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Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine. Zhang Wei,Modén Olof,Mannervik Bengt Chemico-biological interactions Azathioprine has been clinically used for decades in connection with organ transplantation, autoimmune disease, and treatment of cancer. Toxic side-reactions are common and have been linked to the liberation of excessively high concentrations of 6-mercaptopurine and corresponding toxic metabolites. An allelic variant of thiopurine methyltransferase with low activity is associated with elevated concentrations of 6-mercaptopurine. However, other genetic markers remain to be identified in order to fully account for adverse reactions and efficacy failure. In the present study, we studied the five known allelic variants of human glutathione transferase A2-2 (GST A2-2) (EC 2.5.1.18), abundantly expressed in liver and efficiently catalyzing the bioactivation of azathioprine to release 6-mercaptopurine. All five variants exhibited high activity with azathioprine, but allelic variant E of GST A2-2 displayed a 3-4-fold elevated catalytic efficiency compared to the other variants. High GST activity can lead to overproduction of 6-mercaptopurine, and the nature of the multiple forms of GSTs in a patient will obviously affect the metabolism of azathioprine. In addition to GST A2-2, the polymorphic GST M1-1 is also highly active with azathioprine. Considering our findings, it appears that the genotypic and phenotypic variations in the GST complement may influence the presentation of adverse reactions in patients treated with azathioprine. Clinical trials will be required to clarify the impact of the GST expression in comparison with the established biomarker thiopurine methyltransferase as predictors of adverse reactions. 10.1016/j.cbi.2010.04.028
Determination of thiopurine methyltransferase activity in erythrocytes using 6-thioguanine as the substrate. Kröplin T,Weyer N,Iven H Advances in experimental medicine and biology 10.1007/978-1-4615-5381-6_142
Pivotal role of inosine triphosphate pyrophosphatase in maintaining genome stability and the prevention of apoptosis in human cells. PloS one Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer. 10.1371/journal.pone.0032313
Human thiopurine methyltransferase activity varies with red blood cell age. Lennard L,Chew T S,Lilleyman J S British journal of clinical pharmacology AIMS:Inherited differences in thiopurine methyltransferase (TPMT) activity are an important factor in the wide interindividual variations observed in the clinical response to thiopurine chemotherapy. The aim of this study was to establish a population range for red blood cell (RBC) TPMT activity in children with acute lymphoblastic leukaemia (ALL) at disease diagnosis. An additional aim was to investigate factors that can influence TPMT activity within the RBC. METHODS:Blood samples were collected from children with ALL at disease diagnosis, prior to any blood transfusions, as part of the nationwide UK MRC ALL97 therapeutic trial. RBC TPMT activity was measured by h.p.l.c. RBCs were age-fractionated on Percoll density gradients. RESULTS:Pretreatment blood samples were received from 570 children within 3 days of venepuncture. TPMT activities at disease diagnosis ranged from 1.6 to 23.6 units/ml RBCs (median 7.9) compared with 0.654-18.8 units (median 12.9), in 111 healthy control children (median difference 4.5 units, 95% CI 3.9, 5.1 units, P < 0.001). A TPMT quality control sample, aliquots of which were assayed in 60 analytical runs over a 12 month period, contained a median of 11.98 units with a CV of 11.6%. Seven children had their RBCs age-fractionated on density gradients. TPMT activities in the top gradient (young cells) ranged from 4.2 to 14.1 units (median 7.5) and in the bottom gradient (old cells) 1.5-12.6 units (median 4.7 units), median difference 2.3 units, 95% CI 0.7, 4.1, P = 0.035. CONCLUSIONS:Circulating RBCs do not constitute a homogeneous population. They have a life span of around 120 days and during that time undergo a progressive ageing process. The anaemia of ALL is due to deficient RBC production. The results of this study indicate that RBC TPMT activities are significantly lower in children with ALL at disease diagnosis. This may be due, at least in part, to a relative excess of older RBCs. 10.1046/j.0306-5251.2001.01497.x
NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine. Valerie Nicholas C K,Hagenkort Anna,Page Brent D G,Masuyer Geoffrey,Rehling Daniel,Carter Megan,Bevc Luka,Herr Patrick,Homan Evert,Sheppard Nina G,Stenmark Pål,Jemth Ann-Sofie,Helleday Thomas Cancer research Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients. Cancer Res; 76(18); 5501-11. ©2016 AACR. 10.1158/0008-5472.CAN-16-0584
Stability of thiopurine metabolites: a potential analytical bias. de Graaf Peer,de Boer Nanne K H,Jharap Bindia,Mulder Chris J J,van Bodegraven Ad A,Veldkamp Agnes I Clinical chemistry 10.1373/clinchem.2007.092676