6-Methylthioguanylic acid, a metabolite of 6-thioguanine

doi:10.1016/0006-2952(71)90046-3

Biochemical Pharmacology

Volume 20, Issue 4, April 1971, Pages 847-850, IN11-IN12, 851-852

https://doi.org/10.1016/0006-2952(71)90046-3 Get rights and content

Abstract

Human epidermoid carcinoma (H. Ep. No. 2) cells, grown in culture in the presence of 6-thioguanine-³⁵S or methionine[methyl-¹⁴C] plus unlabeled 6-thioguanine, contained a previously undescribed metabolite that was characterized by Chromatographic and electrophoretic migration and by chemical and enzymatic hydrolysis as 6-methylthioguanylic acid. Two other nucleotides present in these cells had the properties expected of the di- and triphosphates of 6-methylthioguanosine. In contrast to previously reported results with 6-mercaptopurine ribonucleotide and its S-methyl derivative, 6-methylthioguanylic acid was inferior to 6-thioguanylic acid as an inhibitor of PP-ribose-P amidotransferase. Thus, the methylated derivatives of 6-thioguanylic acid probably do not contribute significantly to the marked inhibition of purine synthesis de novo produced in mammalian cells by 6-thioguanine

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Cited by (32)

Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells
2013, Biochemical and Biophysical Research Communications
Citation Excerpt :
This can explain why we did not observe more tolerance to cytotoxic effects of 6-MP after treatment with TPMT-targeting siRNA and blockage of methylation pathway which leads to production of higher amounts cytotoxic TGNs. As a matter of fact, the idea that inhibition of DNPS is minimum with 6-TG is further supported by the fact that meTGMP is a twelve-fold less potent inhibitor of the DNPS than meTIMP [25,26]. We concluded that targeting TPMT gene by siRNA can be helpful in understanding possible clinical consequences of TPMT polymorphism.
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 1 μM 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.
The effect of thiopurine drugs on DNA methylation in relation to TPMT expression
2008, Biochemical Pharmacology
Citation Excerpt :
Global DNA methylation of EcR293-TPMT/20 cells was inhibited by 6-MP to a similar extent in conditions of low and high TPMT expression. Also, as 6-TG is not metabolised to MeTIMP, but to MeTGMP, a weak inhibitor of DNPS in contrast to MeTIMP [27], this drug would be expected to have less effect on DNA methylation than 6-MP. This was only the case in high TPMT expressing cells, as 6-TG in low TPMT expressing cells inhibited DNA methylation to a similar extent to 6-MP, at equivalent drug doses.
The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2′-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5’-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.
Inhibitory effect of 8-oxo-7,8-dihydro-2′-deoxyguanosine on the growth of KG-1 myelosarcoma in Balb/c nude mice
2006, Leukemia Research
We previously found that 8-oxo-7,8-dihydro-2′-deoxyguanosine (oh⁸dG) kills KG-1, a human myelocytic leukemic cell line with mutational loss of 8-oxoguanine glycosylase (OGG1) activity in vitro. This observation prompted us to investigate the cytotoxicity of oh⁸dG on KG-1 in vivo. This cytotoxicity was observed by administrating oh⁸dG (3.3–330 mg/kg b.w./day) for 14 days into nude mice bearing a KG-1 myelosarcoma. The results were as follows; oh⁸dG inhibited the growth of KG-1 myelosarcoma dose-dependently in terms of tumor size and weight, but had no effect on the growth of myelosarcoma of U937, a human monocytic leukemic cell line possessing wild-type OGG1. 6-Thioguanine (6-TG), an anticancer drug inhibited the growths of KG-1 and U937 tumors. 2′-Deoxyguanosine (dG) had a statistically insignificant anti-growth effect on both tumors. The oh⁸dG-treated KG-1 tumor showed the increased expression of apoptosis-processing caspases 8, 9 and 3 together with DNA fragmentation, the increased expression of cell cycle inhibitors, p16 and p27, and the decreased expression of cell cycle accelerator, cyclins and cdks, indicating the nature of cytotoxicity is cell cycle arrest and apoptosis. The genomic DNA of oh⁸dG-treated KG-1 tumors showed an increase in OGG1 sensitive sites, which is consistent with an increase in the 8-oxo-7,8-dihydroguanine (oh⁸Gua) level in the DNA of KG-1 treated with oh⁸dG in vitro. Presumably an increased level of oh⁸Gua in DNA may trigger the cytotoxicity. These findings suggest that oh⁸dG is selectively cytotoxic to KG-1 or tumors that are OGG1-deficient.
