6-Mercaptopurine (6-MP) and methylmercaptopurine ribonucleoside (Me-MPR) are purine anti-metabolites which are both metabolized to methylthio-IMP (Me-tIMP), a strong inhibitor of purine synthesis de novo. Me-MPR is converted directly into Me-tIMP by adenosine kinase. 6-MP is converted into tIMP, and thereafter it is methylated to Me-tIMP by thiopurine methyltransferase, an S-adenosylmethionine (S-Ado-Met)-dependent conversion. S-Ado-Met is formed from methionine and ATP by methionine adenosyltransferase, and is a universal methyl donor, involved in methylation of several macromolecules, e.g. DNA and RNA. Therefore, depletion of S-Ado-Met could result in an altered methylation state of these macromolecules, thereby affecting their functionality, leading to dysregulation of cellular processes and cytotoxicity. In this study the effects of 6-MP and Me-MPR on S-Ado-Met, S-adenosylhomocysteine (S-Ado-Hcy), homocysteine and methionine concentrations are determined. Both drugs cause a decrease in intracellular S-Ado-Met concentrations and an increase in S-Ado-Hcy and methionine concentrations in Molt F4 human malignant lymphoblasts. The effects of both 6-MP and Me-MPR can be ascribed to a decreased conversion of methionine into S-Ado-Met, due to the ATP depletion induced by the inhibition of purine synthesis de novo by Me-tIMP. Both 6-MP and Me-MPR thus affect the methylation state of the cells, and this may result in dysregulation of cellular processes and may be an additional mechanism of cytotoxicity for 6-MP and Me-MPR.
The results of this study indicate that sequential administration of MTX before 6MP and of TF before TG may be essential for therapeutic success in the treatment of leukaemia.
SUMMARY. The effects of inhibition of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide de novo synthesis, on cell growth, cell viability, endogenous nucleotide concentrations and concentrations of extracellular nucleosides and bases were studied in Molt F4 human malignant lymphoblasts. Mycophenolic acid (MPA) was used as a specific inhibitor of the enzyme activity. IMPDH activity was maximally inhibited with O·5 J.lM MP A. After a 2 h exposure of the cells to 0·5 J.lM MPA, guanine nucleotides were depleted to approximately 50% of control values, whereas 5-phosphoribosyl-l-pyrophosphate levels increased to approximately 200%. Under these conditions, cytotoxicity became obvious after 24 h. Depletion of guanine nucleotides and cytotoxicity were prevented by addition of guanosine to MP A treatment. Daily supplements of guanosine were required to prevent MPA cytotoxicity during the entire incubation period of 72 h. We conclude that depletion of guanine nucleotides, induced by treatment with MPA, induces a severe and rapid cytotoxicity in Molt F4 cells.
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