DLD-1 and MOLT-4 cell lines were cultured in a Rotating cell culture system to simulate microgravity and mRNA expression profile was observed in comparison to Static controls. Cells were grown in 10mL rotating vessels in an RCCS and in 60mm Petri dishes (test control respectively).Two replicates of test (Microgravity) and control (static) each from DLD-1 and MOLT-4 were analyzed by microarray. Simulated microgravity affected the solid tumor cell line DLD-1 markedly which showed a higher percentage of dysregulated genes compared to the hematological tumor cell line MOLT-4. Microgravity affects the cell cycle of DLD-1 cells and disturbs expression of cell cycle regulatory gene networks. Multiple microRNA host genes were dysregulated and significantly mir-22 tumor suppressor microRNA is highly upregulated in DLD-1.

DLD-1 and MOLT-4 cell lines were cultured in a rotating cell culture system to simulate microgravity and mRNA expression profile in comparison to Static controls. Cells were grown in 10mL rotating vessels in a Rotary Cell Culture System (RCCS) and in 60mm Petri dishes (test control respectively). Two replicates of test (Microgravity) and control (static) each from DLD-1 and MOLT-4 were analyzed by microarray. Simulated microgravity affected the solid tumor cell line DLD-1 markedly which showed a higher percentage of dysregulated genes compared to the hematological tumor cell line MOLT-4. Microgravity affects the cell cycle of DLD-1 cells and disturbs expression of cell cycle regulatory gene networks. Multiple microRNA host genes were dysregulated and significantly mir-22 tumor suppressor microRNA is highly upregulated in DLD-1.

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.

To investigate DNA methylation patterns in rat mammary carcinomas induced by ionizing radiation. DNA methylation patterns were analyzed in radiation-induced rat mammary carcinomas and normal mammary gland tissues using CpG island microarray.

In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a ground-based Rotating Wall Vessel (RWV) bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1g-incubated ones. Among these miR-9-5p miR-9-3p miR-155-5p miR-150-3p and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichments in the biological processes of immune/inflammatory response signal transduction regulation of response to stress regulation of programmed cell death and regulation of cell proliferation. We identified the correlation between miR-9-3p miR-155-5p miR-150-3p and miR-378-3p expression with that of genes involved in immune/inflammatory response (eg. IFNG and IL17F) apoptosis (eg. PDCD4 and PTEN) and cell proliferation (eg. NKX3-1 and GADD45A). Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation. microRNA expression profiling were carried out on total RNA extracted from PBLs of twelve healthy donors at the end of 24h-incubation time in MMG and in 1g conditions. Analyses were performed by using the Human miRNA Microarray kit (V2) (Agilent Technologies) that allows the detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs. By comparing the expression profile of MMG-incubated vs. 1g-incubated PBLs of the same donor we found 42 differentially expressed miRNAs 25 up-regulated and 17 down-regulated.

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.

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.

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity a condition of weightlessness experienced by astronauts during space missions which could have a synergistic action on cells increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of gamma-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover let-7i* miR-7 miR-7-1* miR-27a miR-144 miR-200a miR-598 miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles carried out on PBL of the same donors identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of Response to DNA damage is enriched when PBL are incubated in 1 g but not in MMG. Moreover some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole by integrating the transcriptome and microRNome we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. Overall Design: Gene expression signature was defined in PBL irradiated with gamma-rays (2.0 Gy) and incubated in modeled microgravity (mmg) and in parallel ground conditions (1g) for 24h. Five independent experiments were performed for each donor to address which mRNAs were regulated on IR stress. The level of each transcript was represented as Log2.

Microarray Analysis of Space-flown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors. We used microarrays to detail the gene expression of space-flown thymic tissue and identified distinct classes of up-regulated genes during this process. We report here microarray gene expression analysis in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5 fold or greater change. When these data were averaged (n=4) we identified 12 genes that were significantly up- or down-regulated by at least 1.5 fold after spaceflight (p < 0.05). Together these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress glucocorticoid receptor metabolism and T cell signaling activity. These data explain in part the reported systemic compromise of the immune system after exposure to the microgravity of space.

Accumulating data support the concept that ionizing radiation therapy (RT) has the potential to convert the tumor into an in situ individualized vaccine; however this potential is rarely realized by RT alone. Transforming growth factor xce xb2 (TGF xce xb2) is an immunosuppressive cytokine that is activated by RT and inhibits the antigen-presenting function of dendritic cells and the differentiation of effector CD8+ T cells. Here we tested the hypothesis that TGF xce xb2 hinders the ability of RT to promote anti-tumor immunity. Development of tumor-specific immunity was examined in a pre-clinical model of metastatic breast cancer. Mice bearing established 4T1 mouse mammary carcinoma treated with pan-isoform specific TGF xce xb2 neutralizing antibody 1D11 showed significantly improved control of the irradiated tumor and non-irradiated metastases but no effect in the absence of RT. Notably whole tumor transcriptional analysis demonstrated the selective upregulation of genes associated with immune-mediated rejection only in tumors of mice treated with RT+TGF xce xb2 blockade. Mice treated with RT+TGF xce xb2 blockade exhibited cross-priming of CD8+ T cells producing IFN xce xb3 in response to three tumor-specific antigens in tumor-draining lymph nodes which was not evident for single modality treatment. Analysis of the immune infiltrate in mouse tumors showed a significant increase in CD4+ and CD8+ T cells only in mice treated with the combination of RT+TGF xce xb2 blockade. Depletion of CD4+ or CD8+ T cells abrogated the therapeutic benefit of RT+TGF xce xb2 blockade. These data identify TGF xce xb2 as a master inhibitor of the ability of RT to generate an in situ tumor vaccine which supports testing inhibition of TGF xce xb2 during radiotherapy to promote therapeutically effective anti-tumor immunity. We used genome-wide microarray to depict main biological processes responsibles for the therapeutic benefit of the combination ofTGF-beta blockade and local radiotherapy. To gain a more comprehensice protrait of the effects of RT and TGFbeta blockade on gene expressionin tumors we collected 4T1 tumors 4 days after completion of RT. Three tumors from each group were then subjected to RNA extraction and hybridization on affymetrix array.