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TET1 regulates DNA repair in human glial cells.

ABSTRACT: Glioblastomas are the most aggressive of malignant brain cancers with a median patient survival of approximately 18?months. We recently demonstrated that Tet methylcytosine dioxygenase 1(TET1) is involved in cellular responses to ionizing radiation (IR) in glial-, glioblastoma-, and non-tumor-derived cells. This study used a lentiviral-mediated knockdown of TET1 to further dissect the contribution of TET1 to the DNA damage response in glial cell lines by evaluating its role in DNA repair. TET1-deficient glial cell lines displayed attenuated cytotoxicity compared to non-targeted knockdown after treatment with IR but these differences were not observed between control and TET1 deficient in response to inhibitors of Na+/K+-ATPase. Additionally, the percentage of glial cells displaying ?H2A.x foci was greatly reduced in TET1-deficient glial cells compared to non-targeted knockdown conditions in response to IR and topoisomerase inhibitors. We also observed a lower percentage and a delay in 53BP1 foci formation, a marker of non-homologous end-joining, in response to IR and topoisomerase inhibitors in TET1-deficient glial cells. DNA-PK, another marker of non-homologous end-joining, was also lower in TET1-deficient glial cell lines. Interestingly, TET1-deficient glial cells displayed higher numbers of DNA strand breaks compared to control cells and repaired DNA breaks less efficiently in Comet assays. We suggest that attenuated DNA repair in TET1 deficient gliomas leads to genomic instability, which underlies poor patient survival.

SUBMITTER: Kuhns KJ

PROVIDER: S-EPMC7184935 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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TET1 regulates DNA repair in human glial cells.

Kuhns Katherine J KJ Lopez-Bertoni Hernando H Coulter Jonathan B JB Bressler Joseph P JP

Toxicology and applied pharmacology 20190703

Glioblastomas are the most aggressive of malignant brain cancers with a median patient survival of approximately 18 months. We recently demonstrated that Tet methylcytosine dioxygenase 1(TET1) is involved in cellular responses to ionizing radiation (IR) in glial-, glioblastoma-, and non-tumor-derived cells. This study used a lentiviral-mediated knockdown of TET1 to further dissect the contribution of TET1 to the DNA damage response in glial cell lines by evaluating its role in DNA repair. TET1-d ...[more]

PMID: 31278917

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Project description:Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 ?M, 30 min) or ? irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or ? irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

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TET1 regulates DNA repair in human glial cells.

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TET1 regulates DNA repair in human glial cells.

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