Cell death rates for SF-126 (open bars), SF-767 (striped bars) and SF-763 (solid bars) cells exposed to ACNU at the concentrations of 0.2 (A), 0.6 (B) and 1 (C) mM.

<p>Cell death rates for SF-126 (open bars), SF-767 (striped bars) and SF-763 (solid bars) cells exposed to ACNU at the concentrations of 0.2 (A), 0.6 (B) and 1 (C) mM.</p

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

<div><p>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.</p></div

Comparison of the levels of dG-dC crosslink (fmol/mg DNA) induced by ACNU, BCNU, CCNU and FTMS in L1210 cells, represented by gray bars, open bars, striped bars and solid bars, respectively.

<p>The concentrations of the drugs are 0.025 (A), 0.05 (B), 0.1 (C), 0.2 (D) and 0.4 mM (E).</p

Comparison of the dG-dC crosslinking levels (fmol/mg DNA) in SF-126 (open bars), SF-767 (striped bars) and SF-763 (solid bars) cells treated with ACNU at the concentrations of 0.2 (A), 0.6 (B) and 1 (C) mM.

<p>Comparison of the dG-dC crosslinking levels (fmol/mg DNA) in SF-126 (open bars), SF-767 (striped bars) and SF-763 (solid bars) cells treated with ACNU at the concentrations of 0.2 (A), 0.6 (B) and 1 (C) mM.</p

Supposed mechanisms for the formation of dG-dC crosslinks induced by CENUs and the repair of crosslinks mediated by AGT.

<p>Supposed mechanisms for the formation of dG-dC crosslinks induced by CENUs and the repair of crosslinks mediated by AGT.</p

SRM chromatograms of dG-dC crosslinks in the DNA digestion mixtures from control cells (A) and ACNU-treated SF-126 (B), SF-767 (C), SF-763 (D) and L1210 (E) cells.

<p>SRM chromatograms of dG-dC crosslinks in the DNA digestion mixtures from control cells (A) and ACNU-treated SF-126 (B), SF-767 (C), SF-763 (D) and L1210 (E) cells.</p