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)

Project description:Ionizing radiation (IR) not only affects cells that are directly irradiated but also their non-irradiated neighbors, which show responses known as bystander effects. While bystander and direct responses have several common end points including apoptosis and micronucleation, chromatin remodeling and altered levels or activities of regulatory proteins, they can be quantitatively and qualitatively different. The majority of studies of radiation bystander effects have utilized 2-dimensional in vitro systems, but we have recently demonstrated such effects in EPI-200 (Mat-Tek, Ashland, MA), a 3-dimensional tissue model that precisely imitates the structure and function of human epidermis. Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By exposing only a thin strip across the center of the EPI-200 tissue, we have been able to measure global gene expression responses in directly irradiated and bystander cells located at 0, 0.25, 0.5, 0.75 and 1mm from the irradiation line. The data were analyzed using BRB-Array Tools (NIH), and further network analysis was performed with IPA (Ingenuity). Significantly responding genes were identified at all distances and included sets common to both direct and bystander responses. For instance, all sets demonstrated upregulation of a major component of the drug metabolism pathway, CYP1B1, and downregulation of MMP1, an enzyme involved in degradation of extracellular matrix. In contrast, PTGS2, a gene strongly implicated in the bystander response was upregulated in directly irradiated tissues, but actually downregulated in bystander cells. This effect may be time dependent, but may also suggest activation of bystander signaling mechanisms different from those observed in 2-dimensional cell cultures. According to network analysis of our results, the genes responding in bystander tissue fell into 5 major categories: cell death, cell communication, cell differentiation, stress response, and response to wounding, suggesting active intracellular communication in bystander tissue. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured at 4 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed for the samples collected at different distances from the irradiation line (250-500, 500-750 and 750-1000 micrometers) using three tissue fragments per a data point.

Project description:Ionizing radiation (IR) not only affects cells that are directly irradiated but also their non-irradiated neighbors, which show responses known as bystander effects. While bystander and direct responses have several common end points including apoptosis and micronucleation, chromatin remodeling and altered levels or activities of regulatory proteins, they can be quantitatively and qualitatively different. The majority of studies of radiation bystander effects have utilized 2-dimensional in vitro systems, but we have recently demonstrated such effects in EPI-200 (Mat-Tek, Ashland, MA), a 3-dimensional tissue model that precisely imitates the structure and function of human epidermis. Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By exposing only a thin strip across the center of the EPI-200 tissue, we have been able to measure global gene expression responses in bystander cells located at 0.125 and 0.625 um from the irradiation line, in 16h after irradiation. The data were analyzed using BRB-Array Tools (NIH), and further network analysis was performed with IPA (Ingenuity). Significantly responding genes were identified at the both distances. For instance, all sets demonstrated upregulation of two key enzymes of the lipid biosynthesis, UGT1 and PITPNB, and downregulation of proapoptotic proteins: BAX and ARHGEF5. In contrast, several proteins involved in transcriptional repression (CHD6, CHD8 andWRNIP1) were strongly upregulated suggesting a rearrangement in the gene transcription. These changes suggest an activation of bystander mechanisms different from those observed in 2-dimensional cell cultures. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 16 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed for the samples collected at different distances from the irradiation line (125-625 and 625-1125 um) using three tissue fragments per a data point.

Project description:Background: The radiation bystander response is an important component of the overall response of cells to radiation and critical to understanding health risks of radiation exposure to humans. The mechanism of radiation response includes inter-cellular signaling and intra-cellular communication by which the bystander signal is propagated. Methods: We measured the bystander response to 1Gy a-particle radiation in Mrad9-/- mouse stem cells and H1299shRAD9 cells, using chromosomal aberration and micronucleus formation as DNA damage endpoints. In the H1299 model we used whole genome microarray analyses to profile the transcriptome of irradiated and bystander cells. Results: We investigated the role of RAD9 in the bystander response and showed that depletion or mutation of RAD9 had an effect of increasing chromosomal structural damage as well as micronucleus formation in bystander cells. The enhancement of the damage effect correlated strongly with a transcriptomic response in critical pathways. RAD9 depletion affected many pathways in the cell, including the UV-MAPK pathway, involving p38MAPK members, STAT1 and PARP1 at the mRNA levels. There was an overall reduction of RNA biogenesis of gene members of this pathway suggesting that perhaps these signaling pathways do not function optimally after RAD9 depletion. Using network analysis we found there may be differential activation of transcriptional regulators between the irradiated and bystander cells involving the SP1 and NUPR1 transcription factors. Network analysis also suggested that HIF1a (Hypoxia induced factor 1a) activation could be a negative predictor of the bystander effect and perhaps that local hypoxic stress observed by cells that are directly exposed to radiation may predict whether or not they will elicit a bystander response. Gene expression in H1299 cells was measured at 4 hours after exposure to 1 Gy a-particles. There were two groups based on RAD9 status, RAD9 normal and RAD9 depleted by siRNA. In each of these groups, sham irradiated, direct irradiated cells for positive bystanders, positive bystanders, direct irradiated cells for negative bystanders and negative bystanders; were identified based on micronucleus responses. Five biological replicates were analyzed for each experimental group.

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