High-performance liquid chromatographic assay of methylthioguanine nucleotide
1998, Journal of Chromatography B: Biomedical Applications
This paper describes a specific and sensitive reversed-phase HPLC assay for the measurement of 6-methylthioguanine (methyl-TG) and methyl-TG nucleotides (methyl-TGNs) in red blood cells (RBCs), which is suitable for routine clinical use. Briefly, an ethyl acetate extract of RBCs is evaporated and reconstituted in O.1 M HCl. The methyl-TG is separated from other thiopurines by reversed-phase HPLC and quantitated using UV detection. For the measurement of methyl-TGNs the free base (methyl-TG) is obtained by acid hydrolysis of the nucleotide back to the parent thiopurine. The intra-assay C.V. over the concentration range of 0.055–1.10 nmol methyl-TG per 4×10⁸ (100 μl) RBCs ranged from 2.8 to 8.5%, and the mean recovery of methyl-TG over the calibration range was 61.6% (coefficient of variation, C.V., 3.8%). The lower limit of reproducibility was 0.055 nmol extracted from 100 μl RBCs. Analysis of blood samples from children with leukaemia receiving 6TG chemotherapy, revealed RBC methyl-TGNs at concentrations ranging from 323 to 1365 pmol per 8×10⁸ RBCs. No methyl-TG was detected in any of the patient samples.
Thiopurine methyltransferase: A review and a clinical pilot study
1996, Journal of Chromatography B: Biomedical Applications
Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of 6-mercaptopurine (6MP), which is used in the treatment of acute lymphoblastic leukemia (ALL). TPMT catalyzes the formation of methylthioinosine monophosphate (MetIMP), which is cytotoxic for cultured cell lines, and it plays a role in detoxification of 6MP. Population studies show a genetic polymorphism for TMPT with both high and low activity alleles. About 1 of 300 subjects is homozygous for the low activity. The function TPMT plays in detoxification or therapeutic efficacy of 6MP in vivo is not clear. In this article the genetic polymorphism of TPMT is reviewed and the contribution of TPMT to the cytotoxic action, or detoxification, of 6MP in children with ALL is discussed. Induction of TPMT activity has been described during the treatment for ALL. We performed a pilot study on the influence of high-dose 6MP infusions (1300 mg/m² in 24 h) on TPMT activity of peripheral blood mononuclear cells (pMNC) of eleven patients with ALL. The TPMT activities were in, or, above the normal range. There was no statistically significant difference between the TPMT activities before and after the 6MP infusions. MetIMP levels in pMNC increased during successive courses. This might be explained by TPMT induction, but other explanations are plausible as well. Twenty five percent of the TPMT assays failed, because less than the necessary 5·10⁶ pMNC could be isolated from the blood of leukopenic patients. Red blood cells can not be used for TPMT measurements, since transfusions are frequently required during the treatment with 6MP infusions. Therefore, the influence of high-dose 6MP infusions on TPMT activity can only be investigated further when a TMPT assay which requires less pMNC has been developed.
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1994, Leukemia Research

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^∗: Affiliated with the Sloan-Kettering Institute for Cancer Research, New York, N.Y.

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6-Methylthioguanylic acid, a metabolite of 6-thioguanine

Abstract

Biochem. Pharmac.

Biochim. biophys. Acta

Biochem. Pharmac.

Biochem. Pharmac.

J. biol. Chem.

Cancer Res.

Biochemistry, N.Y.