H1299 cells: p53 transcriptional response in hypoxia
ABSTRACT: Transcriptional profiling of H1299 non-small cell lung carcinoma cells transfected with either wt p53 or mut(175) p53 driven by the 5xHRE promoter (5 repeats of hypoxia-inducible factor response elements) and treated for 16 h with normoxia (21% O2) or hypoxia(<0.1% O2). 5xHRE promoter ensures that p53 expression is induced in hypoxic conditions only. Goal was to determine the transcriptional response of p53 in hypoxia and the 175 p53 mutant was used as a control as it is DNA-binding defective and transcription-incompetent mutant. Overall design: Four-condition experiment: wt p53-transfected H1299 cells treated with normoxia, mut p53-transfected H1299 cells treated with normoxia, wt p53-transfected H1299 cells treated with hypoxia, mut p53-transfected H1299 cells treated with hypoxia. Biological replicates: 1 normoxic sample with wt p53, 1 normoxic sample with mut p53, 3 hypoxic samples with wt p53, 3 hypoxic samples with mut p53.
INSTRUMENT(S): Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Probe Name version)
Project description:Transcriptional profiling of H1299 non-small cell lung carcinoma cells transfected with either wt p53 or mut(175) p53 driven by the 5xHRE promoter (5 repeats of hypoxia-inducible factor response elements) and treated for 16 h with normoxia (21% O2) or hypoxia(<0.1% O2). 5xHRE promoter ensures that p53 expression is induced in hypoxic conditions only. Goal was to determine the transcriptional response of p53 in hypoxia and the 175 p53 mutant was used as a control as it is DNA-binding defective and transcription-incompetent mutant. Four-condition experiment: wt p53-transfected H1299 cells treated with normoxia, mut p53-transfected H1299 cells treated with normoxia, wt p53-transfected H1299 cells treated with hypoxia, mut p53-transfected H1299 cells treated with hypoxia. Biological replicates: 1 normoxic sample with wt p53, 1 normoxic sample with mut p53, 3 hypoxic samples with wt p53, 3 hypoxic samples with mut p53.
Project description:G9a is able to silence gene expression in hypoxic condition by increasing histone H3K9me2. We have identified a set of genes that are negatively regulated by G9a in hypoxia-dependent manner. In this dataset, we include the expression data obtained from MCF7 breast epithelial cells that have been transfected with control (WT) or G9a shRNAs (KD) and exposed to either normoxia or hypoxia. These data are used to obtain 829 genes that are differentially expressed in response to hypoxia, and 205 genes that are sentisive to G9a level. 4 samples were analysed. We generated comparisons between WT and KD in normoxic as well as hypoxic condition. Genes differentially expressed in hypoxic condition were selected followed by selection of genes that lose this differential expression upon G9a knockdown.
Project description:The Hypoxia-Inducible Factors induce the expression of the histone demethylases JMJD1A (KDM3A) and JMJD2B (KDM4B), linking the hypoxic tumor microenvironment to epigenetic mechanisms that may foster tumor progression. Using transcript profiling, we have identified genes that are regulated by JMJD1A and JMJD2B in both normoxic and hypoxic conditions in SKOV3ip.1 ovarian cancer cells. This dataset includes expression data obtained from exposing ovarian cancer cells to hypoxia in combination with siRNA-mediated knockdown of the hypoxia-inducible histone demethylases JMJD1A and JMJD2B. These data were used to both identify functional overlap between each histone demethylase, as well as identify effectors of tumor growth mediated by JMJD2B (KDM4B) in normoxia and hypoxia. Overall design: 18 samples were analyzed. The log2-transformed normalized signal intensities were used for downstream pairwise comparisons (using Partek Genomic Suite): SiControl-Normoxia>SiD1A-Normoxia; SiD1A-Normoxia>SiControl-Normoxia; SiControl-Normoxia>SiD2B-Normoxia; SiD2B-Normoxia>SiControl-Normoxia; SiControl-Hypoxia>SiControl-Normoxia; SiControl-Normoxia>SiControl-Hypoxia; SiControl-Hypoxia>SiD1A-Hypoxia; SiD1A-Hypoxia>SiControl-Hypoxia; SiControl-Hypoxia>SiD2B-Hypoxia; SiD2B-Hypoxia>SiControl-Hypoxia.
Project description:TRAF6 -/- thymi display defects in IR-induced p53 target gene expression and attenuate further enhancement of apoptosis upon IR treatment. TRAF6-mediated K63-linked ubiquitination of p53 at K24 Overall design: 2.5 week WT and TRAF6 null mice were IR (15Gy) treated for 10h, thymi were then isolated and RNA was extracted by standard Trizol methods. H1299 cells were transfected with p53 wt and K24R mutant, RNA was extracted by standard Trizol methods.
