Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.     

Alterations in gene expression in rat skin exposed to 56Fe ions and dietary vitamin A acetate

The purpose of the present work was to examine gene expression patterns in rat skin exposed to a beam of (56)Fe ions, a surrogate for the high-energy, heavy-ion galactic radiation background, as a basis for obtaining a better understanding of the possible mechanism(s) behind the radioprotective activity of vitamin A. A 2 x 4-cm rectangle of dorsal rat skin was exposed to 1.01 GeV/nucleon (56)Fe ions generated by the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Gene expression patterns were monitored in either the presence or absence of a 250-ppm dietary supplement of vitamin A acetate in powdered lab chow. Although vitamin A and other retinoids show anti-carcinogenic activity in several animal models, the underlying changes in gene expression have not been examined extensively. At either 1 or 7 day after irradiation, a 1-cm square of irradiated and control rat skin was excised and analyzed using the Affymetrix rat microarray (RG_U34A) system. Microarray responses were displayed and processed by GeneSpring 7.0 and GOTree software. At 1 day after 3 Gy of (56)Fe-ion irradiation, the expression of 110 genes was significantly up-regulated (P < = 0.05) in comparison to levels in control rat skin, while no genes were altered by the vitamin A acetate supplement alone. Combined with (56)Fe-ion radiation, the vitamin A acetate supplement blocked the expression of 88 (80%) of the 110 genes and eliminated 16 of 18 gene categories that were significantly altered (all increased) by the (56)Fe-ion radiation. Categories with large numbers of genes eliminated by the retinoid included response to stress, 33 genes; response to biotic stimulus, 38 genes; signal transduction, 35 genes; and regulation of cellular/physiological process, 40 genes. Even for immune response and response to biotic stimulus, the only two categories that remained significantly altered in the presence of the vitamin, the combined number of altered genes was reduced from 74 to 13. No significant alterations in gene expression were found at 7 days relative to the numbers in controls. The results indicate that at 1 day dietary vitamin A acetate strongly interfered with (56)Fe-ion-induced gene expression within the broad categories of stimulus- and stress-related genes, implying that the latter gene categories likely play a role in the radioprotective action of the vitamin.     

Early gene expression profile in mouse brain after exposure to ionizing radiation

Acute changes in the gene expression profile in mouse brain after exposure to ionizing radiation were studied using microarray analysis. RNA was isolated at 0.25, 1, 5 and 24 h after exposure to 20 Gy and at 5 h after exposure of the whole brain of adult mice to 2 or 10 Gy. RNA was hybridized onto 15K cDNA microarrays, and data were analyzed using GeneSpring and Significant Analysis of Microarray. Radiation modulated the expression of 128, 334, 325 and 155 genes and ESTs at 0.25, 1, 5 and 24 h after 20 Gy and 60 and 168 at 5 h after 2 and 10 Gy, respectively. The expression profiles showed dose- and time-dependent changes in both expression levels and numbers of differentially modulated genes and ESTs. Seventy-eight genes were modulated at two or more times. Differentially modulated genes were associated with 12 different classes of molecular function and 24 different biological pathways and showed time- and dose-dependent changes. The change in expression of four genes (Jak3, Dffb, Nsep1 and Terf1) after irradiation was validated using quantitative real-time PCR. Up-regulation of Jak3 was observed in another mouse strain. In mouse brain, there was an increase of Jak3 immunoreactivity after irradiation. In conclusion, changes in the gene profile in the brain after irradiation are complex and are dependent on time and dose, and genes with diverse functions and pathways are modulated.     

Detection of oxidative clustered DNA lesions in X-irradiated mouse skin tissues and human MCF-7 breast cancer cells

Bistranded oxidative clustered DNA lesions are closely spaced lesions (1-10 bp) that challenge the DNA repair mechanisms and are associated with genomic instability. The endogenous levels of oxidative clustered DNA lesions in cells of human cancer cell lines or in animal tissues remain unknown, and these lesions may persist for a long time after irradiation. We measured the different types of DNA clusters in cells of two human cell lines, MCF-7 and MCF-10A, and in skin obtained from mice exposed to either 12.5 Gy or sham X radiation. For the detection and measurement of oxidative clustered DNA lesions, we used adaptations of number average length analysis, constant-field agarose gel electrophoresis, putrescine, and the repair enzymes APE1, OGG1 (human) and Nth1 (E. coli). Increased levels of all cluster types were detected in skin tissue from animals exposed to radiation at 20 weeks postirradiation. The level of endogenous (no radiation treatment) oxidative clustered DNA lesions was higher in MCF-7 cells compared to nonmalignant MCF-10A cells. To the best of our knowledge, this is the first study to demonstrate persistence of oxidative clustered DNA lesions for up to 20 weeks in animal tissues exposed to radiation and to detect these clusters in human breast cancer cells. This may underscore the biological significance of clustered DNA lesions.     

