尼莫司汀和卡莫司汀导致细胞中DNA股间交联的高效液相色谱-电喷雾质谱联用研究

Quantitation of DNA Interstrand Crosslinks Induced by ACNU and BCNU in Cells Using HPLC-ESI-MS/MS

氯乙基亚硝基脲（CENUs）是临床常用的烷化剂类抗癌药物,主要通过导致DNA股间横向交联（dG-dC交联）发挥其抗癌作用。为建立高灵敏度的细胞中交联物的检测方法,评价不同种类CENUs的抗癌活性及耐药性,本研究使用尼莫司汀（ACNU）和卡莫司汀（BCNU）对具有不同O6-烷基鸟嘌呤-DNA-烷基转移酶（AGT）活性的NIH/3T3和L1210两种细胞进行药物处理,利用高效液相色谱-电喷雾串联质谱（HPLC-ESI-MS/MS）联用技术对经不同浓度药物处理后细胞中的dG-dC交联进行定量分析。该方法的检测限（S/N=5）和定量限（S/N=17）分别达到2fmol和8fmol,回收率为92.5%~107.4%,灵敏度和准确性均满足定量分析的要求。结果表明：ACNU导致的dG-dC交联率高于BCNU;经相同浓度的同种药物作用后,L1210细胞中交联率显著高于NIH/3T3细胞。这可为新型CENU类烷化剂的抗癌活性评价提供可靠的实验方法。

Chloroethylnitrosoureas （CENUs） are alkylating agents, which are used widely in the clinical treatment of cancers. CENUs exhibit anticancer activity by inducing DNA interstrand crosslinks （ICLs）, mainly dG-dC crosslinks. To evaluate the anticancer activity and drug resistance of CENUs, it is necessary to establish a highly sensitive method for the quantitation of CENU-induced DNA ICLs in cells. In this study, NIH/3T3 fibroblasts cells and L1210 leukemia cells, which had different O6-alkylguanine-DNA alkyltransferase（AGT） activity, were treated with Nimustine （ACNU） and Carmustine （BCNU）. The levels of dG-dC crosslinks in cells were determined by high-performance liquid chromatogra- phy-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS）. The limit of detection （signal to noise （S/N）=5） and limit of quantitation （S/N=IT） achieve to 2 fmol and 8 fmol, respectively. The recovery is range from 92.5% to 107.4%. The sensitivity and accuracy of the quantitative analysis of dG-dC crosslinks in cells are satisfied. The results indicate that the dG-dC level induced by ACNU is higher than that by BCNU. Moreo- ver, the dG-dC level in L1210 cells is obviously higher than that in NIH/3T3 after treated at the same drug concentrations. This work provides a reliable method for evaluating the anticancer activity of novel CENU alkylating agents.