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Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.

ABSTRACT: Elucidating the DNA repair pathways that are activated in the presence of genotoxic agents is critical to understand their modes of action. Although the DT40 cell-based DNA damage response (DDR) assay provides rapid and sensitive results, the assay cannot be used on genotoxic compounds that require metabolic activation to be reactive. Here, we applied the metabolic activation system to a DDR and micronucleus (MN) assays in DT40 cells. Cyclophosphamide (CP), a well-known cross-linking agent requiring metabolic activation, was preincubated with liver S9 fractions. When DT40 cells and mutant cells were exposed to the preactivated CP, CP caused increased cytotoxicity in FANC-, RAD9-, REV3- and RAD18-mutant cells compared to isogenic wild-type cells. We then performed a MN assay on DT40 cells treated with preactivated CP. An increase in the MN was observed in REV3- and FANC-mutant cells at lower concentrations of activated CP than in the parental DT40 cells. These results demonstrated that the incorporation of metabolic preactivation system using S9 fractions significantly potentiates DDR caused by CP in DT40 cells and their mutants. In addition, our data suggest that the metabolic preactivation system for DDR and MN assays has a potential to increase the relevance of this assay to screening various compounds for potential genotoxicity.

SUBMITTER: Hashimoto K

PROVIDER: S-EPMC4723676 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.

Hashimoto Kiyohiro K Takeda Shunichi S Swenberg James A JA Nakamura Jun J

Mutagenesis 20150617 6

Elucidating the DNA repair pathways that are activated in the presence of genotoxic agents is critical to understand their modes of action. Although the DT40 cell-based DNA damage response (DDR) assay provides rapid and sensitive results, the assay cannot be used on genotoxic compounds that require metabolic activation to be reactive. Here, we applied the metabolic activation system to a DDR and micronucleus (MN) assays in DT40 cells. Cyclophosphamide (CP), a well-known cross-linking agent requi ...[more]

PMID: 26085549

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Project description:Rationale: Loss of DNA damage repair (DDR) in the tumor is an established hallmark of sensitivity to DNA damaging agents such as chemotherapy. However, there has been scant investigation into gain-of-function alterations of DDR genes in cancer. This study aims to investigate to what extent copy number amplification of DDR genes occurs in cancer, and what are their impacts on tumor genome instability, patient prognosis and therapy outcome. Methods: Retrospective analysis was performed on the clinical, genomics, and pharmacogenomics data from 10,489 tumors, matched peripheral blood samples, and 1,005 cancer cell lines. The key discoveries were verified by an independent patient cohort and experimental validations. Results: This study revealed that 13 of the 80 core DDR genes were significantly amplified and overexpressed across the pan-cancer scale. Tumors harboring DDR gene amplification exhibited decreased global mutation load and mechanism-specific mutation signature scores, suggesting an increased DDR proficiency in the DDR amplified tumors. Clinically, patients with DDR gene amplification showed poor prognosis in multiple cancer types. The most frequent Nibrin (NBN) gene amplification in ovarian cancer tumors was observed in 15 out of 31 independent ovarian cancer patients. NBN overexpression in breast and ovarian cancer cells leads to BRCA1-dependent olaparib resistance by promoting the phosphorylation of ATM-S1981 and homology-dependent recombination efficiency. Finally, integration of the cancer pharmacogenomics database of 37 genome-instability targeting drugs across 505 cancer cell lines revealed significant correlations between DDR gene copy number amplification and DDR drug resistance, suggesting candidate targets for increasing patient treatment response. Principal Conclusions: DDR gene amplification can lead to chemotherapy resistance and poor overall survival by augmenting DDR. These amplified DDR genes may serve as actionable clinical biomarkers for cancer management.

Dataset Information

Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.

Publications

Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.

Similar Datasets

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