Sensitive liquid chromatography mass spectrometry (LC-MS) assay reveals novel insights on DNA methylation and incorporation of gemcitabine, its metabolite difluorodeoxyuridine, deoxyuridine, and RX-3117 into DNA.

Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6-6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.