Identification of Primary Transcriptional Regulation of Cell-Cycle-Regulated Genes upon DNA Damage
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE7075 | GEO | 2007/08/01
SECONDARY ACCESSION(S): PRJNA98485
REPOSITORIES: GEO
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Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Experiment Overall Design: Normal human fibroblasts were synchronized and harvested at G0, G1, S G2 and M cell cycle phase, and logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâ??s Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScan® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-15 | E-GEOD-7075 | biostudies-arrayexpress
Project description:Cell cycle arrest and transcriptional responses to ionizing radiation (IR)-induced DNA damage were quantified in telomerase-expressing human diploid fibroblasts. Assays of clonal expansion established 1.5 Gy IR as the D0 dose in three fibroblast lines and this dose was used in all subsequent analyses. Fibroblasts exhibited >90% arrest of progression from G2 to M at 2 h and from G1 to S at 6 and 12 h post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 h after irradiation caused the S and M compartments to empty by over 70% at 24 h. Microarray monitored global gene expression in IR-treated cells and a new microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, like CDKN1A, GADD45, BTG2 and PLK3, were significantly up-regulated at 2 h post-IR while many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 h post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile of gene expression 24 h after IR with similarity to that of G0 growth quiescence. These results demonstrate a highly stereotypic pattern of response to IR that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. Keywords: time-course, DNA damgage responses
2007-02-01 | GSE6902 | GEO
Project description:The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. The observation of attenuated and not ablated checkpoint responses in AT cells supports a hypothesis that the ATM- and rad3-related checkpoint kinase ATR also responds to IR-induced DNA damage and complements ATM signaling. Keywords: ATM, cell cycle checkpoint, ionizing radiation, microarray
2007-07-01 | GSE6971 | GEO
Project description:Recently, senescence has been suggested as a defense mechanism to block sporadic induction of cancer cells. Radiation treatment induces proliferating cancer cells to turn into non-proliferating senescent cells in vitro. To characterize transcriptional reprogramming after radiation treatment, we measured the gene expression profiles of MCF7 at different time points after treatment. In these experiments, we found that IR induced premature senescence in MCF7 cells, and IR treatment resulted in significant changes in the expression of 305 marker genes (less than 1% FDR), which were strongly correlated (|r|>0.9) with IR treatment in a time-dependent manner. Functional analysis of these markers indicated that the dynamics of cytoskeletal structure and lysosomal activity gradually increased. The expression of marker genes for modulator proteins, that were responsible for the dynamics of actin stress fibers and focal adhesion, displayed a particularly strong positive correlation with senescence-associated (SA) morphological changes through time. We also observed a strong induction of genes related to lysosomal metabolic activity, which was accompanied by an increase in the number of SA-beta-Gal positive cells. However, the expression of genes for the cell cycle progression, the post-transcription and the translation activities gradually decreased after radiation treatment. Especially, we observed clear cell cycle arrest specifically at the S and G2/M phases with consistent down-regulation of genes for microtubule assembly/disassembly or spindle biogenesis. Gene expression profiles were obtained from human MCF7 cells after ionizing radiation (6 Gy) at day 0 (no ionizing radiation), day 1 (after ionizing radiation), day 2, day 3, and day 4.
2010-06-08 | E-GEOD-21748 | biostudies-arrayexpress
Project description:Humans are exposed to ionizing radiation (IR) from background radiation, medical treatments, occupational and accidental exposures. IR causes profound changes in transcription. Transcription is a primary process where protein amount and function can be regulated. One aspect of the transcriptional IR response that little is known about on a whole genome basis is alternative transcription. These investigations focus on the response to IR at the exon level in human cells but also at the whole gene level. Whole genome exon arrays were utilized to comprehensively characterize radiation-induced transcriptional expression products in two human cell types, namely EBV-transformed lymphoblast and primary fibroblast cell lines. 12 human primary fibroblast cell lines and 12 primary lymphoblast cell line samples were used for two doses (0 and 10 Gy) and two time points (0 and 4hr). Additional doses ( 1 Gy, 2 Gy, 5 Gy, and 20 Gy) and time points ( 1hr, 2hr, 8hr, 24hr and 48hr) were assessed in four lymphoblast cell lines and four primary fibroblasts.
2011-05-02 | E-GEOD-26841 | biostudies-arrayexpress
Project description:Transcription profiling of IMR90 fibroblasts after ionizing radiation. Cells were harvested 30 minutes (acute response) or 5 days (cell cycle arrested) after being irradiated with 5Gy of ionizing radiation.
2014-06-17 | E-MTAB-2088 | biostudies-arrayexpress
Project description:Intraoperative radiotherapy (IOERT) is a high radiation therapeutic technique which administers a single high dose of ionizing radiation (IR) immediately after surgical tumor removal in order to destroy the residual cancer cells in the site at high risk for recurrence. IR is able to regulate several genes and factors involved in cell-cycle progression, survival and/or cell death, DNA repair and inflammation modulating an intracellular response radiation dependent producing an imbalance in cell fate decision. In this study, we examined changes in gene expression in MCF7 breast cancer cell line exposed to 9Gy and 23Gy high single dose of IR delivered by IOERT.
2015-06-11 | GSE63667 | GEO
Project description:Intraoperative radiotherapy (IOERT) is a high radiation therapeutic technique which administers a single high dose of ionizing radiation (IR) immediately after surgical tumor removal in order to destroy the residual cancer cells in the site at high risk for recurrence. IR is able to regulate several genes and factors involved in cell-cycle progression, survival and/or cell death, DNA repair and inflammation modulating an intracellular response radiation dependent producing an imbalance in cell fate decision. In this study, we examined changes in gene expression in MCF7 breast cancer cell line exposed to 9Gy and 23Gy high single dose of IR delivered by IOERT. Changes in gene expression in MCF7 breast cancer cell line exposed to 9Gy and 23Gy high single dose of IR (named MCF7_9Gy and MCF7_23Gy respectively), were analyzed as two-color hybridizations using Agilent Technologies whole human genome 4x44K microarrays
2015-06-11 | E-GEOD-63667 | biostudies-arrayexpress
Project description:Cell cycle arrest and transcriptional responses to ionizing radiation (IR)-induced DNA damage were quantified in telomerase-expressing human diploid fibroblasts. Assays of clonal expansion established 1.5 Gy IR as the D0 dose in three fibroblast lines and this dose was used in all subsequent analyses. Fibroblasts exhibited >90% arrest of progression from G2 to M at 2 h and from G1 to S at 6 and 12 h post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 h after irradiation caused the S and M compartments to empty by over 70% at 24 h. Microarray monitored global gene expression in IR-treated cells and a new microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, like CDKN1A, GADD45, BTG2 and PLK3, were significantly up-regulated at 2 h post-IR while many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 h post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile of gene expression 24 h after IR with similarity to that of G0 growth quiescence. These results demonstrate a highly stereotypic pattern of response to IR that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. Experiment Overall Design: Logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâs Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScan® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-15 | E-GEOD-6902 | biostudies-arrayexpress
Project description:The purpose of the study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. The samples used were Wild type, Pak1 knock out, Wild type treated with ionizing radiation and Pak1 knock out treated with ionizing radiation. Triplicates of each sample type were used for analysis.
2016-07-03 | E-GEOD-47503 | biostudies-arrayexpress