Cesium-137 is a radionuclide of concern in fallout from reactor accidents or nuclear detonations. When ingested or inhaled it can expose the entire body for an extended period of time potentially contributing to serious health consequences ranging from acute radiation syndrome to increased cancer risks. In order to identify changes in gene expression that may be informative for detecting such exposure and to begin examining the molecular responses involved we have profiled global gene expression in mice injected with 137CsCl. We extracted RNA from the blood of control or 137CsCl-injected mice at 2 3 5 20 or 30 days after exposure. Gene expression was measured using Agilent Whole Mouse Genome Microarrays and the data was analyzed using BRB-ArrayTools. Three-month old male C57Bl/6 mice were injected intraperitoneally with 8.0 xc2 xb1 0.3 MBq 137CsCl solution in a volume of 50 xce xbcL or left as controls. Groups of treated and control mice were sacrificed at intervals during the first 2-30 days after exposure and total blood was collected using cardiac puncture. RNA was extracted from the blood globin-transcript reduced and subjected to whole genome expression microarray analysis.

To further development of our gene expression approach to biodosimetry we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish radiation dose across an exposure range relevant for medical decision-making in a radiological emergency. Human peripheral blood from healthy donors was irradiated ex vivo and a 74-gene consensus signature was identified that distinguished between four radiation doses (0.5 2 5 and 8 Gy) and control samples. The same set of genes separated samples by exposure level at both six and 24 hours after treatment with overlap evident only at the highest two doses (5 and 8 Gy). Expression of five genes (CDKN1A FDXR SESN1 BBC3 and PHPT1) from this signature was quantified in the same RNA samples by real-time PCR confirming low variability between donors as well as the predicted radiation response pattern. Experiment Overall Design: Radiation induced gene expression in human blood was measured at 6 and 24 hours after exposure to doses of 0 0.5 2 5 and 8 Gy g-rays. Five independent experiments were performed at each time (6 or 24 hours) using different donors for each experiment

To further development of our gene expression approach to biodosimetry we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish radiation dose across an exposure range relevant for medical decision-making in a radiological emergency. Human peripheral blood from healthy donors was irradiated ex vivo and a 74-gene consensus signature was identified that distinguished between four radiation doses (0.5 2 5 and 8 Gy) and control samples. The same set of genes separated samples by exposure level at both six and 24 hours after treatment with overlap evident only at the highest two doses (5 and 8 Gy). Expression of five genes (CDKN1A FDXR SESN1 BBC3 and PHPT1) from this signature was quantified in the same RNA samples by real-time PCR confirming low variability between donors as well as the predicted radiation response pattern. Experiment Overall Design: Radiation induced gene expression in human blood was measured at 6 and 24 hours after exposure to doses of 0 0.5 2 5 and 8 Gy g-rays. Five independent experiments were performed at each time (6 or 24 hours) using different donors for each experiment

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.

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.

Irradiated cell lines exposed to 1-10 Gy 2 Lymphoblastoid cell lines (GM15510 and GM15036) irradiated 1 2.5 5 7.5 10 Gy RNA is isolated and labeled using a T7 amplification Arcturus kit for hybridization on triplicate arrays.

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.

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

Transforming growth factor beta-1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR) both a carcinogen and a therapeutic agent induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/ml) or double-treated. All double-treated (IR+TGFbeta) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin beta-catenin and ZO-1 remodeling of the actin cytoskeleton and increased expression of mesenchymal markers N-cadherin fibronectin and vimentin. Furthermore double-treatment increased cell motility promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT even though IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this Erk phosphorylation were transiently induced by irradiation persisted in irradiated cells treated with TGFbeta and treatment with U0126 a Mek inhibitor blocked the EMT phenotype. Together these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression. Experiment Overall Design: Nonmalignant human mammary epithelial MCF10A cells (passages 106 and 108) were seeded at cloning density in 35mm dishes (10^5 cells/dish). Cell culture medium consisted of 3ml/dish of MGEM serum free medium (Cambrex Inc.) supplemented or not with 400pg/ml recombinant Transforming Growth Factor-beta. Cells were irradiated or not 5h post plating using 160 KV X-ray with a total dose of 2Gy. Sham IR-treated TGFbeta-treated and double-treated (IR+TGFbeta) MCF10A cells were harvested 8 days post-IR. Briefly cells were washed with PBS denatured in Trizol scraped off the dish and subjected to chloroform extraction. After centrifugation the upper phase was precipitated with an equal volume of isopropanol. RNA precipitates were resuspended in RNase free water and further purified on RNeasy columns (Qiagen Germany). RNA quality was assessed on an Agilent Bio-Analyzer. The dataset analyzed by microarray included biological duplicates for each treatment in two independent experiments and three sham treated samples. Microarray data were generated at the Lawrence Berkeley National Laboratory Molecular Profiling Laboratory (http://hta.lbl.gov) using a high-throughput automated GeneChip system (Affymetrix). Briefly target preparation HT_HG-U133A array plate hybridization setup washing and staining were performed on an Affymetrix robotic system (GCAS) using version 2.1 protocols. Scanning (protocol version 2.2.09) was performed on a CCD-based high throughput scanner (Affymetrix). Samples were analyzed and clustered with the (UNO) One Color GenetrafficTM software version 3.2-12 (Iobion Informatics LLC Stratagene La Jolla CA). Genes whose expression was specifically altered by treatment were defined as those in which dye ratio was more than 1.75-fold (mean log2ratio>0.8) from baseline in at least three out of the four treated samples compared to the three sham samples. Significance analysis tests (p<0.05) were performed using Excel between sham samples and either IR TGFbeta or TGFbeta+IR samples.

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels including miR-92b 137 660 and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579 608 548-3p and 585 are noted for targeting genes involved in radioresponsive mechanisms such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as hub regulators of specific cellular responses immediately down-regulated so as to stimulate DNA repair mechanisms followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together miRNAs may mediate signaling pathways in sequential fashion in response to radiation and may serve as biodosimetric markers of radiation exposure. Overall design: The gene expression patterns in human fibroblasts after 2.0 Gy of low-LET radiation was determined at 2 and 24 hrs post-irradiation time in technical triplicates. Control non-irradiated samples were also prepared in triplicates.