Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Zinc finger nuclease knockouts of human ADP-glucokinase

ABSTRACT: Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

ORGANISM(S): Homo sapiens

SUBMITTER: Cris Print

PROVIDER: E-GEOD-39497 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Expression and role in glycolysis of human ADP-dependent glucokinase.

Richter Susan S Richter Jan P JP Mehta Sunali Y SY Gribble Amanda M AM Sutherland-Smith Andrew J AJ Stowell Kathryn M KM Print Cristin G CG Ronimus Ron S RS Wilson William R WR

Molecular and cellular biochemistry 20120105 1-2

A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP unde ...[more]

PMID: 22219026

Similar Datasets

Project description:The activation of the transcription factor Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small-molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high throughput screen led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations but did not affect expression levels of genes outside of the HIF-1 pathway. Lead structure BAY 87-2243 was found to inhibit HIF-1Î± protein accumulation under hypoxic conditions in NSCLC cell line H460 but had no effect on HIF-1Î± protein accumulation and HIF target gene expression in RCC4 cells lacking VHL activity or in H460 cells after inhibition of HIF prolyl hydroxylase activity. BAY 87-2243 had no effect on HIF-Î±-mRNA levels. Antitumor activity of BAY 87-2243 and suppression of HIF-1 target gene expression in vivo was demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial production of reactive oxygen species (ROS) by blocking complex I activity but has no effect on complex III activity. Lowering of mitochondrial ROS production to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors. We used microarrays to detail the global programme of gene expression that is induced in NSCLC cell line H460 upon hypoxia (16 h incubation at 1 % pO2) and evaluated a dose-dependent effect of our HIF-1-pathway inhibitor BAY 87-2243 on genes tthat are affected by hypoxia. Specificity of BAY 87-2243 for the suppression of HIF-1-mediated gene transcription on a genome-wide scale was evaluated by microarray hybridizations using Affymetrix GeneChip Human Gene 1.0 ST arrays. RNA from normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10 and 100 nM BAY 87-2243 respectively was subjected to array hybridization. Of those 30 genes that were most strongly suppressed by 100 nM BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells, virtually all of them are induced by prior hypoxia and most of these genes have been described in the literature as HIF-1 target genes

Project description:Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which these isoprenoids mediate their effects are not yet fully understood. In this study, we show that FOH is effective inducer of apoptosis in several lung carcinoma cells, including H460 cells. This induction is associated with activation of caspase-3, -9, and -12, and cleavage of PARP. To obtain insight into the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and control H460 cells using microarray analysis. This analysis revealed that many of the genes implicated in endoplasmic reticulum (ER) stress, including ATF3, CHOP/GADD153, HERPUD1, BIP (GRP78), XBP1, PDIA4, and TDAG51, were highly up-regulated within 4 hr of FOH treatment suggesting that FOH-induced apoptosis involves an ER-stress response. This was supported by observations showing that treatment with FOH induces phosphorylation of eIF2ï¡. FOH induces activation of several MAPK pathways, including p38, MEK-ERK, and JNK. Inhibition of MEK1/2 by U0126 inhibited the induction of ER stress-response genes. In addition, knockdown of the MEK1/2 and JNK1/2 expression by short interfering RNA (siRNA) effectively inhibited the induction of apoptosis and activation of caspase-3 and cleavage of PARP by FOH. However, only MEK1/2 siRNAs reduced the expression of ER stress-related genes and inhibited phosphorylation of eIF2ï¡. Our results demonstrate that FOH-induced apoptosis is coupled to ER stress and that activation of MEK1/2 is an upstream event in the FOH-induced ER stress signaling cascade. Vehicle vs. 4h FOH Signature Gene lists (Replicates 1 & 2) are linked as supplementary files to the Series record. Experiment Overall Design: H460 cells were treated for 4 hours with 250 micromolar FOH or vehicle (DMSO) in duplicate experiments. Each FOH treated sample was compared to its matched Vehicle and dye-flips were performed, resulting in 4 arrays (2 replicate sets x 2 technical replicates).

Dataset Information

Zinc finger nuclease knockouts of human ADP-glucokinase

Publications

Expression and role in glycolysis of human ADP-dependent glucokinase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets