Expression data from Glioma-Initiating Cells (GICs) cultured under hypoxia and normoxia
ABSTRACT: Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with glioma initiating cells (GICs) implicated to be critical for tumor progression and resistance to therapy. The hypoxic tumor microenvironment has been shown to play an important role to maintain the GICs; however, the mechanisms regulating responses of GICs to hypoxia remain poorly understood. We used microarray to to detail the global change of gene expression in GICs cultured under hypoxia compared to normoxia and identified de-regulated genes during hypoxia. CD133+ D456MG GICs were cultured under 1% O2 or 20% O2 for 12 hours. Then RNA was extracted and gene expression was profiled by microarray.
Project description:Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with glioma initiating cells (GICs) implicated to be critical for tumor progression and resistance to therapy. The hypoxic tumor microenvironment has been shown to play an important role to maintain the GICs; however, the mechanisms regulating responses of GICs to hypoxia remain poorly understood. Overall design: We used RNA-Sequencing to to detail the global change of microRNA expression in GICs cultured under hypoxia compared to normoxia and identified de-regulated microRNAs during hypoxia. CD133+ 110040 GICs were cultured under 1% O2 for 0h, 6h, 12h and 24 hs. Then RNA was extracted and microRNA expression was profiled by RNA-Sequencing.
Project description:Analysis of expression changes of cultured HepG2 hepatoma, U87 glioma, and MDA-MB231 breast cancer cells subjected to hypoxia (0.5% O2) for 0, 4, 8, 12 hours . Results provide insight to cell type-specific response to hypoxia. HepG2 hepatoma, U87 glioma, and MDA-MB231 breast cancer cells were collected under normoxic conditions (~19% O2, 0 hours) and after 4, 8 and 12 hours of hypoxia treatment (0.5% O2). For each cell line, three replicates of total RNA at each time point were prepared using Trizol and submitted to the DFCI Microarray Core for labeling, hybridization to Affymetrix HG-U133Plus2 oligonucleotide arrays and image scanning.
Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.
Project description:To delineate the role of hypoxia in esophageal epithelial biology, we carried out gene array experiments using a non-transformed immortalized diploid human esophageal cell line, EPC2-hTERT (Mol Cancer Res. 2003;1:729-38). Unlike cancer cell lines, EPC2-hTERT has no genetic alterations at early passages that may affect the cellular response to hypoxia. Experiment Overall Design: EPC2-hTERT cells were exposed to moderate (1% O2) hypoxia in experiment 1 (Exp1) or severe (0.2% O2) hypoxia in experiment 2 (Exp2). Normoxia (21% O2) served as a control in both experiments.
Project description:Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. RNAs derived from human LNCaP cells stably infected with either scrambled control shRNA or a shRNA directed to RB1. Cells were exposed to either 24 h hypoxia (1% O2) or maintained under normoxic conditions. Biological triplicates were tested and compared.
Project description:Analyze miRNA expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for miRNA arrays
Project description:Analysis of 5-hydroxymethylcytosine changes in hypoxia in neuroblastoma cells 5-hmC distribution was analyzed from cells exposed to hypoxia (1% O2) or normoxia (20% O2); mRNA was sequenced in parallel for expression analysis.
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:Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with glioma initiating cells (GICs) implicated to be critical for tumor progression and resistance to therapy. KDM1B is involved in regulating GICs' responses to hypoxia, since over-expression of KDM1B delays the cell growth under hypoxia while knocking-down of KDM1B in GICs promotes their survival and tumorigenic abilities. Overall design: We used RNA-Sequencing to detail the global change of gene expression in GICs with knockdown of KDM1B, and identified de-regulated genes and pathways downstream of KDM1B. CD133+ D456MG GICs were infected with non-targeting control and shRNA of KDM1B. Then RNA was extracted and gene expression was profiled by RNA-Seq.
Project description:Hypoxia plays a key pathogenic role in the outcome of many pathologic conditions. To elucidate how organisms successfully adapt to hypoxia, a population of Drosophila melanogaster was generated, through an iterative selection process, that is able to complete its lifecycle at 4% O2, a level lethal to the starting parental population. Transcriptomic analysis of flies adapted for >200 generations was performed to identify pathways and processes that contribute to the adapted phenotype, comparing gene expression of three developmental stages with generation-matched control flies. A third group was included, hypoxia-adapted flies reverted to 21% O2 for five generations, to address the relative contributions of genetics and hypoxic environment to the gene expression differences. We identified the largest number of expression differences in 0.5-3 hr post-eclosion adult flies that were hypoxia-adapted and maintained in 4% O2, and found evidence that changes in Wnt signaling contribute to hypoxia tolerance in flies. A population of flies able to complete their life cycle at 4% O2 was selected from a starting population of 27 isogenic D. melanogaster lines exposed to increasingly lower O2 levels over many generations. Transcriptomic analysis of adapted flies maintained at 4% O2 or reverted to room air for five generations, and of generation matched naive controls, was performed to better understand changes in gene expression in adapted flies and to investigate the relative contributions of genetics versus environment to these differences.