Effects of lipopolysaccharide on 56Fe-particle radiation-induced impairment of synaptic plasticity in the mouse hippocampus

Space radiation, including high-mass, high-Z, high-energy particles (HZE; e.g. (56)Fe), represents a significant health risk for astronauts, and the central nervous system (CNS) may be a vulnerable target. HZE-particle radiation may directly affect neuronal function, or during immunological challenge, it may alter immune system-to-CNS communication. To test these hypotheses, we exposed mice to accelerated iron particles ((56)Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only) and 1 month later prepared hippocampal slices to measure the effects of radiation on neurotransmission and synaptic plasticity in CA1 neurons. In a model of immune system-to-CNS communication, these electrophysiological parameters were measured in irradiated mice additionally challenged with the peripheral immunological stressor lipopolysaccharide (LPS) injected intraperitoneally 4 h before the slice preparation. Exposure to (56)Fe particles alone increased dendritic excitability and inhibited plasticity. In control mice (0 Gy), LPS treatment also inhibited synaptic plasticity. Paradoxically, in mice exposed to 2 Gy, the LPS treatment restored synaptic plasticity to levels similar to those found in controls (0 Gy, no LPS). Our results indicate that HZE-particle radiation alters normal electrophysiological properties of the CNS and the hippocampal response to LPS.     

Differential expression of oxidative stress and extracellular matrix remodeling genes in low- or high-dose-rate photon-irradiated skin

Changes in the expression of genes implicated in oxidative stress and in extracellular matrix (ECM) remodeling and selected protein expression profiles in mouse skin were examined after exposure to low-dose-rate or high-dose-rate photon irradiation. ICR mice received whole-body γ rays to total doses of 0, 0.25, 0.5 and 1 Gy at dose rates of 50 cGy/h or 50 cGy/min. Skin tissues were harvested for characterization at 4 h after irradiation. For oxidative stress after low-dose-rate exposure, 0.25, 0.5 and 1 Gy significantly altered 27, 23 and 25 genes, respectively, among 84 genes assessed (P < 0.05). At doses as low as 0.25 Gy, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly altered, with changes >2-fold compared to 0 Gy. For an ECM profile, 18-20 out of 84 genes were significantly up- or downregulated after low-dose-rate exposure. After high-dose-rate irradiation, of 84 genes associated with oxidative stress, 16, 22 and 22 genes were significantly affected after 0.25, 0.5 and 1 Gy, respectively. Compared to low-dose-rate radiation, high-dose-rate exposure resulted in different ECM gene expression profiles. The most striking changes after low-dose-rate or high-dose-rate exposure on ECM profiles were on genes encoding matrix metalloproteinases (MMPs), e.g., Mmp2 and Mmp15 for low dose rate and Mmp9 and Mmp11 for high dose rate. Immunostaining for MMP-2 and MMP-9 proteins showed radiation dose rate-dependent differences. These data revealed that exposure to low total doses with low-dose-rate or high-dose-rate photon radiation induced oxidative stress and ECM-associated alterations in gene expression profiles. The expression of many genes was differentially regulated by different total dose and/or dose-rate regimens.     

Expression profile of DNA damage signaling genes in 2 Gy proton exposed mouse brain

Exposure of living systems to radiation results in a wide assortment of lesions, the most significant of is damage to genomic DNA which alter specific cell functions including cell proliferation. The radiation induced DNA damage investigation is one of the important area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes such as damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2 Gy proton exposed mouse brain tissues as compared to control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed cells undergo severe DNA damage which in turn destabilize the chromatin stability.     

Changes in MicroRNA expression patterns in human fibroblasts after low-LET radiation

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.     

