LC-MS/MS Coupled with Stable Isotope Dilution Method for the Quantification of 6-Thioguanine and S6-Methylthioguanine in Genomic DNA of Human Cancer Cells Treated with 6-Thioguanine

摘要

Thiopurines, including mercaptopurine (MP), 6-thioguaninen(SG) and azathioprine, are widely used for thentreatment of many human diseases including acutenlymphoblastic leukemia (ALL). To exert their cytotoxicneffect, these prodrugs need to be metabolically activatednto SG nucleotides and incorporated into nucleicnacids. SG in DNA can be methylated spontaneously tonS6-methylthioguanine (S6mG) in the presence of Sadenosyl-nL-methionine. It was proposed that S6mG,nowing to its high miscoding potential (pairing preferentiallynwith thymine), may induce cell death by triggeringnthe postreplicative mismatch repair pathway.nUnderstanding the implications of this pathway in thencytotoxic effect of thiopurine drugs necessitates annaccurate measurement of the level of S6-methylthio-n2′-deoxyguanosine (S6mdG) in DNA of cells treatednwith thiopurine drugs. Here we developed a sensitivenHPLC coupled with tandem mass spectrometry (LCMSMS) method and measured the level of 6-thio-2′-ndeoxyguanosine (SdG) and S6mdG in genomic DNA ofnfour human leukemia cell lines and one human colorectalncarcinoma cell line. Our results revealed that,nupon treatment with 3 μM SG for 24 h, approximatelyn10, 7.4, 7, and 3% of guanine was replaced with SG innJurkat T, HL-60, CCRF-CEM and K-562 cells, respectively.nHowever, only less than 0.02% of SdG wasnconverted to S6mdG in the above cell lines. HCT-116ncells had the lowest level (0.2%) of guanine beingnreplaced with SG in DNA, and approximately 5 out ofn104 SG was converted to its methylated counterpart.nThis is the first report of the simultaneous and accuratenquantification of SdG and S6mdG in genomic DNA ofncultured human cells treated with SG. In addition, ournresults suggested that DNA SG might trigger mismatchnrepair (MMR) pathway without being converted tonS6mG.