Severe hypoxia increases expression of ATM and DNA-PKcs and it increases their activities through Src and AMPK signaling pathways

Background: Solid tumors often contain hypoxic regions because an abnormal and inefficient tumor vasculature is unable to supply sufficient oxygen. Tissue hypoxia is generally defined as a low oxygen concentration of less than 2%. It is well known that tumor cells under severe hypoxia, where oxygen concentration is less than 0.1%, show radioresistance. It has been reported that cells under severe hypoxia show different responses from those under mild hypoxia, where oxygen concentration is 0.5–2.0%. In the present study, we investigated the effects of severe hypoxia on expression and activities of ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), both of which regulate DNA double-strand breaks (DSBs) repair and radiation sensitivity. Signaling pathways for increasing expression and activities of ATM and DNA-PKcs under severe hypoxia were also investigated. Methods: SV40-transformed human fibroblast cell lines, LM217 and LM205, and normal human dermal fibroblasts (NHDF) were used. Cells were cultured at an oxygen concentration of less than 0.05% for 12 or 24 h. Activities and/or expression of ATM, DNA-PKcs, Src, Caveolin-1, EGFR, HIF-1α, PDK1, Akt, AMPKα, and mTOR were estimated by Western blot analyses. Results: Severe hypoxia increased expression and activities of ATM, DNA-PKcs, Src, Caveolin-1, EGFR, PDK1, Akt, and AMPKα, and decreased expression and activity of mTOR. A specific Src inhibitor, PP2 suppressed activation of ATM, DNA-PKcs, Caveolin-1, EGFR, and Akt under severe hypoxia. Treatment with siRNA for AMPKα suppressed activation of ATM and DNA-PKcs and increase of ATM expression under severe hypoxia. Conclusion: Our data show that severe hypoxia increases activities of ATM and DNA-PKcs through Src and AMPK signaling pathways, and that activation of AMPK under hypoxia causes increase of ATM expression. Since ATM and DNA-PKcs play important roles in DSBs repair induced by ionizing radiation, those data provide novel insights on the molecular mechanism of the cellular radioresistance under severe hypoxia.