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VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells.

ABSTRACT: Purpose:DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported. Experimental Design:The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence assays (IF) evaluated ?-H2AX foci for DSB repair kinetics in human astrocytes and T98G glioma cells. Western blotting was used to evaluate phosphorylation of DNA-PKcs substrates. Results:We found a dose-dependent reduction in CSR efficiency with VX-984, and through the EJ-DR assay, dramatic dose-dependent increases in HR and mNHEJ. Immunofluorescence assays showed an inability of malignant cells to resolve ?-H2AX foci in the presence of VX-984. Radiation-induced phosphorylation of DNA-PK substrates was further reduced by treatment with VX-984. Conclusions:VX-984 efficiently inhibits NHEJ, resulting in compensatory increases in alternative repair pathways, increases DSBs, and appears to affect transformed cells preferentially.

SUBMITTER: Khan AJ

PROVIDER: S-EPMC5995231 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells.

Khan Atif J AJ Misenko Sarah M SM Thandoni Aditya A Schiff Devora D Jhawar Sachin R SR Bunting Samuel F SF Haffty Bruce G BG

Oncotarget 20180525 40

<h4>Purpose</h4>DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported.<h4>Experimental design</h4>The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence ...[more]

PMID: 29899825

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Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Experiment Overall Design: Number of plants pooled:20

Dataset Information

VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells.

Publications

VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells.

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