A New Model of Biodosimetry to Integrate Low and High Doses

Biological dosimetry, that is the estimation of the dose of an exposure to ionizing radiation by a biological parameter, is a very important tool in cases of radiation accidents. The score of dicentric chromosomes, considered to be the most accurate method for biological dosimetry, for low LET radiation and up to 5 Gy, fits very well to a linear-quadratic model of dose-effect curve assuming the Poisson distribution. The accuracy of this estimation raises difficulties for doses over 5 Gy, the highest dose of the majority of dose-effect curves used in biological dosimetry. At doses over 5 Gy most cells show difficulties in reaching mitosis and cannot be used to score dicentric chromosomes. In the present study with the treatment of lymphocyte cultures with caffeine and the standardization of the culture time, metaphases for doses up to 25 Gy have been analyzed. Here we present a new model for biological dosimetry, which includes a Gompertz-type function as the dose response, and also takes into account the underdispersion of aberration-among-cell distribution. The new model allows the estimation of doses of exposures to ionizing radiation of up to 25 Gy. Moreover, the model is more effective in estimating whole and partial body exposures than the classical method based on linear and linear-quadratic functions, suggesting their effectiveness and great potential to be used after high dose exposures of radiation.     

Genome-wide detection of allelic gene expression in hepatocellular carcinoma cells using a human exome SNP chip

Allelic variations in gene expression influence many biological responses and cause phenotypic variations in humans. In this study, Illumina Human Exome BeadChips containing more than 240,000 single nucleotide polymorphisms (SNPs) were used to identify changes in allelic gene expression in hepatocellular carcinoma cells following lipopolysaccharide (LPS) stimulation. We found 17 monoallelically expressed genes, 58 allelic imbalanced genes, and 7 genes showing allele substitution. In addition, we also detected 33 differentially expressed genes following LPS treatment in vitro using these human exome SNP chips. However, alterations in allelic gene expression following LPS treatment were detected in only three genes (MLXIPL, TNC, and MX2), which were observed in one cell line sample only, indicating that changes in allelic gene expression following LPS stimulation of liver cells are rare events. Among a total of 75 genes showing allelic expression in hepatocellular carcinoma cells, either monoallelic or imbalanced, 43 genes (57.33%) had expression quantitative trait loci (eQTL) data, indicating that high-density exome SNP chips are useful and reliable for studying allelic gene expression. Furthermore, most genes showing allelic expression were regulated by cis-acting mechanisms and were also significantly associated with several human diseases. Overall, our study provides a better understanding of allele-specific gene expression in hepatocellular carcinoma cells with and without LPS stimulation and potential clues for the cause of human disease due to alterations in allelic gene expression.     

Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines

In murine macrophages, the anti-tumor agent, paclitaxel, induces expression of a wide variety of inflammatory and anti-inflammatory genes, and causes cytokine secretion via signaling pathways that overlap with those engaged by lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Using semi-quantitative RT-PCR for detection of gene expression, coupled with ELISA for the detection of secreted gene products, we analyzed the responsiveness of an extensive panel of cytokine and non-cytokine genes to induction by paclitaxel and LPS in the murine DA-3 breast cancer line. A subset of the genes examined (e.g., G-CSF, MIP-2, iNOS, and IL-1 beta, and GM-CSF) was upregulated >3-20-fold by both LPS and paclitaxel in the DA-3 cell line, while IP-10 mRNA was induced by paclitaxel, but not by LPS. In the human MDA-MB-231 breast cancer cell line, LPS also increased mRNA levels for both GM-CSF and IP-10 significantly, while, paclitaxel increased IP-10 mRNA levels with delayed kinetics and failed to induce GM-CSF mRNA. Co-cultures of murine breast cancer cells and macrophages, stimulated with IFN-gamma plus either paclitaxel or LPS, resulted in augmented release of nitric oxide. As both GM-CSF and IP-10 have been implicated in tumor rejection in vivo through either indirect actions on the host immune system or by inhibiting tumor angiogenesis, our data strengthen the hypothesis that tumor cell-derived inflammatory mediators may, in part, underlie the anti-tumor efficacy of paclitaxel in breast cancer.     

Gene expression does not change significantly in C3H 10T(1/2) cells after exposure to 847.74 CDMA or 835.62 FDMA radiofrequency radiation

In vitro experiments with C3H 10T(1/2) mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.