Search

Simple Summary: Non-small cell lung carcinoma (NSCLC), similarly to most other solid malignancies, frequently exhibits lack of oxygen (hypoxia), and this has been implicated as a potential cause of treatment resistance. A better understanding of NSCLC behavior under hypoxia in the context of its cellular energetics may lead to developing more effective treatment strategies. In this study, the effects of prolonged hypoxia on the energy metabolism and cellular proliferation of two different NSCLC cell lines were investigated. Glucose consumption and lactate production by cells indicated energy metabolism changes under hypoxia. Cellular proliferation decreased under hypoxia, as indicated by slower cell growth kinetics and cell cycle phase distribution over time. Additionally, RNA sequencing revealed differential expression of genes involved in cell migration under hypoxia. Hypoxia occurs in 80% of non-small cell lung carcinoma (NSCLC) cases, leading to treatment resistance. Hypoxia's effects on NSCLC energetics are not well-characterized. We evaluated changes in glucose uptake and lactate production in two NSCLC cell lines under hypoxia in conjunction with growth rate and cell cycle phase distribution. The cell lines A549 (p53 wt) and H358 (p53 null) were incubated under hypoxia (0.1% and 1% O2) or normoxia (20% O2). Glucose and lactate concentrations in supernatants were measured using luminescence assays. Growth kinetics were followed over seven days. Cell nuclei were stained with DAPI and nuclear DNA content was determined by flow cytometry to determine cell cycle phase. Gene expression under hypoxia was determined by RNA sequencing. Glucose uptake and lactate production under hypoxia were greater than under normoxia. They were also significantly greater in A549 compared to H358 cells. Faster energy metabolism in A549 cells was associated with a higher growth rate in comparison to H358 cells under both normoxia and hypoxia. In both cell lines, hypoxia significantly slowed down the growth rate compared to proliferation under normoxic conditions. Hypoxia led to redistribution of cells in the different cycle phases: cells in G1 increased and the G2 population decreased. Glucose uptake and lactate production increase under hypoxia in NSCLC cells indicated greater shunting of glucose into glycolysis rather than into oxidative phosphorylation compared to normoxia, making adenosine triphosphate (ATP) production less efficient. This may explain the redistribution of hypoxic cells in the G1 cell cycle phase and the time increase for cell doubling. Energy metabolism changes were more prominent in faster-growing A549 cells compared to slower-growing H358 cells, indicating possible roles for the p53 status and inherent growth rate of different cancer cells. In both cell lines, genes associated with cell motility, locomotion and migration were upregulated under chronic hypoxia, indicating a strong stimulus to escape hypoxic conditions. [ABSTRACT FROM AUTHOR]

Abstract: The combined action of ionizing radiation and microgravity will continue to influence future space missions, with special risks for astronauts on the Moon surface or for long duration missions to Mars. It has been estimated that on a 3-year mission to Mars about 3% of the bodies’ cell nuclei would have been hit by one iron ion with the consequence that nuclear DNA will be heavily damaged. There is increasing evidence that basic cellular functions are sensitive not only to radiation but also to microgravity. DNA repair studies in space on bacteria, yeast cells and human fibroblasts, which were irradiated before, flight, gave contradictory results: from inhibition of repair by microgravity to enhancement, whereas others did not detect any influence of microgravity on repair. The space experiment CERASP (CEllular Responses to RAdiation in SPace) to be performed at the International Space Station (ISS) is aimed to supply basic information on the cellular response in microgravity to radiation applied during flight. It makes use of a recombinant human cell line as reporter for cellular signal transduction modulation by genotoxic environmental conditions. The main biological endpoints under investigation will be gene activation based on enhanced green fluorescent protein (EGFP, originally isolated from the bioluminescent jellyfish Aequorea victoria) expression controlled by a DNA damage-dependent promoter element which reflects the activity of the nuclear factor kappa B (NF-B) pathway. The NF-B family of proteins plays a major role in the inflammatory and immune response, cell proliferation and differentiation, anti-apoptosis and tumorgenesis. For radiation exposure during space flight a radiation source has been constructed as damage accumulation by cosmic radiation will certainly be insufficient for analysis. The space experiment specific hardware consists of a specially designed radiation source made up of the -emitter promethium-147, combined with a miniaturized culture vessel and a seeding apparatus. With this prototype hardware, the requirements of CERASP can be fulfilled with cells growing on the polytetrafluoroethylene foil. The radiation source can be enveloped with additional titanium foils for safety issues. The results from the preparatory experimental phase clearly show that the Pm-147 radiation source meets the requirements for the space experiment CERASP. [Copyright &y& Elsevier]

