Abstract
Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we 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 lines (H460 and HCT116). All four clones 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 consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, 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 in these lines.
Highlights
The identification of large numbers of candidate genes through genomic analysis has created a pressing need for new approaches for ascribing biological function
The ability of the zincfinger nucleases (ZFN) to introduce mutations at this site was tested in HCT116 cells using the SurveyorTM mutation detection assay (Figure 1B), following lipid-based co-transfection with a GFP plasmid and FACS sorting 24 h later to enrich for transfected cells
In this study we use a pair of ZFNs, custom-designed to target a genomically unique sequence in the ADPdependent glucokinase (ADPGK) gene (Figure 1A), to generate human tumour cell lines with frameshift mutations at the target site and which completely lack ADPGK protein by immunoblotting
Summary
The identification of large numbers of candidate genes through genomic analysis has created a pressing need for new approaches for ascribing biological function. Dimeric ZFNs capable of recognising 18–42 base pair sequences can be used to introduce double strand DNA breaks at unique locations in the genome. These DNA breaks initiate error-prone non-homologous end joining repair to generate site-specific, heterogeneous mutations (predominantly small indels that disrupt gene function) or, in the presence of a donor DNA sequence, to introduce defined mutations via homology-directed repair. Given extensive investigation of glucose phosphorylation as the central reaction of intermediary metabolism for many decades [11], mammalian ADPGK was discovered only recently through its sequence to archaeal ADP-dependent glucokinases [12]. The biochemical properties of ADPGK, its ability to utilise ADP, led us to hypothesise that it may play a role in priming glycolysis under stress conditions where ATP becomes limiting [12], such as under hypoxia when cells become highly dependent on glycolytic ATP generation [19]
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