Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells

• Published in: Biochemical and biophysical research communications 2013-07, Vol.437 (2), p.280-286
• Main Authors: Karim, Hazhar, Ghalali, Aram, Lafolie, Pierre, Vitols, Sigurd, Fotoohi, Alan K.
• Format: Article
• Language: English
• Subjects: 6-Mercaptopurine; 6-Thioguanine; Antineoplastic Agents - pharmacology; Cancer och onkologi; Cell Line, Tumor; Farmakologi och toxikologi; Humans; Klinisk medicin; Leukemia; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Mercaptopurine - pharmacology; Methyltransferases - genetics; Methyltransferases - metabolism; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma - enzymology; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma - pathology; RNA, Small Interfering; Thioguanine - pharmacology; Thiopurine methyltransferase
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2013.06.067

•We knocked down the TPMT gene using specifically designed siRNA in MOLT4 cells.•Our results show increased sensitivity of the cells to 6-TG, as compared to 6-MP.•The sensitivity of the cells toward 6-MP was not affected by TPMT knockdown.•Targeting TPMT by siRNA reflects the clinical consequences of TPMT polymorphism.•TPMT provides a differential contribution to the cytotoxic effects of the two drugs. The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.