Translatome and transcriptome profiling of estradiol response in MCF7 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Toma Tebaldi
PROVIDER: E-GEOD-22012 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
2014-04-17 | E-GEOD-50650 | biostudies-arrayexpress
Project description:Translatome analysis by sucrose gradient centrifugation of cell lysates followed by microarray profiling of the polysomal and subpolysomal RNA fractions represents a way of both studying translational control networks and better approximating the proteomic representation of cells. It is an established notion that translational control takes place essentially at the translation initiation level, therefore the variation in abundance of a given mRNA species on polysomes can be directly related to the variation in abundance of the corresponding protein. Comparison of translatome profile changes with corresponding transcriptome profile changes can provide a measure of the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. To provide a direct experimental evaluation of the phenomenon, we decided to study a classical example of transcriptional reprogramming of gene expression: Epidermal Growth Factor (EGF) treatment. This stimulus triggers a well known chain of intracellular transduction events, ultimately resulting in a multifaceted phenotypic spectrum of changes with prevalent induction of cell growth and proliferation. We subjected HeLa cells to serum starvation for 12h and then we added EGF at final concentration of 1 μg/ml, profiling before and after 40 minutes of treatment the transcriptome, the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation, and also the mRNA content of the subpolysomal pool, expected not to be actively translated. Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, EGF starvation release. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved HeLa cells transcriptome in response to EGF stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA, subpolysomal RNA and total RNA were isolated from HeLa cells serum starved and treated with EGF. Cells lysates were collected before (t = 0 min) and after (t = 40 min) EGF treatment. All experiments were run in triplicates.
2010-05-27 | E-GEOD-20277 | biostudies-arrayexpress
Project description:Neuroblastoma (NB) is the most frequent extracranial solid tumour of childhood. Clinical courses are highly variable, ranging from spontaneous regression/maturation to rapid progression despite intensive multimodal therapy. The estimation of 5-year event free survival in high-risk patients of only about 40 % stresses an importance of novel therapeutic strategies. A number of iron chelators have demonstrated marked in vitro and in vivo anti-tumor activity and are currently being developed as novel anti-cancer agents. Therefore, the potential antitumor effect of iron chelators in NB cancer was investigated. Among the compounds tested, ciclopirox olamine (CPX) was shown to be one of the most effective intracellular iron chelators in NB cells. To unveil the molecular mechanisms underlying the effects of CPX on viability of NB cells, microarray analysis was performed in CHP134 control cells and cells treated with 5 M-BM-5M CPX for 90 minutes. Inclusion of both total RNA (reflecting transcriptional status of the cells) and polysomal RNA (approximating the proteomic representation of the cells) provided us with a deeper understanding of changes in the cells upon CPX treatment. Keywords: ciclopirox olamine, iron chelator, neuroblastoma, translatome profiling, transcriptome profiling. Keywords: ciclopirox olamine, iron chelator, neuroblastoma, translatome profiling, transcriptome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling Microarray analysis was performed to enable a comprehensive view of the changes in the transcriptional and translational status of the cells in response to the treatment with an iron chelator CPX. Polysomal RNA and total RNA were isolated from vehicle-treated CHP134 cells and CHP134 cells treated with 5 M-BM-5M CPX for 90 minutes. Each condition is represented by three biological replicates, i.e. the experiment was repeated starting each time from cells seeding yielding an independent RNA sample. In total, 12 samples corresponding to 4 conditions were subjected to microarray analysis.
2015-02-04 | E-GEOD-60675 | biostudies-arrayexpress
Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to staphylococcal α-hemolysin oligomers (rAHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with 3nM of extracted oligomers (rAHL) for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.
2015-04-01 | E-GEOD-60543 | biostudies-arrayexpress
Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. Informations about the translational state of mRNAs or the activity of RNA binding proteins and ncRNAs after treatment with sublytic doses of pore forming toxins are completely missing. This study uncovers the reshaping undergoing in the translational control system of the host in response to sublytic doses of staphylococcal α-hemolysin (AHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to sublytic doses of staphylococcal α-hemolysin (AHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with sublytic doses (3nM) of AHL for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.
2015-04-01 | E-GEOD-50652 | biostudies-arrayexpress
Project description:We explored the combinatorial interactions between p53, estrogen receptors and NFkB using the breast adenocarcinoma-derived MCF7 cells. Recently, we have established that p53 can modulate transcription also through non-canonical response elements (REs), consisting of half-sites and ¾ sites. In particular, we identified a half-site p53 response element in the promoter of FLT1/VEGFR-I gene that was required but not sufficient for the p53 responsiveness of the promoter. The transcriptional control required also estrogen receptor (ER) half-sites (EREs) located in close proximity to the p53 RE. Further studies led us to establish that p53-mediated transcription from the FLT1 half site RE is dependent on ER binding at the EREs and strongly influenced by the level of ER proteins, and the specific nature of the p53-inducing stress as well as ER ligand. In another study we found an enrichment of NFkB REs nearby p53 REs in p53 target genes, suggesting an additional mode of functional interactions between these two transcription factors. To identify at a genome scale the transcriptional network that selectively responds to the concomitant activation of p53, ER and/or NFkB, we measured gene expression upon treatment with specific chemotherapeutic agents combined with 17-β estradiol (E2) and/or the inflammatory cytokine TNFα. Firstly we measured using MCF7 cells growing in estrogen-depleted media the transcriptome changes induced by doxorubicin (doxo) and 5-fluorouracil (5FU), two different drugs both able to stabilize and activate the p53 protein. We next obtained the expression profiles in the presence of E2 with or without doxo or 5FU. We also measured transcriptome changes in response to TNFαalone or in combination with doxo and/or E2. All these analyses were conducted using the same batch of MCF7 cells (wild type for p53, ERα, ERβ -weakly-, and NFkB positive) that we had previously used to characterize the cis-mediated transcriptional cooperation between p53 and ER at the FLT1 gene. Keywords: transcription factor, transcriptional networks, p53, ER, NFkB, p53-ER-NFkB crosstalk, doxorubicin, 5-fluorouracil, 17-β estradiol, TNFα, combined genotoxic, estrogenic and inflammatory responses, composite DNA binding site signatures, MCF7. The cooperation at the transcriptional level among p53, ERs and NFkB was analyzed by transcriptome analysis in response to different stimuli (doxo or 5FU, E2, TNFα). To identify mRNA molecules that would be modulated by the coordinated activity of the three TFs, gene expression signals derived from the combinatorial treatment were compared by microarrays analyses to those obtained from each drug when individually administered. Total RNA was isolated from MCF7 cells grown in estrogen-depleted medium and treated so as to stimulate the activity of the three different transcription factors (p53, ERs and NFkB). Cells lysates were collected after 10 hours from the treatments. All experiments were run from triplicate to septuples.