Project description:The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. Studies of the regulation of the expression of many of these genes by oxygen has implicated a role for the heterodimeric transcription factor hypoxia inducible factor (HIF). The mechanism of oxygen sensing which controls this heterodimeric factor is via oxygen dependent prolyl and asparaginyl hydroxylation by specific 2-oxoglutarate dependent dioxygenases (PHD1, PHD2, PHD3 and FIH-1). Whilst HIF appears to have a major role in hypoxic regulation of gene expression, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. The extent to which 2-oxoglutarate dependent dioxygenases are responsible for the oxygen sensing mechanism in HIF-independent hypoxic gene regulation is also unclear. Both the prolyl and asparaginyl hydroxylases can be inhibited by dimethyloxalylglycine (DMOG). Such inhibition can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischaemic disease. We have examined the extent to which the HIF system contributes to the regulation of gene expression by hypoxia, to what extent 2-oxoglutarate dependent dioxygenase inhibitor can mimic the hypoxic response and the nature of the global transcriptional response to hypoxia. We have utilised microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high level of mimicry of the hypoxic response by use of the 2-oxoglutarate dependent dioxygenase inhibitor, dimethyloxalylglycine. We have also used microarray analysis of cells treated with small interfering RNA (siRNA) targeting HIF-1alpha and HIF-2alpha to demonstrate the differing contributions of each transcription factor to the transcriptional response to hypoxia. Candidate transcripts were confirmed using an independent microarray platform and real-time PCR. The results emphasise the critical role of the HIF system in the hypoxic response, whilst indicating the dominance of HIF-1alpha and defining genes that only respond to HIF-2alpha. Keywords: Hypoxia response, gene knockdown, chemical treatment Overall design: MCF7 breast cancer cell lines were grown under conditions of either normoxia (21% oxygen) or hypoxia (1% oxygen) for 16 hours in an Invivo2 Hypoxia Workstation (Ruskin Technologies, UK). All culture media comprised DMEM, 2mM L-Glutamine and 10% Fetal Bovine Serum. Total RNA was extracted from each sample using the Absolutely RNA RT-PCR Miniprep kit (Stratagene). In total, 7 different types of sample were analysed by microarray technology. These were: ‘normoxia’ - cells grown in normoxic (21% oxygen) conditions; ‘hypoxia’ - cells grown in hypoxic conditions (1% oxygen) for 16 hours; ‘DMOG’ - cells grown in normoxic conditions and exposed to the 2-oxoglutarate dependent dioxygenase inhibitor dimethyloxalylglycine, DMOG (2 mM) for 16 hours; ‘OF’ - cells grown in hypoxic conditions as above and exposed to oligofectamine transfection reagent (Invitrogen) alone; ‘HIF1’ - cells grown in hypoxic conditions as above and exposed to HIF-1alpha siRNA; ‘HIF2’ - cells grown in the same hypoxic conditions with HIF-2alpha siRNA and ‘HIF12’ - cells grown in the same hypoxic conditions with both HIF-1alpha and HIF-2alpha siRNA. Three independent samples were analysed for each experimental condition. All of the analyses for the hypoxia and DMOG samples were compared to the normoxia samples whilst the siRNA transfected samples were compared against the OF samples. The normoxia, hypoxia and DMOG samples (replicates 1-3) were arrayed to HG-U133A Genechips and sample types OF, HIF1, HIF2 and HIF12 (replicates 1-3) were arrayed to HG-U133 plus 2 Genechips (Affymetrix). The normoxia, hypoxia and DMOG samples (replicates 2-3) along with the samples OF, HIF1, HIF2 and HIF12 (replicates 4-6) were arrayed to the ‘whole genome’ Sentrix Human-6 Expression BeadChips (Illumina). All labelling, hybridisation and scanning steps were performed according to the manufacturers’ instructions.
Project description:Microarrray experiments were performed in order to identify genes whose expression is altered under hypoxic conditions and to determine whether these changes are dependant on the p53 and HIF tanscription factors. In order to determine HIF dependance, isogenic RCC4 cell lines were used that were either wildtype of null for vhl, the gene responsible for degrading HIF under normoxic conditions. In order to determine p53 dependance, isogenic HCT116 and H1299 cell lines that were wildtype or null for p53 were used. Gene expression was measured after exposure of cells in monolayer culture to hypoxia (<0.01% O2) for various times. Keywords: hypoxia, time course, genetic modification, cell line comparison Overall design: Affymetrix genechips were used to measure expression in cells exposed to hypoxia in monolayer culture for different times. One replicate was analysed at each time point, except for the control (0 hr) and 18 hr time points in the RCC4 cells, where two independant replicates were performed.
Project description:To evaluate the effect on gene expression by p53 family members and AKR1B10, we overexpressed p53 family members in H1299 cells or knocked down AKR1B10 in HCT116 cells and evaluated the gene expression by microarray analysis. Gene expression was measured in H1299 cells infected with adenovirus expressing p53, p63g and p73b, and in HCT116 cells transfected with siRNAs targeting AKR1B10 and then treated with adriamycin.
Project description:Cartilage endplate-derived stem cells (CESCs) with chondro-osteogenic differentiation capacity may be responsible for the balance of chondrification and ossification in cartilage endplate (CEP). CEP is an avascular and hypoxic tissue, and hypoxia could inhibit the osteogenic differentiation of CESCs. We used high-throughput scanning to identify differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) during osteogenic differentiation of CESCs under hypoxia compared to those induced under normoxia. Human cartilage endplate-derived stem cells (CESCs) were treated with osteogenic differentiation medium under normoxia and hypoxia for 21 days respectively.