Andrea Arenz

Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of Space Environmental Radiation

Abstract Baumstark-Khan, C., Hellweg, C. E., Arenz, A. and Meier, M. M. Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of Space Environmental Radiation. Radiat. Res. 164, 527–530 (2005). A screening assay for the detection of NF-κB-dependent gene induction using the destabilized variant of the reporter protein enhanced green fluorescent protein (d2EGFP) is used for assessing the biological effects of accelerated heavy ions as a model of space environmental radiation conditions. The time course of d2EGFP expression and therefore of activation of NF-κB-dependent gene expression was measured after treatment with TNFA or after heavy-ion exposure using flow cytometry. The reported experiments clearly show that accelerated argon ions (95 MeV/nucleon, LET 230 keV/μm) induce the NF-κB pathway at low particle densities (1–2 particle hits per nucleus), which result in as few as 5–50 induced DSBs per cell.

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.

Gene Expression Modulation in A549 Human Lung Cells in Response to Combustion Generated Nano-Sized Particles

Abstract:  High levels of ambient air pollution are associated in humans with aggravation of asthma and of respiratory and cardiopulmonary morbidity; long‐term exposures to particulate matter (PM) have been linked to possible increases in lung cancer risk, chronic respiratory disease, and increased death rates. The Biodiagnostics Group of the DLR Institute of Aerospace Medicine develops cellular test systems capable of monitoring the biological consequences of environmental conditions on humans already on cellular and molecular level. Such bioassays rely on the receptor–reporter principle, where cell lines are transfected with plasmids carrying a reporter gene under control of environment‐dependent promoters (receptor), which play a key role in regulating gene expressions in response to extracellular signals. We developed the recombinant human lung epithelial cell line A549‐NF‐κB‐EGFP/Neo carrying a genetically encoded fluorescent indicator for monitoring activation of the NF‐κB signaling pathway in living cells in response to genotoxic and cytotoxic environmental influences. With this cell line we screened several candidate human radiation‐responsive genes (GADD45β, CDKN1A) and NF‐κB‐dependent genes (IL‐6, NFκBIA, and pNF‐κB‐EGFP) for gene expression changes by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) assay, using cDNA obtained from total RNA isolated at various time points after exposure to combustion generated nano‐sized particle samples.

Getting ready for the manned mission to Mars: bioassays for space research

Harmful environmental factors - namely ionizing radiation - will continue to influence future manned space missions. The Cellular Biodiagnostic group at the German Aerospace Center (DLR) develops cellular monitoring systems, which include bacterial and mammalian cell systems capable of recognizing DNA damage as a consequence of the presence of genotoxic conditions. Such bioassay or biosensor systems will complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. Furthermore, synergistic mutagenic and cancerogenic impacts of the radiation environment together with other potentially genotoxic constituents of the space habitat can be quantified using such systems, whose signals are especially relevant for the molecular damage to the DNA or the chromosomes. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA to be performed on the International Space Station. It will supply basic information on the cellular response to radiation applied in microgravity. One of the biological end-points under investigation will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP), originally isolated from the bioluminescent jellyfish Aequorea victoria. A second end-point will be gene activation by space flight conditions in mammalian cells, based on fluorescent promoter reporter systems using the destabilized EGFP variant (d2EGFP). The promoter element to be investigated will reflect the activity of the NF-kappaB stress response pathway as an anti-apoptotic radiation response. DNA damage will be measured by fluorescent analysis of DNA unwinding (FADU). The systems have worked properly for terrestrial applications during the first experiments. Experiments using accelerated particles produced at the French heavy ion accelerator GANIL have given insights into cellular mechanisms relevant for the exceptional radiation field in space.