Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human fibroblasts subjected to DNA damage reveals ATM-dependent regulation of gene expression

ABSTRACT: 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. 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 Âµg 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 SureScanÂ® 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Tong Zhou

PROVIDER: E-GEOD-6971 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts.

Zhou Tong T Chou Jeff J Zhou Yingchun Y Simpson Dennis A DA Cao Feng F Bushel Pierre R PR Paules Richard S RS Kaufmann William K WK

Molecular cancer research : MCR 20070801 8

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 with those previously recognized ...[more]

PMID: 17699107

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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 Âµg 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 SureScanÂ® 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.

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 Âµg 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 SureScanÂ® 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.

Project description:Damage of the intestinal epithelial barrier by xenobiotics or reactive oxygen species and a dysregulated immune response are both factors involved in the pathogenesis of inflammatory bowel diseases (IBD). Curcumin and rutin are polyphenolic compounds known to have anti-oxidant and anti-inflammatory activities, but their mechanism(s) of action are yet to be fully elucidated. Mdr1a-/- mice spontaneously develop intestinal inflammation, predominantly in the colon, with pathology similar to IBD, so this mouse model is relevant for studying diet-gene interactions and potential effects of foods on remission or development of IBD. This study tested whether the addition of curcumin or rutin to the diet would alleviate colonic inflammation in mdr1a-/- mice. Using whole-genome microarrays, the effect of dietary curcumin on gene expression in colon tissue was also investigated. Twelve mice were randomly assigned to each of three diets; control (AIN-76A), control + 0.2% curcumin or control + 0.1% rutin and monitored from the age of 7 to 24 weeks. Curcumin, but not rutin, significantly reduced histological signs of colonic inflammation in mdr1a-/- mice. Microarray and pathway analyses suggested that the effect of dietary curcumin on colon inflammation could be via an up-regulation of xenobiotic metabolism and a down-regulation of pro-inflammatory pathways probably mediated by PXR and PPARï¡ activation of RXR. These results reveal the potential of global gene expression and pathway analyses to study and better understand the effect of foods in colonic inflammation. Experiment Overall Design: Twelve mice were randomly assigned to each of three diets; control (AIN-76A), control + 0â¢2% curcumin or control + 0â¢1% rutin and monitored from the age of 7 to 24 weeks. As only curcumin significantly reduced colonic HIS, comparison of the gene expression levels in colon was carried out using total RNA from colon tissue of four mdr1a-/- mice from the control group (high HIS) and four mdr1a-/- mice from the curcumin group (low HIS). A reference design with eight arrays was used for this comparison, where each individual RNA sample was hybridized in the array with the reference RNA, totalizing 4 biological replicates per treatment.

Project description:VEGF165 is one of the key regulators of tumor development. Using HCT116 cells transfected with full-length vegf mRNA, full-length vegf mRNA with mutations of H9D and L14E, mutated vegf mRNAs lacking untranslated regions (UTRs), and 5â??UTR mutated between nt 591 and nt 746, we found that vegf 5â??UTR resided an anti-apoptotic activity against chemotherapy independently of VEGF165. We next established HCT116 clones stably expressing vegf 5â??UTR or the mutated vegf 5â??UTR. The cells expressing 5â??UTR, but not the mutated UTR, showed the drug-resistant phenotype, anchorage-independent growth, and rapid tumor growth when implanted in athymic nude mice. Microarray and real-time PCR showed that the vegf 5â??UTR-expressing tumors up-regulated anti-apoptotic genes including mia and down-regulated pro-apoptotic genes including fas, pdcd1, nrg1, and bax. Microarray analysis also revealed specific down-regulation of IFN-inducible genes (43 genes) in the growing tumors. HCT116 cells stably transcribing vegf 5â??UTR decreased STAT1 expression and IFN alpha-STAT1 pathway. In addition to 5-fluorouracil treatment, the vegf 5â??UTR-expressing tumors did not respond to IFN alpha therapy at all. This novel tumor-promoting function in the vegf 5â??UTR may partly explain the insufficiency of the VEGF-VEGFR strategies and suggest a vegf-targeted silencing strategy as a more effective anti-tumor therapy. Experiment Overall Design: We established HCT116 clones stably expressing vegf 5â??UTR (vegf 5â??-G5) and vegf 5â??UTR mutated between nt 591 and nt 746 (vegf 5â?? mut-D5) and implanted into the left and the right flanks of the identical mouse, respectively. The generated tumors on day 14 after injection were removed and total RNA was extracted from each tumor using RNeasy mini (Qiagen). After removing contaminated DNA by DNaseI treatment, 10 micro grams each of RNA of vegf 5â??-G5-derived tumor from 3 individual mice was mixed and prepared for microarray analysis as sample RNA. The reference RNA of vegf 5â?? mut-D5-derived tumor was arranged in the same manner. The quality of both RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA (vegf 5â??-G5) and reference RNA (vegf 5â?? mut-D5) were converted to cDNA with oligo dT conjugated T7 promoter primer and reverse transcriptase. These cDNAs were amplified using T7 RNA polymerase while labeling with either Cy5-CTP or Cy3-CTP (Low RNA Fluorescent Linear Amplification Kit, Agilent Technologies). 750 ng of Cy3 and Cy5-labeled cRNA were mixed and hybridized on an Agilent 22k human 1A array in a hybridization oven at 65oC for 17 h as rotating. Following hybridization and washing of a slide according to manufactureâ??s protocol, the arrays were scanned using the Agilent DNA Microarray Scanner (G2565BA) and microarray images were analyzed using Agilent Feature Extraction Software (v7.5). The final signal intensity was resulted from subtracting background signal from row spot signal. The gene expression level was presented as ratio of sample intensity against reference intensity.

Project description:Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoan, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known virulence determinant used by L. pneumophila to infect host cells is a Type IVb translocation system named Icm/Dot, which is used to modify the host cell functions to the benefit of the bacteria. To date the Icm/Dot systeme is known to translocate more than 100 effectors. While the transcriptional response of Legionella to the intracellular environement of A. castelannii as already been investigated, much less is known of how Legionella reacts transcriptionnally inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth and during infection of human cultured macrophages by using microarray and a RNA amplification procedure called SCOTS to allow for the study of conditions of low bacterial loads. Among the genes induced intracellularly are those involved in amino acid synthesis pathway leading to L-arginine, L-histidne and L-proline as well as many transport system involved in amino acid and iron uptake. The Icm/Dot systems is not differentially expressed inside cells compare to the E phase control but the effectors are strongly induced. The intracellular transcriptome was further used to identify putative new Icm/Dot effectors and translocation was show to occur for 3 of them. This study provides a comprehensive view of how L. pneumophila react to the human macrophages intracellular environment. To validate the use of SCOTS, we compare the expression patterns obtained by SCOTS to those obtained when a standard microarray protocol was used. RNA from bacteria in E phase was treated using SCOTS or a standard microarray protocol where the cDNA is labeled during the reverse transcription reaction as previously published. The resulting cDNA was hybridized to the microarray slides as described previously and data analysis was carried out the same way as for the SCOTS treated samples.

Dataset Information

Transcription profiling of human fibroblasts subjected to DNA damage reveals ATM-dependent regulation of gene expression

Publications

Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts.

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