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Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.

ABSTRACT: Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O⁶-methylguanine (O⁶mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O⁶mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O⁶mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O⁶mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O⁶mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

SUBMITTER: Fuchs RP

PROVIDER: S-EPMC8289412 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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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Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.

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