2748

Hypermethylation of gene promoters has been described as a common mechanism of gene silencing in cancer. The adenosine analogues, fludarabine and cladribine, have been shown to inhibit DNA methylation, believed to be related to alterations in the S-adenosylmethionine: S-adenosylhomocysteine (SAM/SAH) ratio (Wyczechowska et al., 2003). SAM is used as the methyl-donor for methylation of DNA with its subsequent conversion to SAH, an inhibitor of DNA methyltransferases. 6-mercaptopurine (MP) has also been shown to alter the SAM/SAH ratio in the leukaemic cell line MOLT-4, with a concomitant decrease in global methylation (Lambooy et al., 1998). These experiments did not take into account the level of thiopurine methyltransferase (TPMT), which uses SAM as the methyl-donor for the methylation of MP and its metabolites. Therefore, we investigated the effect of TPMT on SAM/SAH levels and global methylation on exposure of cells to 6-MP and 6-thioguanine (TG), both, used in the treatment of acute lymphoblastic leukaemia, using EcR 293 cells transfected with TPMT under an inducible vector system. For MP, induction of TPMT led to a 1.2 to 1.9 fold increase in SAH for doses between 1.8 and 80μM, respectively, whereas MP with low TPMT and TG had no significant effect on SAH levels. Increase in SAH with MP appeared not to be caused by the MP metabolite, methylthioinosine monophosphate (MeTIMP), the potent purine de novo synthesis inhibitor, as exposure of cells to methylmercaptopurine riboside (MeMPR), which is converted directly to MeTIMP intracellularly, did not significantly alter SAH levels. There was a decrease in SAM levels for both drugs, which was more pronounced with MP compared to TG. Global methylation after 1.8μM MP unexpectedly resulted in increased DNA methylation with both high and low TPMT levels to approximately 150% of control. However, at higher doses of 8, 20 and 80μM, with high TPMT, there was a decrease in methylation to a maximum of 70% of control. This decrease was not due to MeTIMP production as exposure of cells to MeMPR caused no decrease in global methylation. When cells were treated with TG a dose dependent decrease in methylation was observed to about 55% of control, with low TPMT at a dose of 4μM and above. With high TPMT decrease in DNA methylation was significantly less up to a dose of at least 4μM. Changes in SAM and SAH levels did not reflect the different effects seen with MP compared to TG on DNA methylation, suggesting other mechanisms are involved. Interestingly, when cells were exposed to 1-20μM of the de-methylating agent, decitabine, cells showed on average a 1.5-fold greater de-methylation with high compared to low TPMT. TG was shown to have a de-methylating effect comparable to decitabine, with low TPMT, at pharmacologically relevant doses. The re-expression of genes silenced by promoter methylation is currently being investigated.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]