Project description:The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. It raises concerns for the interpretation of risks from exposure to low doses of ionizing radiation. Sparse data exists about the bystander signaling mechanisms and the ability to transmit damaging effects both spatially and temporally. To understand early signaling and cellular changes in bystanders, we have measured global gene expression 30 minutes after direct and bystander exposure to alpha particle in primary human lung fibroblasts. Gene ontology and pathway analyses suggested that the earliest measured changes at 30 minutes after treatment are in cell structure, motility and adhesion categories and a significant number of genes belong to the category of inflammation and cell-to-cell communication. We investigated time course gene expression profiles of matrix metalloproteinases 1 and 3 (MMP1 and MMP3), chemokine ligands 2, 3 and 5 (CXCL2, CXCL3 and CXCL5), interleukins 1a, 1b, 6 and 33 (IL1A, IL1B, IL6 and IL33) growth differentiation factor 15 (GDF15) and superoxide dismutase2 (SOD2) by real time quantitative PCR. These encode proteins involved in cellular signaling via the NFkappaB pathway and time course of mRNA levels revealed an increased response at 30 minutes after irradiation followed by another wave at 4 to 6 hours. We also investigated protein modifications in the AKT-GSK-3 signaling pathway and found that in irradiated cells AKT and GSK3beta are hyper-phosphorylated at 30 minutes and this effect is maintained until 4 hours after exposure. In bystanders there is a similar response with a delay of 30 minutes. In irradiated cells, inactivated GSK3beta led to decreased phosphorylation of beta-catenin. Our results are the first to show that the radiation induced bystander signal can induce a widespread gene expression response as early as 30 minutes after exposure and that these changes are accompanied by protein modification of signaling modules such as AKT and GSK3beta. There are 12 total samples, 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C), irradiated (alpha=A) and bystander (B), cells were harvested 0.5 hr after treatment

Project description:The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. It raises concerns for the interpretation of risks from exposure to low doses of ionizing radiation. Sparse data exists about the bystander signaling mechanisms and the ability to transmit damaging effects both spatially and temporally. To understand early signaling and cellular changes in bystanders, we have measured global gene expression 30 minutes after direct and bystander exposure to alpha particle in primary human lung fibroblasts. Gene ontology and pathway analyses suggested that the earliest measured changes at 30 minutes after treatment are in cell structure, motility and adhesion categories and a significant number of genes belong to the category of inflammation and cell-to-cell communication. We investigated time course gene expression profiles of matrix metalloproteinases 1 and 3 (MMP1 and MMP3), chemokine ligands 2, 3 and 5 (CXCL2, CXCL3 and CXCL5), interleukins 1a, 1b, 6 and 33 (IL1A, IL1B, IL6 and IL33) growth differentiation factor 15 (GDF15) and superoxide dismutase2 (SOD2) by real time quantitative PCR. These encode proteins involved in cellular signaling via the NFkappaB pathway and time course of mRNA levels revealed an increased response at 30 minutes after irradiation followed by another wave at 4 to 6 hours. We also investigated protein modifications in the AKT-GSK-3 signaling pathway and found that in irradiated cells AKT and GSK3beta are hyper-phosphorylated at 30 minutes and this effect is maintained until 4 hours after exposure. In bystanders there is a similar response with a delay of 30 minutes. In irradiated cells, inactivated GSK3beta led to decreased phosphorylation of beta-catenin. Our results are the first to show that the radiation induced bystander signal can induce a widespread gene expression response as early as 30 minutes after exposure and that these changes are accompanied by protein modification of signaling modules such as AKT and GSK3beta.

Project description:The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is now well established. It raises concerns for the interpretation of risks arising from exposure to low doses of ionizing radiation. However, the regulatory mechanisms involved in the bystander response have not been well elucidated. To provide insight into the signaling pathways responding in bystanders, we have measured global gene expression four hours after bystander and direct alpha particle exposure of primary human lung fibroblasts.Although common p53-regulated radiation response genes like CDKN1A were expressed at elevated levels in the directly exposed cultures, they showed little or no change in the bystanders. In contrast, genes regulated by NFkappaB, such as PTGS2 (cyclooxygenase-2), IL8 and BCL2A1, responded nearly identically in bystander and irradiated cells. This trend was substantiated by gene ontology and pathway analyses of the microarray data, which suggest that bystander cells mount a full NFkappaB response, but a muted or partial p53 response. In time-course analyses, quantitative real-time PCR measurements of CDKN1A showed the expected 4-hour peak of expression in irradiated but not bystander cells. In contrast, PTGS2, IL8 and BCL2A1 responded with two waves of expression in both bystander and directly irradiated cells, one peaking at half an hour and the other between four and six hours after irradiation.Two major transcriptional hubs that regulate the direct response to ionizing radiation are also implicated in regulation of the bystander response, but to dramatically different degrees. While activation of the p53 response pathway is minimal in bystander cells, the NFkappaB response is virtually identical in irradiated and bystander cells. This alteration in the balance of signaling is likely to lead to different outcomes in irradiated cells and their bystanders, perhaps leading to greater survival of bystanders and increased risk from any long-term damage they have sustained.

