{"owner": "ArrayExpress Uploader", "pop_total": 0, "species": "human", "factors": [{"GSM1020347": {"VARIATION": "WT"}}, {"GSM1020347": {"VARIATION": "WT"}}, {"GSM1020347": {"VARIATION": "WT"}}, {"GSM1020347": {"VARIATION": "WT"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}, {"GSM1020347": {"VARIATION": "WT"}}, {"GSM1020347": {"VARIATION": "WT"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}, {"GSM102035": {"VARIATION": "eIF4G1 KD"}}], "id": 4596, "ownerprofile_id": "arrayexpress_sid", "platform": 4, "summary_wrapped": "The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest  and...", "geo_gse_id": "E-GEOD-41627", "owner_profile": "/profile/8773/arrayexpressuploader", "factor_count": 1, "sample_count": 12, "tags": ["brca1", "breast", "breast cancer", "cancer", "cell", "heparin", "internal", "protein", "ribosome", "volume"], "lastmodified": "Dec.12, 2014", "is_default": false, "geo_gds_id": "", "slug": "dna-damage-and-eif4g1-in-breast-cancer-cells-repro", "geo_id_plat": "E-GEOD-41627_A-AFFY-44", "name": "DNA damage and eIF4G1 in breast cancer cells reprogram translation for survival and DNA repair mRNAs", "created": "Sep.19, 2014", "summary": "The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest  and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer  cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1,  common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing  autophagy and apoptosis (Survivin, HIF1\u03b1, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1,  BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to  DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci  with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found  to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced  dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore  promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair  following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers,  in response to DNA damage caused by ionizing radiation, Polysome isolation was performed by separation of ribosome-bound mRNAs in sucrose gradients.  Beckman Ultra-Clear centrifuge tubes were loaded with 5.5 ml of 50% sucrose in low salt buffer (LSB: 200 mM Tris pH7.4 in DEPC H2O, 100 mM NaCl, 30 mM MgCl2) with 1:1000 RNasin (Fermentas) and 100 \u03bcg/mL cycloheximide (CHX) in ethanol and incubated at 4\u00b0C horizontally overnight.  Media was removed from cell cultures, replaced with media containing 100 \u03bcg/mL CHX, cells incubated at 37\u00b0C for 10 min to halt protein synthesis, and trypsinized in trypsin/EDTA containing 100 \u03bcg/mL CHX.  Cells were washed twice in PBS containing CHX, RNasin and Roche complete EDTA-free protease inhibitor tablet, lysed in LSB with CHX and RNasin.  Lysates were added to a Dounce homogenizer and incubated on ice for 3 min before addition of Triton detergent buffer (1.2% Triton N-100, 0.2M sucrose in LSB) and homogenization.  Samples were transferred to cold sterile Eppendorf centrifuge tubes and centrifuged at 13,000 x RPM for 10 min at 4\u00b0C.  Post-nuclear supernatants were transferred to centrifuge tubes containing 100 \u03bcL Heparin solution (10 mg/mL heparin, 1.5M NaCl in LSB) with RNasin and CHX, applied to a sucrose gradient.  Gradients were centrifuged at 36K RPM at 4\u00b0C for 2 h, and supernatants recovered with an ISCO-UV fractionator.  Samples were recovered into Eppendorf centrifuge tubes containing 40 \u03bcL RNase-free 0.5M EDTA and kept on ice.  One volume acidic phenol-chloroform was added to each sample, which were then mixed and centrifuged at 10,000 x g for 15 min.  The acidic phenol-chloroform extraction was repeated with the aqueous phase, which was then extracted twice via chloroform extraction.  RNA was precipitated at -20\u00b0C overnight by addition of isopropanol with 0.1 vol NaOAc, pH5.2.  The following day, RNA pellets were recovered by centrifugation, washed with 70% ethanol, air-dried and resuspended in sterile H2O.  RNA from fractions was then pooled based on their relative rate of translation, with fractions containing 4 or more ribosomes considered heavily translated and used for gene chip analysis.  The RNA quality was examined via the Agilent Technologies kit.", "source": "http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-41627", "sample_source": "http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-41627/samples/"}