Project description:Direct irradiation of 3-dimensional skin model, Epi-200, with alpha-particles led to differential regulation of 545 genes at 4 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 (Applied Biosystems). The responding pathways were identified jointly and separately for up- and downregulated genes. Receptors (12 genes, p=0.0467), including nuclear hormone receptors (3 genes, p=8.2 x E-04) and Ser/Thr receptors (3 genes, p=5.3 x E-03), chemokines (2 genes, p=0.0155) and signaling molecules were overrepresented among 123 upregulated genes while DNA binding proteins (27 genes, p=5.07 x E-04), particularly helicases (6 genes, p=3.61 x E-03) and, G-protein modulators (16 genes, p=4.4 x E-03) were overrepresented among 403 downregulated genes. Remarkably, 6 differentially regulated genes in this group were represented by helicases and methyltransferases (6 genes, p=0. 0534) involved in methylation of histones and DNA. These changes confirmed an importance in the biological response of two its basic components: receptor-regulated pathways and chromatin remodeling. Upregulation the first group supposes to increase sensitivity of irradiated cells to chemokines and cytokines while downregulation of the second group would likely affect the DNA methylation pattern and stimulate the transcription. 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 using one tissue sample per a data point.

Project description:Bystander mechanisms that originate in the areas surrounding a tissue damage presumably play an important role participating in wound healing and tissue remodeling. Thus, identification and characterization of bystander mechanisms will help to development of new treatments of patients with a radiation exposure. In the present study, we irradiated 3-dimensional tissue model of human epidermis, Epi-200 (Mat-Tek, Ashland, MA), with 2.5 Gy protons. 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 0.125-0.375, 0.375-0.625, 0.625-875 mm from the irradiation line. The data were analyzed using BRB-Array Tools (NIH), and further gene ontology analysis and network analysis was performed with Panther (Applied Biosystems) and IPA (Ingenuity), accordingly. Significantly responding genes were identified at all distances and included sets common to both direct and bystander responses. False discovery rate in bystander samples did not exceed 20% (p=0.001) and was sufficiently low in the samples obtained after the whole tissue exposure (0.06-1.16%). Analysis of the fragments cut at the same distance revealed 52, 54 and 88 differentially expressed genes. These gene lists overlapped each other had from 3 to 12 genes in common including CLED2, S100A7A. Samples obtained after the whole tissue exposure discovered 949 differentially expressed genes. Moreover, the performed gene ontology analysis showed there overrepresentation of TP53 pathway (pathways, p=2.04E-02), a common marker of direct irradiation response, and also overrepresentation of the following groups of genes: signal transduction (p=4.52E-04), cell communication (p=1.24E-04) and cell cycle in the category of biological processes; DNA helicase activity (p=2.54E-07), receptor binding (p=6.19E-04), calcium ion binding proteins (p=2.57E-03) as the molecular functions. Differentially expresses genes of bystander samples had few categories in common such as cell communication (p=2.36E-03) and signal transduction (p=2.42E-03) among the biological processes and receptor activity (p=4.54E-03) among the molecular functions. Categories specific for the bystander samples included G-protein coupled receptors (p=7.24E-03) and ligand-gated ion channels (p=4.16E-03) suggesting a role of external stimulation and ion trafficking in bystander mechanisms. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 16 hours after exposure to 2.5 Gy of protons. Four independent experiments were performed for the samples collected at different distances from the irradiation line (125-375, 375-625 and 625-875 micrometers) using three tissue fragments per a data point. Moreover, three sets of whole tissue irradited samples were also generated for 0 and 2.5 Gy (6 samples total) and used for comparison of bystander and direct responses.

Project description:Bystander mechanisms that originate in the areas surrounding a tissue damage presumably play an important role participating in wound healing and tissue remodeling. Thus, identification and characterization of bystander mechanisms will help to development of new treatments of patients with a radiation exposure. In the present study, we irradiated 3-dimensional tissue model of human epidermis, Epi-200 (Mat-Tek, Ashland, MA), with 2.5 Gy protons. 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 0.125-0.375, 0.375-0.625, 0.625-875 mm from the irradiation line. The data were analyzed using BRB-Array Tools (NIH), and further gene ontology analysis and network analysis was performed with Panther (Applied Biosystems) and IPA (Ingenuity), accordingly. Significantly responding genes were identified at all distances and included sets common to both direct and bystander responses. False discovery rate in bystander samples did not exceed 20% (p=0.001) and was sufficiently low in the samples obtained after the whole tissue exposure (0.06-1.16%). Analysis of the fragments cut at the same distance revealed 52, 54 and 88 differentially expressed genes. These gene lists overlapped each other had from 3 to 12 genes in common including CLED2, S100A7A. Samples obtained after the whole tissue exposure discovered 949 differentially expressed genes. Moreover, the performed gene ontology analysis showed there overrepresentation of TP53 pathway (pathways, p=2.04E-02), a common marker of direct irradiation response, and also overrepresentation of the following groups of genes: signal transduction (p=4.52E-04), cell communication (p=1.24E-04) and cell cycle in the category of biological processes; DNA helicase activity (p=2.54E-07), receptor binding (p=6.19E-04), calcium ion binding proteins (p=2.57E-03) as the molecular functions. Differentially expresses genes of bystander samples had few categories in common such as cell communication (p=2.36E-03) and signal transduction (p=2.42E-03) among the biological processes and receptor activity (p=4.54E-03) among the molecular functions. Categories specific for the bystander samples included G-protein coupled receptors (p=7.24E-03) and ligand-gated ion channels (p=4.16E-03) suggesting a role of external stimulation and ion trafficking in bystander mechanisms.

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.

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Bystander responses to 0.5Gy of alpha-particles in a human 3-dimensional skin model in 4h after exposure to ionizing radiation

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