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IFN signaling after proton and carbon ion irradiation is enhanced by ATR inhibition
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International audience. BackgroundExternal beam irradiation with x-rays is part of the standard treatment for glioblastoma, but is limited by tumor radioresistance and damage to the normal brain. A lower radiation dose to healthy tissue may be obtained by irradiating with charged particles, due to their beneficial depth-dose distribution. Furthermore, protons and heavier ions have an increased linear energy transfer (LET) as compared to x-rays, particularly at the Bragg peak, causing clustered and more-difficult-to-repair DNA- damage. To further challenge the repair capacity of tumor cells, radiation can be combined with DNA-damage response inhibitors. Such combinations may cause cells to enter mitosis with higher amount of damaged DNA leading to more micronuclei. DNA from these micronuclei may be exposed to the cytosol and trigger an innate antitumor immune response. However, to what extent such immune responses are induced by protons and heavy ions, is largely unknown.MethodsHere we have compared the DNA-damage response and immune signaling in cells exposed to high or low LET charged particle irradiation in combination with inhibitors of the DNA-damage response proteins ATM and ATR. T98G or U-251 glioblastoma cell lines were irradiated (2-6 Gy) with experimental proton and carbon ion beams having energies of 17 MeV and 95 MeV, respectively. LETs for protons were 7 and 38 keV/µm and for carbon ions 28 and 73 keV/µm. DNA-damage and immune signaling was measured by immunoblotting, and flow cytometry was applied to assess cell cycle progression and induction of the DNA-damage marker yH2AX.ResultsIrradiation with high LET protons induced more damage and a stronger G2 arrest, as measured by flow cytometry, than low LET protons for the same radiation dose. Immunoblotting for phosphorylated CHK1 S345, and ATM S1981 also showed a more robust activation of ATR and ATM in response to high compared to low LET protons. Similar differences were obtained for carbon ions. Furthermore, co-treatment with an ATR inhibitor (VE822) abrogated the G2 checkpoint and led to increased type I interferon production at both high and low LET, as measured by immunoblotting of pSTAT1 three days after treatment in U-251. Secretion of IFN-β from x-ray irradiated cells was also increased by ATR inhibition. In contrast, co-treatment with an ATM inhibitor (AZD1390) caused a prolonged G2 checkpoint arrest and gave little increase in interferon levels. ConclusionThese results show an LET-dependent DNA-damage response in glioblastoma, and suggest that ATR inhibition can increase interferon signaling in both high and low LET particle irradiated cells, likely through abrogation of the G2 checkpoint.
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