Project description:The radiation bystander effect is an important component of the overall biological response of tissues and organisms to ionizing radiation. Little is known about the contribution of genome level changes in neighboring bystander cells to tissue and organ stress after irradiation. The timing of these changes is critical in the physiological context and these questions can only be answered by studying signaling and global transcriptomics in a chronological way. Here, we present a strategy to identify different biologically important signaling modules that act in concert in the radiation and bystander responses. We used time series gene expression analysis of normal human fibroblast cells measured at 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours after exposure to radiation coupled with a novel clustering method targeted to short time series, Feature Based Partitioning around medoids Algorithm (FBPA), to look for genes that were potentially co-regulated. This method uses biologically meaningful features of the expression profile and dimension augmentation to address the analysis of sparse data sets such as ours. We applied FBPA and Short Time series Expression Miner (STEM) to the same datasets and present the results of our comparisons using computational metrics as well as biological enrichment. Enrichment showed that gene expression in irradiated cells fell into broad categories of signal transduction, cell cycle/cell death and inflammation/immunity; but only FBPA clustered functions well. In bystander cells, the gene expression response was also broadly categorized into functions associated with cell communication and motility, signal transduction and inflammation; but neither STEM nor FBPA separated biological functions as well as in irradiated samples. Network analysis revealed that p53 and NF-kappaB were central players in gene expression in both irradiated and bystander gene clusters. Analysis of individual clusters also suggested new regulators of gene expression in the radiation and bystander response that may act at the epigenetic level such as histone deacetylases (HDAC1 and HDAC2) and methylases (KDM5B) that can act as strong transcription repressors. Based on these results, we propose a novel time series clustering method, FBPA, as a powerful approach that can be applied to sparse data sets (including genomic profiling data), where the choice of features selected for clustering and stringent statistical outcome analysis can augment our knowledge of the underlying cellular mechanisms in biological processes. There are 72 total samples, 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C), irradiated (alpha=A) and bystander (B), cells were harvested at 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours after treatment

Project description:The radiation bystander effect is an important component of the overall biological response of tissues and organisms to ionizing radiation. Little is known about the contribution of genome level changes in neighboring bystander cells to tissue and organ stress after irradiation. The timing of these changes is critical in the physiological context and these questions can only be answered by studying signaling and global transcriptomics in a chronological way. Here, we present a strategy to identify different biologically important signaling modules that act in concert in the radiation and bystander responses. We used time series gene expression analysis of normal human fibroblast cells measured at 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours after exposure to radiation coupled with a novel clustering method targeted to short time series, Feature Based Partitioning around medoids Algorithm (FBPA), to look for genes that were potentially co-regulated. This method uses biologically meaningful features of the expression profile and dimension augmentation to address the analysis of sparse data sets such as ours. We applied FBPA and Short Time series Expression Miner (STEM) to the same datasets and present the results of our comparisons using computational metrics as well as biological enrichment. Enrichment showed that gene expression in irradiated cells fell into broad categories of signal transduction, cell cycle/cell death and inflammation/immunity; but only FBPA clustered functions well. In bystander cells, the gene expression response was also broadly categorized into functions associated with cell communication and motility, signal transduction and inflammation; but neither STEM nor FBPA separated biological functions as well as in irradiated samples. Network analysis revealed that p53 and NF-kappaB were central players in gene expression in both irradiated and bystander gene clusters. Analysis of individual clusters also suggested new regulators of gene expression in the radiation and bystander response that may act at the epigenetic level such as histone deacetylases (HDAC1 and HDAC2) and methylases (KDM5B) that can act as strong transcription repressors. Based on these results, we propose a novel time series clustering method, FBPA, as a powerful approach that can be applied to sparse data sets (including genomic profiling data), where the choice of features selected for clustering and stringent statistical outcome analysis can augment our knowledge of the underlying cellular mechanisms in biological processes.

Project description:Radiation affects tissue and cellular integrity at the level of DNA, protein and metabolites of the cell and extracellular space. The effects of radiation are not limited to targeted cells and tissue and radiation induced bystander effects are significant to exposed individuals in accidental or therapeutic situations. These non-targeted effects of radiation have been studied extensively at the low dose range where they appear to have adverse effects on cells and surrounding environments. The requirement of cellular contact and shared fluid media has been established as critical to the bystander effect yet there is not much known about the actual signaling mechanism and its ability to transmit the damaging effect over space and time. Experimental cell types and context within the tissue are also quite important to the nature and extent of this bystander effect and must be considered when drawing parallels at the organismal level. Our approach was to use a genomic level analysis of global mRNA expression in primary lung fibroblast cells to understand the cellular triggers and mechanism of the bystander effect. Gene ontology and pathway analyses suggested that the p53 induced transcriptional response appears muted in bystanders while cytokine and cell signaling mechanisms such as those controlled by NFkB and p38 MAPK are highly active in both populations. We validated a large number of genes that are significantly changed at 4hrs after irradiation in both irradiated and bystander populations. We investigated time course gene expression profiles of cyclooxygenase2 (PTGS2), interleukin 8 (IL8) and BCL2 related protein 2 (BCL2A1), as genes that are involved in cellular signaling via the NFkB pathway, which revealed that there is a dramatic response at 0.5hr after irradiation followed by another wave at 4hr in both populations. The induction of interleukins such as cytokine IL8 and chemokine IL6 at the transcriptional level is both early and amplified and if followed by translation and secretion of these proteins could explain the concerted response seen in bystander cells. Our results are the first to show that there is a significant and distinct global response of cellular signaling genes in bystander cells with some genes showing a response as early as 0.5hr after irradiation which implies a fast moving intercellular signal that leads to a concerted response in the irradiated and bystander populations. Keywords: gene expression fold change There are 12 total samples, 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C), irradiated (alpha=A) and bystander (B)

Dataset Information

Regulation of early signaling and gene expression in the alpha-particle and bystander response of IMR-90 human fibroblasts.

Publications

Regulation of early signaling and gene expression in the alpha-particle and bystander response of IMR-90 human fibroblasts.

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