8 research outputs found
Modulation of Gene Expression in Human Lung Carcinoma Cells by Hypoxia and Exposure to Ionizing Radiation
Carbon ion (C-ion) therapy is a relatively new alternative to classical X-ray treatment in
cancer radiation therapy. It has multiple benefits from causing less damage to the
surrounding tissues to lowering metastatic responses and being more effective against
hypoxic tumors, where classical X-ray therapy often struggles. While the physical
differences of these radiation therapy settings are well understood, knowledge about changes
in gene regulation after exposure to these two radiation types is incomplete, especially
concerning the radiation exposure under hypoxic conditions.
To tackle this problem, A549 human lung carcinoma cells were exposed to these two
different radiations under normoxia and hypoxia. RNA was extracted four hours after
exposure and gene expression was then performed by RNA sequencing. In this work, a
comprehensive bioinformatic analysis of the obtained data was performed, including the
following steps: Running it through a DeSeq2 pipeline, then identification and removal of
outliers. This process was followed by data exploration to assess the data without outliers.
A GSEA analysis was used to identify key GO term groups and interesting core genes,
followed with an indebt analysis of the selected gene sets.
Three GSEA groups were identified that under all conditions were affected in Cell Cycle
Regulation, RNA- and Protein Regulation, Cellular Responses and Development. While
these groups were the same between X-ray treated samples and C-ion treated samples, the
specific affected genes differ. Twenty genes were identified that in the selected gene sets
behaved differently between the radiation types. Among them is a group of genes coding for
heat shock proteins (HSP), behaving specifically different after C-ion radiation treatment
combined with hypoxia. This points to a connection between these conditions and the
regulation of HSP expression. The analysis also revealed that genes involved in the AKT
pathway and NF-κB pathway are over-represented in genes that differ between X-rays and
C-ions exposure under normoxia and hypoxia, with the AKT pathway being more prevalent.
However, genes that differ specifically under hypoxia tend to only be involved in the NF-
κB pathway. While lastly identifying potential target candidate genes for combined chemo
and C-ion radiation therapy, PVRL4, PDGFRB NEFL, DRAXIN were found to be
potentially promising knockdown targets, as well as EPHB3 as a target for enhancement.For best results from the auspicious method of C-ion radiation in cancer treatment, it must
be adjusted for its exact biological effects, the discovery of which this thesis provides a
baseline point for
NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia
International audienceCellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes’ upregulation, as well as its target cytokines’ synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells
Hypoxia Changes Energy Metabolism and Growth Rate in Non-Small Cell Lung Cancer Cells
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
Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells
Hypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance
through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during
irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions
due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or
clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity
of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (¹²C) ions.
The mechanisms responsible for ¹²C ions’ potential to overcome hypoxia-induced radioresistance
are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells
following exposure to X-rays or ¹²C ions was investigated along with cell cycle progression and gene
expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated
under normoxia (20% O₂) or hypoxia (1% O₂) for 48 h and then irradiated with X-rays (200 kV) or 12C
ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA)
assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were
analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined
by flow cytometry of 4′,6-diamidino-2-phenylindole-stained cells. The global transcription profile
post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during
and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of ¹²C ions was much higher than that of X-rays. Cell survival under
hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to ¹²C
ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged.
DSB induction and resolution following irradiation were not significantly different under normoxia
and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for
both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration
and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance
may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility
and migration in A549 cells rather than less DNA damage or faster repair
Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells
International audienceHypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (12C) ions. The mechanisms responsible for 12C ions’ potential to overcome hypoxia-induced radioresistance are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells following exposure to X-rays or 12C ions was investigated along with cell cycle progression and gene expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h and then irradiated with X-rays (200 kV) or 12C ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA) assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined by flow cytometry of 4′,6-diamidino-2-phenylindole-stained cells. The global transcription profile post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of 12C ions was much higher than that of X-rays. Cell survival under hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to 12C ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged. DSB induction and resolution following irradiation were not significantly different under normoxia and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility and migration in A549 cells rather than less DNA damage or faster repair
Minimum Information about an Uncultivated Virus Genome (MIUViG)
International audienceWe present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere