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: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:Direct irradiation of 3-dimensional skin model, Epi-200, with alpha-particles led to differential regulation of 166 genes: 16 and 150 genes were differentially expressed at 1 and 16 h postirradiation. Unlike the traditional 2-dimensional in vitro systems, Epi-200 made of the primary cells, epidermal human keratinocytes. It mimics the structure of the 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 comparing irradiated tissues with non-irradiated control, we have been able to measure global gene expression responses and reveal the affected biological pathways and molecular functions. The data were analyzed using BRB-Array Tools (NIH), and further gene ontology analysis was performed with Panther database (Applied Biosystems). Gene ontology analysis of the samples harvested in 16h after exposure showed that irradiation presumably affected the genes involved in cell-cell signaling (15 genes, , p=9.0 x E-04) ion transport (10 genes, p=0.00189) and amino acid metabolism (5 genes, p=0.0258). Among 16 genes differentially expressed in 1h after exposure we found NOTCH2 (ENST00000401649) and methyltransferase AOF1 (KDM1B). In the mammalian cells, NOTCH signaling pathway has a role in differentiation and intracellular communication. Moreover the intercellular domain of NOTCH regulates gene expression acting as a transcription factor. In turn, AOF1 affects the transcription via histone demethylation. Thus, irradiation with alpha-particles caused predominant downregulation of multiple genes in 1 and 16h after exposure. It also suggested that changes in cell metabolism initially affected transcriptional regulation and finally led to the rearrangement in expression of genes playing a role in biosynthesis and ion trafficking. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 1 and 16 h hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed using one tissue sample per a data point.

Project description:Biological response X-rays traditionally serves as a standard in comparative analysis of different qualities of ionizing radiation. Most such studies have utilized 2-dimensional culture systems, which may not fully represent responses in 3-dimensional tissues. To gain insight into biological responses to X-rays in tissue, we have profiled global gene expression in EPI-200, a 3-dimensional tissue model that imitates the structure and function of human epidermis, at 4, 16 and 24 hours after exposure to 2.5 Gy of X-rays. The most significant gene ontology groups were associated with cell cycle, cytokinesis, establishment and maintenance of chromatin architecture. Remarkably, genes with a role in cell cycle were predominantly downregulated at all time points while genes involved in the cell defense response - upregulated. Methyltransferases were predominantly upregulated at 4 and 16h while transcription factors - at 16h and 24h suggesting remodeling of the chromatin. Among the genes with a role in signal transduction, irradiation affected kinase modulators and microtubule binding motor proteins at all time points. Kinases, especially non-receptor serine/threonine protein kinases were predominantly upregulated at 16 and 24h suggesting a rearrangement in the signaling pathways. The results also confirm involvement in the biological response of the genes participating in p53 pathway that were overrepresented at 4 and 16h in the set of upregulated genes and ubiquitin proteasome pathway that were predominantly downregulated at 16h and 24h In the same time, growing numbers of altered in expression genes: 449 at 4h; 720 and 3986 genes - at 16 and 24h postirradiation, accordingly suggested that ionizing radiation caused long term changes in the gene expression the exceeded the time frame proposed for this study. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured at 4, 16 and 24 hours after exposure to the dose of 2.5 Gy of X-rays. Three independent experiments were performed in the each time point using one tissue sample per a data point.

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