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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
Author(s) -
Li Lili,
Zhao Lijiao,
Zhong Rugang
Publication year - 2014
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6800
Subject(s) - chemistry , electrospray ionization , chromatography , tandem mass spectrometry , extractive electrospray ionization , mass spectrometry , electrospray , direct electron ionization liquid chromatography–mass spectrometry interface , protein mass spectrometry , liquid chromatography–mass spectrometry , ionization , chemical ionization , ion , organic chemistry
RATIONALE Chloroethylnitrosoureas (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. METHODS In 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). RESULTS The 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 12 h. 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. CONCLUSIONS 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. Copyright © 2014 John Wiley & Sons, Ltd.