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Inhibition of DNA Repair by Inappropriate Activation of ATM, PARP, and DNA-PK with the Drug Agonist AsiDNA.

ABSTRACT: AsiDNA is a DNA repair inhibitor mimicking DNA double-strand breaks (DSB) that was designed to disorganize DSB repair pathways to sensitize tumors to DNA damaging therapies such as radiotherapy and chemotherapy. We used the property of AsiDNA of triggering artificial DNA damage signaling to examine the activation of DSB repair pathways and to study the main steps of inhibition of DNA repair foci after irradiation. We show that, upon AsiDNA cellular uptake, cytoplasmic ATM and PARP are rapidly activated (within one hour) even in the absence of irradiation. ATM activation by AsiDNA leads to its transient autophosphorylation and sequestration in the cytoplasm, preventing the formation of ATM nuclear foci on irradiation-induced damage. In contrast, the activation of PARP did not seem to alter its ability to form DNA repair foci, but prevented 53BP1 and XRCC4 recruitment at the damage sites. In the nucleus, AsiDNA is essentially associated with DNA-PK, which triggers its activation leading to phosphorylation of H2AX all over chromatin. This pan-nuclear phosphorylation of H2AX correlates with the massive inhibition, at damage sites induced by irradiation, of the recruitment of repair enzymes involved in DSB repair by homologous recombination and nonhomologous end joining. These results highlight the interest in a new generation of DNA repair inhibitors targeting DNA damage signaling.

SUBMITTER: Berthault N

PROVIDER: S-EPMC9320633 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Non?small cell lung cancer (NSCLC) is radioresistant to X?rays due to powerful cellular DNA damage repair mechanisms. DNA?dependent protein kinase (DNA?PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA?PK activity. In the present study A549 cells were treated with the DNA?PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X?ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, ?H2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c?nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X?ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X?ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased ?H2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X?rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full?length transactivating (TA) p73 and an N?terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab?like GTPase activators and myotubularins domain?containing 4 (GRAMD4) protein to reduce the Bcl?2/Bax protein ratio. In addition, ATM siRNA efficiently prevented ?H2AX foci formation, and enhanced NU7026?induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA?PK activity increased the radiosensitivity of A549 cells to X?ray irradiation. NU7026 treatment activated the ATM?dependent DNA damage response and induced p73 apoptosis pathway. DNA?PK inhibitor may be an effective constituent of radiosensitization products. DNA damage repair pathway could be a potential target for radiosensitization.

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Inhibition of DNA Repair by Inappropriate Activation of ATM, PARP, and DNA-PK with the Drug Agonist AsiDNA.

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