LC-MS/MS coupled with stable isotope dilution method for the quantification of 6-thioguanine and S(6)-methylthioguanine in genomic DNA of human cancer cells treated with 6-thioguanine.

Thiopurines, including mercaptopurine (MP), 6-thioguanine ((S)G) and azathioprine, are widely used for the treatment of many human diseases including acute lymphoblastic leukemia (ALL). To exert their cytotoxic effect, these prodrugs need to be metabolically activated to (S)G nucleotides and incorporated into nucleic acids. (S)G in DNA can be methylated spontaneously to S(6)-methylthioguanine (S(6)mG) in the presence of S-adenosyl-l-methionine. It was proposed that S(6)mG, owing to its high miscoding potential (pairing preferentially with thymine), may induce cell death by triggering the postreplicative mismatch repair pathway. Understanding the implications of this pathway in the cytotoxic effect of thiopurine drugs necessitates an accurate measurement of the level of S(6)-methylthio-2'-deoxyguanosine (S(6)mdG) in DNA of cells treated with thiopurine drugs. Here we developed a sensitive HPLC coupled with tandem mass spectrometry (LC-MS/MS) method and measured the level of 6-thio-2'-deoxyguanosine ((S)dG) and S(6)mdG in genomic DNA of four human leukemia cell lines and one human colorectal carcinoma cell line. Our results revealed that, upon treatment with 3 muM (S)G for 24 h, approximately 10, 7.4, 7, and 3% of guanine was replaced with (S)G in Jurkat T, HL-60, CCRF-CEM and K-562 cells, respectively. However, only less than 0.02% of (S)dG was converted to S(6)mdG in the above cell lines. HCT-116 cells had the lowest level (0.2%) of guanine being replaced with (S)G in DNA, and approximately 5 out of 10(4 S)G was converted to its methylated counterpart. This is the first report of the simultaneous and accurate quantification of (S)dG and S(6)mdG in genomic DNA of cultured human cells treated with (S)G. In addition, our results suggested that DNA (S)G might trigger mismatch repair (MMR) pathway without being converted to S(6)mG.