2011-06-01 | E-GEOD-24065 | biostudies-arrayexpress
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency. Gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from total RNAs (derived from the sum of all the fractions in the polysomal gradient). Polysomal RNA, subpolysomal RNA and total RNA were isolated from Jurkat cells treated with FK866 5 nM or DMSO (mock, control treatment) for 48 hr. Cells lysates were collected from control cells (mock) and from treated cells (FK866). All experiments were run in biological quadruplicates.
2015-04-01 | E-GEOD-53772 | biostudies-arrayexpress
Project description:Neuroblastoma (NB) is the most frequent extracranial solid tumour of childhood. Clinical courses are highly variable, ranging from spontaneous regression/maturation to rapid progression despite intensive multimodal therapy. The estimation of 5-year event free survival in high-risk patients of only about 40 % stresses an importance of novel therapeutic strategies. A number of iron chelators have demonstrated marked in vitro and in vivo anti-tumor activity and are currently being developed as novel anti-cancer agents. Therefore, the potential antitumor effect of iron chelators in NB cancer was investigated. Among the compounds tested, ciclopirox olamine (CPX) was shown to be one of the most effective intracellular iron chelators in NB cells. To unveil the molecular mechanisms underlying the effects of CPX on viability of NB cells, microarray analysis was performed in CHP134 control cells and cells treated with 5 µM CPX for 24 hours. Inclusion of both total RNA (reflecting transcriptional status of the cells) and polysomal RNA (approximating the proteomic representation of the cells) provided us with a deeper understanding of changes in the cells upon CPX treatment. Keywords: ciclopirox olamine, iron chelator, neuroblastoma, translatome profiling, transcriptome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling Microarray analysis was performed to enable a comprehensive view of the changes in the transcriptional and translational status of the cells in response to the treatment with an iron chelator CPX. Polysomal RNA and total RNA were isolated from vehicle-treated CHP134 cells and CHP134 cells treated with 5 µM CPX for 24 hours. Each condition is represented by three biological replicates, i.e. the experiment was repeated starting each time from cells seeding yielding an independent RNA sample. In total, 12 samples corresponding to 4 conditions were subjected to microarray analysis.
2016-06-01 | E-GEOD-53187 | biostudies-arrayexpress
Project description:We subjected MCF7 cells to starvation with 0.5% charcoal treated serum for 48h and then we added 17-beta estradiol (E2) at final concentration of 10 nM, profiling before and after 60 minutes of treatment the transcriptome and the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation. Comparison of translatome profile changes with corresponding transcriptome profile changes represents a way of studying translational control networks and the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. It is well known that E2 is a strong transcriptional regulator, while its translational control activity is less characterized. To provide a direct experimental evaluation of E2 induced translational regulation, we compared translatome and transcriptome profiles of E2 treated cells. Keywords: polysomal profiling, translatome profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, estradiol stimulation, estrogen receptor.
2010-12-31 | GSE22012 | GEO
Project description:The purpose of the study was to identify mRNA bound to HuR in the presence of doxorubicin in MCF7 cells. We collected cytoplasmic RNA from untreated and treated cells and detected differentially expressed genes (DEGs). We also coimmunoprecipitated HuR and IgG (as control) from doxorubicin treated cells. Comparison between HuR RIP and IgG RIP signals was used to discriminate specific mRNA bound to HuR. HuR coimmmunoprecipitated material was hybridized together with cytoplasmic mRNA of doxorubicin treated cells, enabling the fold enrichment calculation and the selection of mRNAs bound to HuR. Keywords: RIP-Chip, HuR, doxorubicin, MCF7, HuR consensus binding, post-transcriptional regulation. We subjected MCF7 cells to starvation for 24h and then we added doxorubicin at final concentration of 10 uM, profiling before and after 4 hours of treatment in biological quadruplicate (only on cytoplasmic mRNAs, as HuR was found in the cytoplasm). Differentially expressed genes, altered during the treatment, were identified. Data derived from HuR RIP-Chip and IgG RIP-Chip (in biological quadruplicate) allowed the identification of specific mRNAs bound to HuR. The comparison between HuR RIP-Chip and cytoplasmic extracts from doxorubicin treated cells (in biological triplicate) identified those genes that were more strictly bound to HuR independently from their expression levels.
2011-12-31 | E-GEOD-33055 | biostudies-arrayexpress