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.

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.

Age plays a major role in tumor incidence and is an important consideration when modeling the carcinogenesis process or estimating cancer risks. Epidemiological data show that from adolescence through middle age cancer incidence increases with age. This effect is commonly attributed to a lifetime accumulation of cellular particularly DNA damage. However during middle-age the incidence begins to decelerate and for many tumor sites it actually decreases at sufficiently advanced ages. We investigated if the observed deceleration and potential decrease in incidence could be attributed to a decreased capacity of older hosts to support tumor progression and whether HZE (high atomic number (Z) high energy (E)) radiation differentially modulates tumor progression in young versus middle-age hosts issues relevant to estimating carcinogenesis risk for astronauts. Lewis lung carcinoma (LLC) cells were injected into syngeneic mice (143 and 551 days old) which were then subject to whole-body 56Fe irradiation (1GeV/amu). Three findings emerged: 1) among unirradiated animals substantial inhibition of tumor progression and significantly decreased tumor growth rates were seen for middle-aged mice compared to young mice; 2) whole-body 56Fe irradiation (1GeV/amu) inhibited tumor progression in both young and in middle-aged mice (with greater suppression seen in case of young animals) with little effect on tumor growth rates; and 3) 56Fe irradiation (1GeV/amu) suppressed tumor progression in young mice to a degree not significantly different than transiting from young to middle-aged. Thus 56Fe irradiation (1GeV/amu) acted similar to aging with respect to tumor progression. We further investigated the molecular underpinnings driving the radiation modulation of tumor dynamics in young and middle-aged mice. Through global gene expression analysis the key players FASN AKT1 and the CXCL12/CXCR4 complex were determined to be contributory. In sum these findings demonstrate a reduced capacity of middle-aged hosts to support the progression phase of carcinogenesis and identify molecular factors contributory to HZE radiation modulation of tumor progression as a function of age. For genome-wide expression profiling of tumor tissue Mouse WG-6 BeadArray chips (Illumina San Diego CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion Austin TX) and labeled from all replicate biological samples for each condition. The number of tumor sample replicates used from each condition is as follows: 10 samples from young unirradiated mice 8 samples from young irradiated mice 7 samples from middle-aged unirradiated mice 5 samples from middle-aged irradiated mice. Total RNA was isolated and purified using Trizol (Invitrogen) or RNeasy (Qiagen) quantified and qualified using Agilent Bioanalyzer (Agilent) and samples were deemed suitable for amplification and hybridization if they had O.D. 260/280 = 1.7 - 2.1 28s/18s = 2:1 RIN (RNA integrity number) >7. Total RNA of 500ng per sample was amplified using Ambion TotalPrep (Ambion) and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C the chips were washed and then scanned using the Illumina iScan (Illumina) and the data were analyzed using GenomeStudio (Illumina). Data were first analyzed for gene expression and then culled for present genes (genes that meet the criteria of detection p-value < 0.05). Expression above background was included in an expressed genes working data set for further analyses. Rank variant normalization was applied to the data before extensive analysis. Differential gene expression analysis was used to compare to the reference group young unirradiated mice and genes were then evaluated and validated.

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.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi and tongue (as a new potential in-flight negative control) adaptation to microgravity at global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice (False Discovery Rrate < 0,05 and fold change < -2 and > +2) while only a small number of genes were found differentially regulated in tongue muscle ( BF vs. BG = 27 genes). Overall Design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF) mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG) mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC) mice housed in a standard animal facility.

We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b CD31 CD44 Ly6C Ly6G F4/80 Mac2 c-Fos as well as c-Fms. In addition RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b CD31 F4/80 Mac2 Ly6C and c-Fos. However there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.

Transcriptional profiling of mammary tissue irradiated at 10 weeks of age with either 100 cGy sparsely ionizing gamma-rays or 10 cGy or 30 cGy densely ionizing radiation (350 MeV/amu Si). Mammary tissue was collected 1 weeks 4 weeks and 12 weeks post-irradiation. Four radiation treatment groups: sham 100 cGy sparsely ionizing gamma-rays 10 cGy or 30 cGy densely ionizing radiation (350 MeV/amu Si). Three time points post-irradiation (1 4 and 12 weeks). Three or four replicates per time point.

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.

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.

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