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.

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

Accumulating data suggest that the biological responses to high and low doses of radiation are qualitatively different necessitating the direct study of low dose responses. Most such studies have utilized 2-dimensional culture systems which may not fully represent responses in 3-dimensional tissues. To gain insight into low dose responses in tissue we have profiled global gene expression in EPI-200 a 3-dimensional tissue model from MatTek that imitates the structure and function of human epidermis at 4 16 and 24 hours after exposure to high (2.5 Gy) and low (0.1 Gy) doses of low LET protons. Untreated controls and samples exposed to 10 cGy or to 2.5 Gy were analyzed at three different times (4 16 or 24 hours after exposure). Three biological repeats were performed for each condition

Here we present a whole-genome survey of the murine transcriptomic response to physiologically-relevant radiation doses 2 and 8 Gy. There are 18 distinct biological samples here. Mice were exposed to ionizing radiation (Cesium-138 source) and whole blood was collected by cardiac puncture 6 hours post treatment. Doses were 0 (7 samples) 2 (5 samples) and 8 (6 samples) gy.

This is a genome-wide approach to identifying genes persistently induced in the mouse mammary gland by acute whole body low dose ionizing radiation (10cGy) 1 and 4 weeks after exposure. Gene expression that is modified under these parameters were compared between Tgfb1 wild type and heterozygote littermates in order to determine which genes induced or repressed by radiation were mediated via Tgfb1 status. Differential gene expression was analyzed in Tgfb1 heterozygote and wild type littermate 4th mammary glands after whole body exposure to an acute dose of 10cGy ionizing radiation. Estrus cycle was normalized in all mice two days prior to irradiation by injection with an estrogen and progesterone mixture. It is widely believed that the carcinogenic action of ionizing radiation is due to targeted DNA damage and resulting mutations but there is also substantial evidence that non-targeted radiation effects alter epithelial phenotype and the stromal microenvironment. Activation of transforming growth factor beta 1 (TGFbeta) is a non-targeted radiation effect that mediates cell fate decisions following DNA damage and regulates microenvironment composition; it could either suppress or promote cancer. Gene expression profiling shown herein demonstrates that low dose radiation (10 cGy) elicits persistent changes in Tgfb1 wild type and heterozygote murine mammary gland that are highly modulated by TGFbeta. We asked if such non-targeted radiation effects contribute to carcinogenesis by using a novel radiation chimera model. Unirradiated Trp53 null mammary epithelium was transplanted to the mammary stroma of mice previously exposed to a single low (10 -100 cGy) radiation dose. By 300 days 100% of transplants in irradiated hosts at either 10 or 100 cGy had developed Trp53 null breast carcinomas compared to 54% in unirradiated hosts. Tumor growth rate was also increased by high but not low dose host irradiation. In contrast irradiation of Tgfb1 heterozygote mice prior to transplantation failed to decrease tumor latency or increase growth rate at any dose. Host irradiation significantly reduced the latency of invasive ductal carcinoma compared to spindle cell carcinoma as well as those tumors negative for smooth muscle actin in wild type but not Tgfb1 heterozygote mice. However irradiation of either host genotype significantly increased the frequency of estrogen receptor negative tumors. These data demonstrate two concepts critical to understanding radiation risks. First non-targeted radiation effects can significantly promote the frequency and alter the features of epithelial cancer. Second radiation-induced TGFbeta activity is a key mechanism of tumor promotion. Keywords: Differential gene expression after low dose irradiation Two genotypes: TGBbeta1 heterozygote and wildtype mouse mammary glands. Two time points post-10cGy-irradiation per genotype (1 week 4 weeks); control time point was 1 week post-sham-irradiation. Two or three replicates per time point.

Transcriptional profiling of human lymphoblastoid TK6 cells comparing mock irradiated cells with cells exposed 24 hours previously to 1.67 Gy HZE (1 GeV/amu iron ions accelerated at the NASA Space Research Laboratory (NSRL) of Brookhaven National Laboratory) or 2.5 Gy 137Cs gamma rays. TK6 cells were mock irradiated or exposed to HZE or gamma-rays and RNA was harvested 24 hours later. 3 biological replicates were independently grown and harvested during three different runs at the NSRL. One replicate per array.

One of the most likely risks astronauts on long duration space missions face is exposure to ionizing radiation associated with highly energetic and charged heavy (HZE) particles. Since access to medical expertise on such a mission is limited at best early diagnosis and mitigation of such exposure is critical. In order to accurately determine the dosage within 1 hour post-exposure dose-dependent biomarkers are needed. Therefore we performed a dose-course transcriptional analysis for radiation exposure at 0 0.3 1.5 and 3.0 Gy with corresponding time point at 1 hour (hr) post-exposure using Affymetrix GeneChip Human Gene 1.0 ST v1 Array chips. The analysis of our data suggests a set of sensitive genetic biomarkers specific to each radiation level as well as generic radiation response biomarkers. Upregulated biomarkers can then be used within lab-on-a-chip (LOC) systems to detect exposure to ionizing radiation. A total of sixteen human samples representing radiation exposure at levels 0 Gy 0.3 Gy 1.5 Gy and 3.0 Gy at time point 1 hour (hr) post-exposure were constructed. Blood samples were extracted from four human volunteers and were irradiated. Leukocytes were extracted and gene expression was measured. Samples for all four volunteers were measured at 1 hr for all four dose levels resulting in four replicates at each dose level. Thus a total of 4 samples at each of the four radiation levels were sampled yielding the total of 16 samples.

Analysis of human peripheral blood 48 hours after irradiation ex vivo with graded doses of gamma rays. Results have been used in building and testing classifiers to predict exposure dose for use in radiological triage and also provide insight into immune cell responses. Results were compared with those from earlier times and from patients exposed in vivo. Peripheral blood from 5 healthy donors was exposed ex vivo to 0. 0.5 2 5 or 8 Gy gamma-rays and gene expression was analyzed up to 48 hours after exposure.

Understanding the possible impact of potential confounding factors is necessary for any approach to radiation biodosimetry. Potential confounding factors have not been fully addressed for gene expression-based biodosimetry approaches such as we are developing. To begin addressing this need we have used an ex vivo irradiated peripheral blood cell model to investigate the potential effect of smoking on the global radiation gene expression response and looked for genes that respond to radiation differently in smokers and non-smokers and also in males and females. The results indicate that only a small number of genes may be significantly confounded by either factor supporting the idea of developing peripheral blood gene expression strategies for radiation biodosimetry. Blood from each of 24 different donors was exposed to four doses of ionizing radiation (0 0.1 0.5 or 2 Gy) and analyzed using single-color microarray hybridization. The donors represented equal numbers of male and female smokers (1 or more packs a day) and non-smokers. There are 95 data sets in the study as the sample from one of the female smokers exposed to 2 Gy was lost.

Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body xce xb3 radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. 5 samples were analyzed: 2 controls at 2 months 1 2 Gy at 2 months and 2 7 Gy at 2 months