Transcription profiling by array of mouse macrophage cell cultures from C57BL/6 or CBA/Ca strains after exposure to 4 Gy irradiation
Ontology highlight
ABSTRACT: Macrophages were produced from bone marrow cell suspensions from the femurs of five C57BL/6 or CBA/Ca mice by culture for 7 days in modified Eagles medium supplemented with 25% pre-tested horse serum and 25% pre-tested conditioned medium from the L929 cell line as a source of CSF-1. For each experiment, five flasks were exposed to 4Gy irradiation at a dose rate of 0.5Gy/min at room temperature using a CIS Bio International 637 Caesium irradiator and five flasks were sham-irradiated. RNA was extracted 24 hours later for analysis on MOE430-2 affymetrix arrays
Project description:Single cell RNA-seq of SVZ lateral ventricle walls from non-irradiated mice brains and 5 days post-irradiation 4Gy-irradiated mice brains (according to the model SVZ regeneration after a moderate dose of irradiation that we previously described (Daynac et al. 2013). We used this model of SVZ reconstitution to gain insights on the annotation and and temporal ordering of neural progenitors clusters.
Project description:We performed bulk RNA-Seq to investigate global transcriptional changes upon overexpression of the centromeric H3 variant CenH3/CENP-A in p53 wild-type and defective cells, and after X-irradiation treatment. We established clonal MCF-10-2A TetOn-CENPA-FLAG-HA cell lines where CENP-A can be reversibly induced by Doxycycline (Dox) treatment. Upon CENP-A overexpression, these cells exhibit different radiosensitivity depending on p53 status. In order to profile global changes in expression, we compared MCF-10-2A TetOn-CENPA-FLAG-HA cells expressing either empty vector (p53-WT) or dominant-negative p53 (p53-DN) with 0X Dox (no Dox), 1X Dox (10 ng/ml), or 10X Dox (100 ng/ml) for 24h. At time 0, we irradiated one set of cells by X-ray generator (4gy) while a control set remained un-irradiated (0gy). 6h later, we extracted RNA for RNA-seq.
Project description:The p53 protein is a pivotal tumor suppressor that is frequently mutated in many human cancers, although precisely how p53 prevents tumors is still unclear. To add to its complexity several isoforms of human p53 have now been reported. The Δ133p53α isoform is generated from an alternative transcription initiation site in intron 4 of the p53 gene (TP53) and lacks the N-terminus. Elevated expression of Δ133p53α has been observed in a variety of tumors. To explore the functions of Δ133p53α, we created a mouse expressing an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53α. Δ122p53 mice show decreased survival and a different and more aggressive tumor spectrum compared to p53 null mice, implying that Δ122p53 is a dominant oncogene. Consistent with this, Δ122p53 also confers a marked proliferative advantage on cells and reduced apoptosis. In addition to tumor development Δ122p53 mice show a profound pro-inflammatory phenotype having increased serum concentrations of interleukin (IL)-6 and other pro-inflammatory cytokines and lymphocyte aggregates in the lung and liver as well as other pathologies. Based on these observations, we propose that human Δ133p53α also functions to promote cell proliferation and inflammation, one or both of which contribute to tumor development. Total RNA was isolated from pooled splenocytes from four 5-6 week old mice with different p53 status (wild type, p53-/-, or Δ122p53) subjected to DNA damaging or control treatments.
Project description:Ionizing radiation (IR) not only affects cells that are directly irradiated but also their non-irradiated neighbors, which show responses known as bystander effects. While bystander and direct responses have several common end points including apoptosis and micronucleation, chromatin remodeling and altered levels or activities of regulatory proteins, they can be quantitatively and qualitatively different. The majority of studies of radiation bystander effects have utilized 2-dimensional in vitro systems, but we have recently demonstrated such effects in EPI-200 (Mat-Tek, Ashland, MA), a 3-dimensional tissue model that precisely imitates the structure and function of human epidermis. Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By exposing only a thin strip across the center of the EPI-200 tissue, we have been able to measure global gene expression responses in directly irradiated and bystander cells located at 0, 0.25, 0.5, 0.75 and 1mm from the irradiation line. The data were analyzed using BRB-Array Tools (NIH), and further network analysis was performed with IPA (Ingenuity). Significantly responding genes were identified at all distances and included sets common to both direct and bystander responses. For instance, all sets demonstrated upregulation of a major component of the drug metabolism pathway, CYP1B1, and downregulation of MMP1, an enzyme involved in degradation of extracellular matrix. In contrast, PTGS2, a gene strongly implicated in the bystander response was upregulated in directly irradiated tissues, but actually downregulated in bystander cells. This effect may be time dependent, but may also suggest activation of bystander signaling mechanisms different from those observed in 2-dimensional cell cultures. According to network analysis of our results, the genes responding in bystander tissue fell into 5 major categories: cell death, cell communication, cell differentiation, stress response, and response to wounding, suggesting active intracellular communication in bystander tissue. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured at 4 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed for the samples collected at different distances from the irradiation line (250-500, 500-750 and 750-1000 micrometers) using three tissue fragments per a data point.
