S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts

Abstract

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity. We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10-50microM) rescues cells from the toxic effects of 6-MP (5microM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor. This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

“…A study by Brouwer et al 2005 [15], showed that TPMT enzyme activity increased in MOLT4 cells after incubation with MTX, however, in their study, cells were simultaneously treated with methionine, thus creating increased amounts of SAM in the cells. As SAM stabilises TPMT it could be that the increase in enzyme activity is due to SAM, not to MTX [21].…”

Pharmacogenetics of thiopurine therapy: from thiopurine S-methyltransferase to S-adenosylmethionine

“…We herein present evidence of a novel TPMT-mediated mechanism of SAM-specific effects on 6-MP-induced cytotoxicity [2]. Our results show that exogenous SAM rescues cells from the toxic effects of 6-MP by restoring cell proliferation and delaying the onset of apoptosis.…”

One amino acid makes a difference–Characterization of a new TPMT allele and the influence of SAM on TPMT stability

“…The importance of studying the influence of SAM on TPMT on a molecular level becomes even more clear by the fact that studies on cultured leukemic cells show that the cytotoxic effect of 6-MP can be reduced by the addition of exogenous SAM. The addition of exogenous SAM lowered the concentration of TGNs and methyl thioinosine monophosphate (MeTIMP)23, cytotoxic metabolites of 6-MP. In the same study, the addition of exogenous SAM reversed the 6-MP induced reduction of TPMT activity and protein levels.…”

“…However, the finding that polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) gene could indirectly influence TPMT activity22, by altering intracellular SAM levels as a result of the affected folate metabolism, leads to the question of whether SAM could function as a predictor of the enzymatic activity. By studying SAM levels and TPMT activity in a leukemic cell line, it has been shown that adding exogenous SAM to culturing media could sustain the intracellular levels of adenosine triphosphate (ATP) and SAM and delay the cytotoxic mechanisms, induced by 6-MP23. Earlier studies comparing the intrinsic stability, at 37 °C, of TPMT*1, TPMT*3B and TPMT*3C revealed that, despite the fact that no loss of immunodetectable protein occurred, the addition of SAM partially stabilized the catalytic activity of TPMT*3C24.…”