Quantification of DNA interstrand crosslinks induced by ACNU in NIH/3T3 and L1210 cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Li, Lili; Zhao, Lu; Zhong, Rugang

doi:10.1002/rcm.6800

Cited by 11 publications

(14 citation statements)

References 38 publications

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“…Moreover, the high AGT activity appeared to correlate well with the elevated cell resistance to chloroethylating agents, which were also in good agreement with the obtained results that low AGT level potentiated the loss of cellular viability in human glioma cell lines. 22,33 Similar results were also obtained in previous studies. Bodell and colleagues quantified seven DNA alkylation products formed in different human glioma cell lines treated with CENU.…”

Section: ■ Results and Discussionsupporting

confidence: 93%

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents

Wang

Ding

et al. 2016

Anal. Chem.

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Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.

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Section: ■ Results and Discussionsupporting

confidence: 93%

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents

Wang

Ding

et al. 2016

Anal. Chem.

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“…In our previous study [ 32 ], the HPLC-ESI-MS/MS method was successfully employed in the determination of dG-dC crosslinks in mouse leukemia L1210 cells and fibroblast NIH/3T3 cells treated with ACNU. In the present study, the method was used for quantifying the dG-dC crosslinks in human glioma SF-126, SF-763 and SF-767 cells treated with ACNU and in L1210 cells treated with four types of CENUs.…”

Section: Resultsmentioning

confidence: 99%

“…The purity of DNA was confirmed by measuring UV absorption at 230, 260 and 280 with the ratios of 260/230 and 260/280 at 2.4 and 1.8, respectively. Following the previously reported protocols [ 32 ], DNA samples were digested with four enzymes, including DNase I, nuclease S1, alkaline phosphatase and snake venom phosphodiesterase. Briefly, the solutions were heated at 98°C for 5 min and promptly chilled in an ice-bath for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) is considered to be a robust technique with higher sensitivity and specificity compared with chromatographic, electrophoretic and fluorescent assays in the quantification of DNA adducts. In our previous studies, HPLC-MS/MS was employed to determine the yield of dG-dC crosslinks induced by MeCCNU in calf thymus DNA [ 31 ], and induced by ACNU in NIH/3T3 and L1210 cells [ 32 ]. In this work, we reported an HPLC-MS/MS quantification of dG-dC crosslink induced by ACNU, BCNU, CCNU and FTMS in SF-763, SF-767, SF-126 and L1210 cells containing different levels of AGT.…”

Section: Introductionmentioning

confidence: 99%

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Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry

Sun

et al. 2015

PLoS ONE

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Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.

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“…TMZ also acts as an inhibitor of DNA mismatch repair and can induce apoptosis. 22) ACNU forms a chloroethyl adduct at the O6 position of guanine, 10,23,24) whereas TMZ appears to act by methlyation at this site. Both the methyl and chloroethyl adducts are repaired by the DNA repair protein O6-alklyguanine-DNA alkytransferase (AGT).…”

Section: Discussionmentioning

confidence: 99%

Phase I/II Study of Temozolomide Plus Nimustine Chemotherapy for Recurrent Malignant Gliomas: Kyoto Neuro-oncology Group

Aoki

Arakawa

Ueba

et al. 2017

Neurol. Med. Chir.(Tokyo)

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The objective of this phase I/II study was to examine the efficacy and toxicity profile of temozolomide (TMZ) plus nimustine (ACNU). Patients who had received a standard radiotherapy with one or two previous chemo-regimens were enrolled. In phase I, the maximum-tolerated dose (MTD) by TMZ (150 mg/m2/day) (Day 1–5) plus various doses of ACNU (30, 35, 40, 45 mg/m2/day) (Day 15) per 4 weeks was defined on a standard 3 + 3 design. In phase II, these therapeutic activity and safety of this regimen were evaluated. Forty-nine eligible patients were enrolled. The median age was 50 years-old. Eighty percent had a KPS of 70–100. Histologies were glioblastoma (73%), anaplastic astrocytoma (22%), anaplastic oligodendroglioma (4%). In phase I, 15 patients were treated at four cohorts by TMZ plus ACNU. MTD was TMZ (150 mg/m2) plus ACNU (40 mg/m2). In phase II, 40 patients were treated at the dose of cohort 3 (MTD). Thirty-five percent of patients experienced grade 3 or 4 toxicities, mainly hematologic. The overall response rate was 11% (4/37). Sixty-eight percent (25/37) had stable disease. Twenty-two percent (8/37) showed progression. Progression-free survival (PFS) rates at 6 and 12 months were 24% (95% CI, 12–35%) and 8% (95% CI, 4–15%). Median PFS was 13 months (95% CI, 9.2–17.2 months). Overall survival (OS) at 6 and 12 were 78% (95% CI, 67–89%) and 49% (95% CI, 33–57%). Median OS was 11.8 months (95% CI, 8.2–14.5 months). This phase I/II study showed a moderate toxicity in hematology and may has a promising efficacy in OS, without inferiority in PFS.

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Quantification of DNA interstrand crosslinks induced by ACNU in NIH/3T3 and L1210 cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract: This work will contribute to the further understanding of the drug resistance of CENUs, and will provide a means to evaluate the anticancer activity of new bifunctional anticancer agents.

Cited by 11 publications

References 38 publications

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents

Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry

Phase I/II Study of Temozolomide Plus Nimustine Chemotherapy for Recurrent Malignant Gliomas: Kyoto Neuro-oncology Group

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