Unknown,Transcriptomics,Genomics,Proteomics

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Radiation-Induced Changes in Gene Expression Involve Recruitment of Existing Messenger RNAs to and from Polysomes

ABSTRACT: Although ionizing radiation has been shown to influence gene transcription, little is known about the effects of radiation on gene translational efficiency. To obtain a genome-wide perspective of the effects of radiation on gene translation, microarray analysis was done on polysome-bound RNA isolated from irradiated human brain tumor cells; to allow for a comparison with the effects of radiation on transcription, microarray analysis was also done using total RNA. The number of genes whose translational activity was modified by radiation was f10-fold greater than those whose transcription was affected. The radiation-induced change in a gene’s translational activity was shown to involve the recruitment of existing mRNAs to and away from polysomes. Moreover, the change in a gene’s translational activity after irradiation correlated with changes in the level of its corresponding protein. These data suggest that radiation modifies gene expression primarily at the level of translation. In contrast to transcriptional changes, there was considerable overlap in the genes affected at the translational level among brain tumor cell lines and normal astrocytes. Thus, the radiation-induced translational control of a subset of mRNAs seems to be a fundamental component of cellular radioresponse. (Cancer Res 2006; 66(2): 1052-61) There are 32 samples in total in this study. Each sample has a technical duplicate (16samples x2). Comparison of radiation-induced gene expression profiles generated from microarray analysis of total and polysome-bound RNA isolated from three brain tumor cell lines U87, SF126, SF539 and normal astrocytes. Cells were irradiated at the dose of 7gy and collected 6 hours later for isolation of total cellar RNA or polysome-bound RNA. Gene expression was directly compared between irradiated and unirradiated control cells for total and polysome RNA. The array labeling is two-dye method. Labeling two dye samples are labeled using CY3 dye. All the human reference RNA from Stratagene are labeled using CY5 dye.

ORGANISM(S): Homo sapiens

SUBMITTER: Shuping Zhao

PROVIDER: E-GEOD-11517 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Radiation-induced changes in gene expression involve recruitment of existing messenger RNAs to and away from polysomes.

Lü Xing X de la Peña Lorena L Barker Christopher C Camphausen Kevin K Tofilon Philip J PJ

Cancer research 20060101 2

Although ionizing radiation has been shown to influence gene transcription, little is known about the effects of radiation on gene translational efficiency. To obtain a genome-wide perspective of the effects of radiation on gene translation, microarray analysis was done on polysome-bound RNA isolated from irradiated human brain tumor cells; to allow for a comparison with the effects of radiation on transcription, microarray analysis was also done using total RNA. The number of genes whose transl ...[more]

PMID: 16424041

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Project description:Samples and RNA preparation: The series is composed of 4 hybridizations for analysis of differentially expressed genes in the liver of ciprofibrate dosed vs. control rats. Rats (200-250 g) were given (by gastric intubation) tap water (control group, n=5) or ciprofibrate (50 mg/kg body weight, once daily for 60 days, n=4). Liver total RNA extraction was performed first by ultracentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: One microgram of total RNA from the control pool (n=5), and from each ciprofibrate dosed rat was reverse transcribed and labeled with Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array Detection Submicro kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at expected Cy5/Cy3 ratios of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 0.5, 0.33, 0.25 and 0.125 for control spike 1, 2, 3, 4, 5, 6, 8, 9, 10 and 7, respectively. The Cy3 and Cy5 labeled samples (combined volume 5 microliter) were mixed with 30 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution, 0.25 microliter antifade reagent (Genisphere) and 0.2 microgram mouse COT-1 DNA (GIBCO BRL Life Technologies), added onto the array and covered with 22 x 50mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 65oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55oC for 15 min, then in 2 x SSC at room temperature for 15 min, and finally with 0.2 x SSC at room temperature for 15 min. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a confocal laser scanner constructed in-house in collaboration with NEMKO (Trondheim, Norway) according to a prototype developed at NHGRI (http://www.nhgri.gov/DIR/LCG/15K(HTML/). Microarray image analysis was done with MicroArray Suite software version 2.1 with globally normalization (Scanalytics, Inc., Fairfax VA). First, spots undetected (target intensity=1) in at least one array were excluded. Then, spots with signal intensity (averages of the 4 arrays) less than 300 in both channels were removed. The fluorescence intensity ratios (Cy5/Cy3) for genes measured from probes spotted in duplicate were averaged for each slide. Then, mean ratios of the 4 hybridizations were calculated for each gene, and differentially regulated genes were determined using 1.4 fold change cutoff values (1.4 for up-regulated or 0.7 for down-regulated genes).

Dataset Information

Radiation-Induced Changes in Gene Expression Involve Recruitment of Existing Messenger RNAs to and from Polysomes

Publications

Radiation-induced changes in gene expression involve recruitment of existing messenger RNAs to and away from polysomes.

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