Claudia Schmitz

High sensitivity of Deinococcus radiodurans to photodynamically-produced singlet oxygen.

PURPOSE To study the sensitivity of two bacterial cell systems to photodynamic treatment and X-ray irradiation as part of a project to establish efficient procedures for waste water disinfection. MATERIALS AND METHODS Stationary-phase cells of Deinococcus radiodurans (Gram-positive) and Escherichia coli (Gram-negative) were exposed to visible light in a buffer solution containing up to 5 microg/ml sensitizer rose bengal (RB) and to X-rays at dose rates of 32.8 Gy/min or 14.6 Gy/min, respectively. RESULTS Survival of both cell types decreased with increasing exposure time to visible light and increasing concentration of RB, and therefore with an increase in singlet oxygen production. Surprisingly, D. radiodurans, the most resistant cell system to ionizing radiation, was more sensitive to photodynamic treatment than E. coli by about a factor of 100. CONCLUSIONS The main target of singlet oxygen reaction is the cell membrane. The repair of such damage in D. radiodurans is less effective than in E. coli.

The Influence of Microgravity on Repair of Radiation Induced DNA Damage in Bacteria and Human Fibroblasts

The influence of the space flight environment, above all microgravity, on the repair of radiation-induced DNA damage was examined during the Spacelab mission IML-2 as (1) rejoining of DNA strand breaks induced by X irradiation in cells of Escherichia coli B/r (120 Gy) and (2) in human fibroblasts (5 and 10 Gy); (3) induction of the SOS response after gamma irradiation (300 Gy) of cells of Escherichia coli PQ37; and (4) survival of spores of Bacillus subtilis HA 101 after UV irradiation (up to 340 J m(-2)). Cells were irradiated prior to the space mission and were kept frozen (E. coli and fibroblasts) until incubation for defined periods (up to 4.5 h) in orbit; thereafter they were frozen again for laboratory analysis. Germination and growth of spores of B. subtilis on membrane filters was initiated by humidification in orbit. Controls were performed in-flight (1g reference centrifuge) and on the ground (1g and 1.4g). We found no significant differences between the microgravity samples and the corresponding controls in the kinetics of DNA strand break rejoining and of the induction of the SOS response as well as in the survival curves (as proven by Students t test, P < or = 0.1). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage almost normally. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

Activation of Nuclear Factor κB by Different Agents

Abstract:  The transcription factor nuclear factor κB (NF‐κB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF‐κB in epithelial cells in response to different stresses, a cell‐based screening assay for activation of NF‐κB‐dependent gene transcription in human embryonic kidney cells (HEK/293) was developed . This assay allows detection of NF‐κB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell‐based assay, activation of the pathway by several agents, for example, tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF‐κB activation by TNF‐α were examined. NF‐κB was activated by TNF‐α, IL‐1β, PMA, and camptothecin in a dose‐dependent manner, but not by LPS. TNF‐α results in the strongest induction of NF‐κB‐dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20–35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF‐κB activation or direct communication via gap junctions in the cell layer diminishing the TNF‐α response.

Carbon-Ion-Induced Activation of the NF-κB Pathway

Carbon-ion cancer therapy offers several physical and radiobiological advantages over conventional photon cancer therapy. The molecular mechanisms that determine cellular outcome, including the activation of transcription factors and the alteration of gene expression profiles, after carbon-ion exposure are still under investigation. We have previously shown that argon ions (LET 272 keV/µm) had a much higher potential to activate the transcription factor nuclear factor &kgr;B (NF-&kgr;B) than X rays. NF-&kgr;B is involved in the regulation of cellular survival, mostly by antiapoptosis and cell cycle-regulating target genes, which are important in the resistance of cancer cells to radiotherapy. Therefore, activation of the NF-&kgr;B pathway by accelerated carbon ions (LET 33 and 73 keV/µm) was examined. For comparison, cells were exposed to 150 kV X rays and to accelerated carbon ions. NF-&kgr;B-dependent gene induction after exposure was detected in stably transfected human 293 reporter cells. Carbon ions and X rays had a comparable potential to activate NF-&kgr;B in human cells, indicating a comparable usefulness of pharmacological NF-&kgr;B inhibition during photon and carbon-ion radiotherapy.

Activation of the Nuclear Factor κB pathway by heavy ion beams of different linear energy transfer

Abstract Purpose: Risk assessment of radiation exposure during long-term space missions requires the knowledge of the relative biological effectiveness (RBE) of space radiation components. Few data on gene transcription activation by different heavy ions are available, suggesting a dependence on linear energy transfer. The transcription factor Nuclear Factor κB (NF-κB) can be involved in cancerogenesis. Therefore, NF-κB activation by accelerated heavy ions of different linear energy transfer (LET) was correlated to survival. Materials and methods: NF-κB-dependent gene induction after exposure to heavy ions was detected in stably transfected human embryonic kidney 293 cells (HEK-pNF-κB-d2EGFP/Neo cells carrying a neomycin resistance), using the destabilized Enhanced Green Fluorescent Protein (d2EGFP) as reporter. Results: Argon (LET 272 keV/μm) and neon ions (LET 91 keV/μm) had the highest potential to activate NF-κB, resulting in a RBE of 8.9 in comparison to 150 kV X-rays. The RBE for survival also reached its maximum in this LET range, with a maximal value of 2. Conclusions: NF-κB might be involved in modulating survival responses of cells hit by heavy ions in the LET range of 91–272 keV/μm and could therefore become a factor to be considered for risk assessment of radiation exposure during space travel.

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).

The Role of the Nuclear Factor κB Pathway in the Cellular Response to Low and High Linear Energy Transfer Radiation

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality.