effective in inducing reproductive cell killing by causing cell cycle arrest. Expression analyses indicated that cells exposed to ionizing radiation exhibited significantly up-regulated p21(CDKN1A) gene expression. In conclusion, our findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression.

Radiation response of bone cells, especially the bone-forming osteoblasts, is an important issue for radiotherapy in young age. A radiation-induced cell cycle arrest may enhance or accelerate osteoblastic differentiation. To analyze radiation response of osteoblastic cells, the correlation between DNA double-strand break induction (DSB), cell cycle alterations and gene expression modifications after X-irradiation was investigated in the osteoblast-like cell line OCT-1. As marker of the cellular response to DSB, the temporal appearance of gamma-H2AX foci after X-irradiation was visualized. Gene expression profiles of the key cell cycle regulatory protein p21 (CDKN1A), and the most abundant growth factor in human bone, transforming growth factor beta 1 (TGF-beta1) were recorded using quantitative real-time reverse transcription PCR (qRT-PCR). The distribution of cells in the cell cycle phases G1, S and G2 was determined by propidium iodide (PI) staining and flow cytometry. Initial studies show a strong dose dependency in the number of gamma-H2AX foci shortly after X-irradiation. Exposure to 1 Gy yields approximately 36 small foci in OCT-1 cells after 30 min that became larger after 1 h of incubation; after 24 h most of the foci had disappeared. X-rays provoked a dose-dependent arrest in G2 phase of the cell cycle, accompanied by a dose-dependent gene expression regulation for p21 and TGF-beta1. As TGF-beta1 is known to affect osteoblast differentiation, matrix formation and mineralization, modulation of its expression could influence the expression of the main osteogenic transcription factor Runx2 (Cbfa1) and other osteoblast differentiation markers.

The cellular protection reaction known as ultraviolet (UV) response leads to increased transcription of several genes. Parts of this transcriptional response are transmitted via activation of the Nuclear factor kappaB (NF-kappaB). The contribution of different UV radiation qualities to this process is not yet known. In a previous work, a stably transfected human cell line was developed which indicates activation of the NF-kappaB pathway by fluorescence of the reporters Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) thereby allowing a fast and reliable monitoring of UV effects on the NF-kappaB pathway. Cells were exposed to a mercury low-pressure lamp or to simulated sunlight of different wavelength ranges and subjected to flow cytometric analysis after different post-irradiation periods. Growth capacity of cells after UV irradiation was quantified using a luminance measurement of crystal violet stained cell layers. In contrast to UVC and UVB, UVA radiation induced d2EGFP expression and NF-kappaB activation in a non-cytotoxic dose range. These results show that NF-kappaB plays a role in the UVA-induced gene activation in a non-cytotoxic dose range in a human epithelial cell line.

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.

X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using calcofluor white, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis.

Thin sections of budding yeast cells and giant gells grown after X-irradiation have been examined by electron microscopy. The different steps of cross-wall formation during budding were documented with unirradiated cells. With X-ray induced giant cells cytokinesis was shown to be absent. Neither primary nor secondary septae appeared thus cell separation did not occur. Despite this fact both macromolecular synthesis and bud growth continued, giving rise to the formation of giant cells.

Spindle formation and nuclear division of budding and irradiated yeast cells (Saccharomyces uvarum) was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with antitubulin antibodies and DAPI, a fluorescent dye staining DNA. In budding yeast cells, duplication of spindle pole bodies as well as formation of complete 1-micron spindles and elongated 8-micron spindles were documented. In X-irradiated cells, spindle pole bodies were duplicated as well, forming the complete 1-micron spindle. Nuclei of giant cells have lost the elongation ability and remain in a "normal" G2-phase state, thus preventing nuclear as well as cellular division.