Abstract: Cellular bioassays for detection of cyto- and genotoxicity are useful in the risk assessment of space environmental factors. Such bioassay systems have the potential complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA/ESA 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 endpoints 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. In this ground based study, the usefulness of this approach in comparison to standard techniques (colony forming ability test, MTT test) is shown. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: High LET radiations, such as heavy ions or neutrons, have an increased biological effectiveness compared to X-rays for gene mutation, genomic instability and carcinogenesis. Estimating the biological risks from space radiation encountered by cosmonauts will continue to influence long term duration in space, such as the planned mission to Mars. The human radiation responsive genes CDKN1A (p21/WAF), GADD45α (GADD45), GADD45β (MyD118), RRM2b (p53R2) and BRCA2 (FancD1), involved in cell cycle control or damage repair, were screened for gene expression changes in MCF-7 cells by quantitative real-time reverse transcription PCR (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after irradiation with accelerated doses of 36-argon ions and X-rays. Examination of the expression profiles 2 and 12 h after exposure reveals a pattern consistent with a population of cells in the early response to DNA damage and invoking cell stress responses. Interesting new data showing different expression patterns according to the gene and the type of ionizing radiation used could be obtained. Results show, that the signaling and repair activities induced after heavy ion or X-ray exposure are not the same and gene expression patterns may become useful indicators for distinguishing different types of radiation in relation to their biological effects. [Copyright &y& Elsevier]

The use of high linear energy transfer (LET) ionizing radiation (IR) is progressively being incorporated in radiation therapy due to its precise dose localization and high relative biological effectiveness. At the same time, these benefits of particle radiation become a high risk for astronauts in the case of inevitable cosmic radiation exposure. Nonetheless, DNA Damage Response (DDR) activated via complex DNA damage in healthy tissue, occurring from such types of radiation, may be instrumental in the induction of various chronic and late effects. An approach to elucidating the possible underlying mechanisms is studying alterations in gene expression. To this end, we identified differentially expressed genes (DEGs) in high Z and high energy (HZE) particle-, γ-ray- and X-ray-exposed healthy human tissues, utilizing microarray data available in public repositories. Differential gene expression analysis (DGEA) was conducted using the R programming language. Consequently, four separate meta-analyses were conducted, after DEG lists were grouped depending on radiation type, radiation dose and time of collection post-irradiation. To highlight the biological background of each meta-analysis group, functional enrichment analysis and biological network construction were conducted. For HZE particle exposure at 8–24 h post-irradiation, the most interesting finding is the variety of DNA repair mechanisms that were downregulated, a fact that is probably correlated with complex DNA damage formation. Simultaneously, after X-ray exposure during the same hours after irradiation, DNA repair mechanisms continue to take place. Finally, in a further comparison of low- and high-LET radiation effects, the most prominent result is that autophagy mechanisms seem to persist and that adaptive immune induction seems to be present. Such bioinformatics approaches may aid in obtaining an overview of the cellular response to high-LET particles. Understanding these response mechanisms can consequently aid in the development of countermeasures for future space missions and ameliorate heavy ion treatments. [ABSTRACT FROM AUTHOR]

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies. [ABSTRACT FROM AUTHOR]