RATIONALEChloroethylnitrosoureas　(CENUs)　are　important　alkylating　agents　employed　for　the　clinical　treatment　of　cancer.　The　cellular　toxicity　of　CENUs　is　primarily　due　to　induction　of　DNA　interstrand　crosslinks　(ICLs),　which　has　been　characterized　as　l-(3-deoxycytidyl),　2-(l-deoxyguanosinyl)ethane　(dG-dC).　However,　the　formation　of　dG-dC　crosslinks　can　be　prevented　by　O-6-alkylguanine-DNA　alkyltransferase　(AGT),　which　removes　the　O-6-chloroethyl　group　from　O-6-chloroethylguanine　(O-6-ClEt-Gua),　and　ultimately　its　increased　expression　can　result　in　drug　resistance.　Differing　levels　of　AGT　expression　can　lead　to　varying　amounts　of　dG-dC　crosslinking,　which　influences　the　sensitivity　of　cells　to　CENUs.　METHODSIn　this　work,　a　sensitive　method　for　the　quantitation　of　dG-dC　crosslinks　in　cellular　DNA　has　been　established　using　high-performance　liquid　chromatography/electrospray　ionization　tandem　mass　spectrometry　(HPLC/ESI-MS/MS).　RESULTSThe　limit　of　detection　(LOD)　and　limit　of　quantitation　(LOQ)　of　the　method　were　determined　to　be　2　fmol　and　8　fmol　on-column,　respectively,　and　the　recovery　ranged　from　96%　to　105%　with　the　relative　standard　deviation　(RSD)　below　5%.　Using　this　method,　the　levels　of　dG-dC　crosslink　induced　by　1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea　hydrochloride　(ACNU)　were　determined　in　NIH/3T3　fibroblasts　cells　(high　level　of　expression　of　AGT)　and　L1210　leukemia　cells　(low　level　of　expression　of　AGT).　The　time-course　profile　indicated　that　the　levels　of　dG-dC　crosslink　uniformly　increased　in　the　early　incubation　period　and　reached　the　maximum　at　12h.　Subsequently,　the　amount　of　dG-dC　crosslinking　decreased　to　very　low　levels　presumably　owing　to　the　repair　of　O-6-ClEt-Gua　by　AGT.　The　crosslinking　levels　in　L1210　cells　were　significantly　higher　than　those　in　NIH/3T3　cells　at　each　time　point.　This　provides　strong　evidence　that　high　express　of　AGT　in　CENU-resistant　cells　inhibits　the　formation　of　dG-dC　crosslinks.　CONCLUSIONSThis　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.　Copyright　(c)　2014　John　Wiley　&　Sons,　Ltd.