On the paradoxically concentration-dependent metabolism of 6-mercaptopurine in WEHI-3b murine leukemia cells (original) (raw)
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Biochemical and Biophysical Research Communications, 2013
The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive prodrugs 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 lM 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.
Cancer Research, 2001
Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT؉) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT؉ versus the MOCK cell lines (P < 0.001). TPMT؉ cells were less sensitive to thioguanine than MOCK cells (IC 50 ؍ 1.10 ؎ 0.12 M versus 0.55 ؎ 0.19 M; P ؍ 0.02); in contrast, TPMT؉ cells were more sensitive to mercaptopurine than MOCK cells (IC 50 ؍ 0.52 ؎ 0.20 M versus 1.50 ؎ 0.23 M; P < 0.01). The lower sensitivity of TPMT؉ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 ؎ 282 versus 1515 ؎ 183 pmol/5 ؋ 10 6 cells; P ؍ 0.01), higher methylthioguanine nucleotide concentrations (252 ؎ 34 versus 27 ؎ 10 pmol/5 ؋ 10 6 cells; P ؍ 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 ؎ 0.6% versus 7.2 ؎ 2.0%; P < 0.001). The higher sensitivity of TPMT؉ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 ؎ 1055 versus 174 ؎ 77 pmol/5 ؋ 10 6 cells; P ؍ 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.
Cancer research, 1969
Human and animal leukemias, varying widely in responsive ness to 6-mercaptopurine, were examined to seek determinants of drug sensitivity. Drug-responsive rodent leukemias showed a comparatively high capacity for in vitro conversion of 6-mer captopurine into nondiffusible cellular metabolites, predomi nantly drug nucleotides. A similar study of human leukemia cells indicated that the in vitro capacity for 6-mercaptopurine anabolism can also be a useful predictive measure of drug responsiveness in man.
Brazilian Journal of Medical and Biological Research, 2004
A procedure is described for the rapid determination of the intraerythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10 8 cells) in 350 µl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 µl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100°C for 45 min. After cooling, 100 µl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r 2 > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra-and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10 8 erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10 8 erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10 8 erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10 8 erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.
Cancer research, 2001
Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus ...
2011
Introduction 6-Mercaptopurine (6MP) is an antimetabolite with antineoplastic and immunosuppressive activities used, usually in combination with other drugs, for treatment of leukemia (Schmiegelow et al., 1994). The 6MP is also active metabolite of the immunosuppressive drug, azathioprine (Dubinsky, 2004). In fact, 6MP is a prodrug, which is activated to 6-thioguanosine-5'phosphate and 6-thioinosine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) inside the cell. These metabolites inhibit de novo synthesis of purine, thereby blocking the formation of purine nucleotide and inhibiting DNA synthesis. In addition, 6MP exerts its effect through incorporation into DNA in the form of deoxythioguanosine which results in the disruption of DNA replication (Lennard et al., 1989, Zimm et al., 1984). Apart from the above metabolic pathway, 6MP could also enter the catabolic pathway in which the drug is degraded to its inactive forms. In this pathway, 6MP is oxidized to 6-thiouric acid (6TUA) by xanthine oxidase as the major enzyme and aldehyde oxidase through either 8-hydroxo-6-mercaptopurine (8OH6MP) intermediate or 6-thioxanthine (6TX) intermediate (Fig.
Biochemical and Biophysical Research Communications, 2011
Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP-and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122-and 93-fold higher in 6-TG-and 6-MP-resistant cells, respectively. The varying responses to 6-MP-and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.