Project description:Ionizing radiation (IR) not only affects cells that are directly irradiated but also their non-irradiated neighbors, which show responses known as bystander effects. While bystander and direct responses have several common end points including apoptosis and micronucleation, chromatin remodeling and altered levels or activities of regulatory proteins, they can be quantitatively and qualitatively different. The majority of studies of radiation bystander effects have utilized 2-dimensional in vitro systems, but we have recently demonstrated such effects in EPI-200 (Mat-Tek, Ashland, MA), a 3-dimensional tissue model that precisely imitates the structure and function of human epidermis. Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By exposing only a thin strip across the center of the EPI-200 tissue, we have been able to measure global gene expression responses in bystander cells located at 0.125 and 0.625 um from the irradiation line, in 16h after irradiation. The data were analyzed using BRB-Array Tools (NIH), and further network analysis was performed with IPA (Ingenuity). Significantly responding genes were identified at the both distances. For instance, all sets demonstrated upregulation of two key enzymes of the lipid biosynthesis, UGT1 and PITPNB, and downregulation of proapoptotic proteins: BAX and ARHGEF5. In contrast, several proteins involved in transcriptional repression (CHD6, CHD8 andWRNIP1) were strongly upregulated suggesting a rearrangement in the gene transcription. These changes suggest an activation of bystander mechanisms different from those observed in 2-dimensional cell cultures. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 16 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed for the samples collected at different distances from the irradiation line (125-625 and 625-1125 um) using three tissue fragments per a data point.
Project description:Direct irradiation of 3-dimensional skin model, Epi-200, with alpha-particles led to differential regulation of 545 genes at 4 h postirradiation. Unlike the traditional 2-dimensional in vitro systems, Epi-200 made of the primary cells, epidermal human keratinocytes. It mimics the structure of the human epidermis Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By comparing irradiated tissues with non-irradiated control, we have been able to measure global gene expression responses and reveal the affected biological pathways and molecular functions. The data were analyzed using BRB-Array Tools (NIH), and further gene ontology analysis was performed with Panther (Applied Biosystems). The responding pathways were identified jointly and separately for up- and downregulated genes. Receptors (12 genes, p=0.0467), including nuclear hormone receptors (3 genes, p=8.2 x E-04) and Ser/Thr receptors (3 genes, p=5.3 x E-03), chemokines (2 genes, p=0.0155) and signaling molecules were overrepresented among 123 upregulated genes while DNA binding proteins (27 genes, p=5.07 x E-04), particularly helicases (6 genes, p=3.61 x E-03) and, G-protein modulators (16 genes, p=4.4 x E-03) were overrepresented among 403 downregulated genes. Remarkably, 6 differentially regulated genes in this group were represented by helicases and methyltransferases (6 genes, p=0. 0534) involved in methylation of histones and DNA. These changes confirmed an importance in the biological response of two its basic components: receptor-regulated pathways and chromatin remodeling. Upregulation the first group supposes to increase sensitivity of irradiated cells to chemokines and cytokines while downregulation of the second group would likely affect the DNA methylation pattern and stimulate the transcription. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured at 4 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed using one tissue sample per a data point.
Project description:Direct irradiation of 3-dimensional skin model, Epi-200, with alpha-particles led to differential regulation of 166 genes: 16 and 150 genes were differentially expressed at 1 and 16 h postirradiation. Unlike the traditional 2-dimensional in vitro systems, Epi-200 made of the primary cells, epidermal human keratinocytes. It mimics the structure of the human epidermis Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By comparing irradiated tissues with non-irradiated control, we have been able to measure global gene expression responses and reveal the affected biological pathways and molecular functions. The data were analyzed using BRB-Array Tools (NIH), and further gene ontology analysis was performed with Panther database (Applied Biosystems). Gene ontology analysis of the samples harvested in 16h after exposure showed that irradiation presumably affected the genes involved in cell-cell signaling (15 genes, , p=9.0 x E-04) ion transport (10 genes, p=0.00189) and amino acid metabolism (5 genes, p=0.0258). Among 16 genes differentially expressed in 1h after exposure we found NOTCH2 (ENST00000401649) and methyltransferase AOF1 (KDM1B). In the mammalian cells, NOTCH signaling pathway has a role in differentiation and intracellular communication. Moreover the intercellular domain of NOTCH regulates gene expression acting as a transcription factor. In turn, AOF1 affects the transcription via histone demethylation. Thus, irradiation with alpha-particles caused predominant downregulation of multiple genes in 1 and 16h after exposure. It also suggested that changes in cell metabolism initially affected transcriptional regulation and finally led to the rearrangement in expression of genes playing a role in biosynthesis and ion trafficking. Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 1 and 16 h hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed using one tissue sample per a data point.
Project description:MicroRNA expression was assessed in human cancer cells (K562, Me45, HCT116 wt and HCT116 p53-/-) treated with 4Gy of ionizing radiation. RNA was extracted from the cells 12 hours after irradiation and after 1h from non-treated controls.
Project description:Following radiation damage, cells resume ribosome biogenesis for survival. We combined chromatin immunoprecipitation (ChIP) with mass spectrometry to identify key binding partners of RPA194 (the major subunit of RNA polI) at the rDNA loci in response to irradiation.