83008-59-7Relevant academic research and scientific papers
Quantification of meccnu-induced dg-dc crosslinks in oligonucleotide duplexes by liquid chromatography/electrospray ionization tandem mass spectrometry
Bai, Baoqing,Zhao, Lijiao,Zhong, Rugang
, p. 2027 - 2034 (2011)
Chloroethynitrosoureas (CENUs) are important alkylating agents widely used in the treatment of cancers. Decomposition of CENUs generates active electrophilic ions that damage DNA, including the formation of dG-dC crosslinks which represents the most important cytotoxic mechanism of CENUs. In this work, a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method was employed to analyze the dG-dC crosslinks induced by 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (meCCNU, Semustine). The direct quantitation of dG-dC crosslinks in oligonucleotide duplexes was achieved by the selected reaction monitoring (SRM) mode using synthesized 15N3-labeled dG-dC as an internal standard. Methods of enzymatic digestion and HPLC separation were developed for obtaining separation and reproducibility of the dG-dC peak in chromatograms. The limit-of-detection (LOD) was determined to be 0.08 nM and the limit-of-quantification (LOQ) was determined to be 0.16 nM. The linearity of the calibration curve was 0.9997 over the range of 0.08 to 32 nM. The precision and accuracy of the method ranged from 1.1 to 6.6% and 96 to 109%, respectively. The recovery of the dG-dC crosslink in the enzymatic hydrolysates from the oligonucleotide duplex was determined to be from 91 to 106%. The results of the validation study indicate that the method is suitable for quantifying dG-dC crosslinks in DNA. Consequently, this method was used to determine meCCNU-induced dG-dC crosslinks in four duplexes with different GC contents. The results showed that the crosslinking fraction (CF) increased as the GC content in the duplex increased, and a relatively low CF was observed in the early period of the reaction. Copyright
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, Lijiao,Zhong, Rugang
, p. 439 - 447 (2014/03/21)
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 O6-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 O6-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
Comparative investigation of the DNA inter-strand crosslinks induced by ACNU, BCNU, CCNU and FTMS using high-performance liquid chromatography- electrospray ionization tandem mass spectrometry
Zhao, Lijiao,Li, Lili,Xu, Jie,Zhong, Rugang
, p. 30 - 36 (2014/06/23)
Chloroethylnitrosoureas (CENUs) are an important family of alkylating agents employed in the clinical treatments of cancer. They exert cytotoxicity by inducing DNA interstrand crosslinks (ICLs) between guanine and the complimentary cytosine, namely dG-dC crosslink. Many investigations have been performed on the DNA ICLs involved in the anticancer efficacy of CENUs, but no conclusive comparisons between these agents have been published. In this work, the levels of dG-dC crosslink in calf thymus DNA induced by four CENUs, including nimustine (ACNU), carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS), were quantitatively determined using HPLC-ESI-MS/MS. The obtained time-courses for the dG-dC crosslinking levels indicated that there is an induction period with very low crosslinking activity at the initial stage of the treatment by BCNU and CCNU. The induction period provides a convincing evidence for the presumed mechanism that the formation of dG-dC crosslinks was initiated by the monoalkylation of guanine followed by the second alkylation of the complimentary cytosine. The crosslinking activity of ACNU is remarkably higher than those of BCNU, CCNU and FTMS at all time points. The crosslinking activities of CENUs were found to be related to their stability in aqueous solution. ACNU has the shortest half-life among the four CENUs, but has highest crosslinking levels; on the contrary, CCNU has the lowest crosslinking activity with the longest half-life. Moreover, a correlation was found between the crosslinking activity and the anticancer efficiency. ACNU with the highest crosslinking activity showed the better survival gain for high-grade glioma than BCNU, CCNU and FTMS as reported in an epidemiological study. This suggests that dG-dC crosslink can possibly be employed as a potential biomarker for evaluating the anticancer efficiency of novel CENU drugs.
