A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia

Abstract

SummaryDNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O 2 ) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK . To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways. Journal of Cell Sciencesee Brahimi-Horn and Pouyssegur, 2009;Semenza, 2007;Yee Koh et al., 2008). Therefore, the activity of this complex is exquisitely dependent upon the limiting expression of the a-subunit. Under hypoxia, HIF-1a is stabilised, enters the nucleus and heterodimerises with HIF-1. The heterodimer binds to hypoxia responsive elements (HREs) to transactivate a variety of hypoxiaresponsive genes (Yee Koh et al., 2008), therefore contributing to the adaptative response to hypoxic conditions. Our experiments are designed to determine whether DNA-PK is activated by hypoxic stress in human cells, the mechanisms of its activation and the biological consequences for cells of this stress-response pathway. We demonstrate that DNA-PK is activated under hypoxic conditions. This cellular stress response favours hypoxia adaptation by protecting HIF-1a from degradation. Importantly, our data are consistent with the new hypothesis of a DNA-dependent stress response initiated by chromatin modifications. Results DNA-PK is activated in hypoxic cellsDuring the processing of DNA lesions, DNA-PK is phosphorylated, partly by autophosphorylation. Among the sites identified, Ser2056 appears to be phosphorylated only in response to DNA DSBs in a strictly DNA-PK-dependent manner (Chen et al., 2005). To assess DNA-PK activation, cells were exposed to hypoxia (0.1 or 1% O 2 ) at different times. As a control for DNA-PK activation, we used a well-known DNA-strand-breaking agent, calicheamicin gamma-1 (CLg1) (Fig. 1A, left) (Bouquet et al., 2006). In the presence of DSBs, we detected a strong and early (1 hour) accumulation of phosphorylated forms of DNA-PKcs (P-DNAPKcs) and AT...

HIF mediated and DNA damage independent histone H2AX phosphorylation in chronic hypoxia

“…We previously reported that DNA-PK expression was strongly reduced by the absence of HIF-1 α under both normoxic and hypoxic conditions (Wirthner et al , 2008 ), raising the possibility that DNA-PK might be the responsible kinase for H2AX phosporylation in chronic hypoxia. In line with this hypothesis, accumulation of DNA-PKcs, Ku70 and Ku80 following hypoxia and iron chelation have been demonstrated in a number of different cell lines (Ginis and Faller , 2000 ;Lynch et al , 2001 ;Um et al , 2004 ;Bouquet et al , 2011 ). DNA-PK has been shown to phosphorylate H2AX in different cell lines and in vivo in response to DNA damage (Stiff et al , 2004 ;Koike et al , 2008 ;An et al , 2010 ) under hypertonic conditions (Reitsema et al , 2005 ) and during apoptotic DNA fragmentation (Mukherjee et al , 2006 ).…”

“…DNA-PK has been shown to phosphorylate H2AX in different cell lines and in vivo in response to DNA damage (Stiff et al , 2004 ;Koike et al , 2008 ;An et al , 2010 ) under hypertonic conditions (Reitsema et al , 2005 ) and during apoptotic DNA fragmentation (Mukherjee et al , 2006 ). Of note, the hypoxic DNA-PK activation resulted in increased HIF-dependent gene expression (Bouquet et al , 2011 ). These data suggest that DNA-PK might be both upstream and downstream of HIF.…”

RON Nuclear Translocation under Hypoxia Potentiates Chemoresistance to DNA Double-Strand Break–Inducing Anticancer Drugs

“…A recent study (47) showed that DNA-PK was important for regulating HIF-1 stability and its target gene, GLUT1, under hypoxia (47). Taken together, these findings suggest that the tyrosine kinase domain of RON not only activates the intracellular signaling pathway, but also interacts with other nuclear proteins, HIF-1a, Ku70, and DNA-PKcs, when cancer cells are exposed to hypoxiainduced stress.…”

Inhibition de la réparation des cassures double-brin de l'ADN: quel intérêt en radiothérapie et pharmacologie antitumorale?

“…(i) La DNA-PK est impliquée dans des voies biochimiques autres que la réparation des cassures: maintien de la longueur des télo-mères, rôle dans le point de contrôle G2 après irradiation, méta-bolisme en réponse à l'insuline, phosphorylation de substrats qui ne participent pas à la réparation de l'ADN, contrôle de la dégradation du facteur de régulation de l'hypoxie, HIF-1␣ (Bouquet et al 2011 ;Kong et al 2011). Ces nouvelles données renforcent l'importance de la DNA-PK dans le maintien de l'homéostasie cellulaire et la réponse à divers stress mais paradoxalement compromettent l'intérêt thérapeutique d'un blocage de cette activité non spécifique aux cellules tumorales.…”