L. H. J. Lambooy

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 microM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP from Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.

Genetic polymorphism of thiopurine S-methyltransferase in Argentina

Background: Thiopurine methyltransferase (TPMT) catalyses the S-methylation of 6-thiopurine drugs, which are commonly used in the treatment of autoimmune diseases, leukaemia and organ transplantation. TPMT activity is polymorphic as a result of gene mutations. Ethnic variations in phenotype and genotype have been identified in previous population studies, but no information was available within Latin-American populations. Aim: To establish the genetic polymorphism of TPMT in an Argentine population. Methods: TPMT enzymatic activity of 147 healthy Argentine subjects was measured using a high-performance liquid chromatography method. The genotyping assay for nine defective alleles (TPMT*2 - *8) was based on restriction fragment length polymorphism polymerase chain reaction and allele-specific polymerase chain reaction methods. Results: All subjects had detectable TPMT activity. Twelve individuals with low to intermediate activity were heterozygous for one of the mutant alleles: nine were TPMT*1/*3A, two TPMT*1/*2 and one TPMT*1/*4. All examined subjects with normal activity had wild-type genotype (TPMT*1/*1). Conclusion: Variant TPMT alleles were present in 8·2% of the examined subjects, which is in accordance with other studies. The frequency of TPMT*3A, TPMT*2 and TPMT*4 was 3·1%, 0·7% and 0·3%, respectively. TPMT*3A was the most prevalent allele, which is in accordance with results from Caucasian populations. This study provides the first analysis of TPMT activity and allele frequency distribution in Argentina, South America.

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL)

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5′ region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5′ region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (Pu2009<u20090.01). each of six all patients who were investigated at initial diagnosis and at relapse showed alterations in dna methylation between the two stages. these six cases were also investigated by southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in dna methylation in only four of the six cases. the dna sequencing method thus appears to be better suited to assess alterations of dna methylation than southern blot analysis. there are marked regional differences in the frequency of methylation of individual cpg sites and in the frequency of alterations between the two stages. our results show that alterations in dna methylation continue to occur from the initial stage to the relapse stage of all, suggesting that aberrant dna methylation may play a role in tumor progression.

Reversal of methylmercaptopurine ribonucleoside cytotoxicity by purine ribonucleosides and adenine

6-Methylmercaptopurine ribonucleoside-5-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.

Pitfalls in the determination of mutant alleles of the thiopurine methyltransferase gene.

Thiopurine methyltransferase (TPMT) is a methylating enzyme which is involved in the metabolism of thiopurines. The enzyme is known to exhibit an autosomal codominant polymorphism with 89–94% of all individuals studied having a high activity, 6–11% an intermediate activity and 0.3% having a very low or non-detectable activity. The gene encoding TPMT is localized on chromosome 6. Eight variant alleles of the gene are reported to be associated with decreased TPMT enzyme activity, and TPMT*3 mutations are the most prevalent.1,2 Heterozygotes and homozygotes for these alleles are at moderate and high risk, respectively, for thiopurine drug toxicity. In Caucasians and South West Asians, TPMT*3A (transitions G460A and A719G) is the most prevalent mutant allele whereas in a Chinese, a Korean and an African population, as well as an African-American population the TPMT*3C (A719G) mutation is more common. The TPMT*3A allele is believed to have arisen as a result of a TPMT*3B (G460A) mutation on an ancestral TPMT*3C variant allele.1 From reviewing the literature on TPMT populations studies and from our own genotyping, we note that 19 cases of TPMT*3B have been reported from a total of 12208 alleles, a frequency of 0.16%.3–7 The mutations TPMT*3A and TPMT*3C accounted for 4.00% (488 alleles) and 1.38% (168 alleles), respectively, of the total amount of alleles published. The A719G (TPMT*3C) mutation creates an Accl recognition sequence and the G460A (TPMT*3B) mutation destroys a Mwol site.2 Using the method described by Yates et al we have screened 116 children who were or had been suffering from acute lymphoblastic leukemia (ALL); eight of these children were screened after presentation of toxicologic features during thiopurine treatment. We also genotyped 43 control children not suffering from any hematological disorders. Twenty children were found to carry one or two mutant alleles. Unexpectedly, we found TPMT*3B heterozygotes and one patient homozygous for TPMT*3B (Table 1). In view of the low occurrence of the TPMT*3B allele, we expected that these alleles were ‘misdiagnosed’ and resulted from incomplete restriction endonuclease digestion. These patients were re-examined using a stringent restriction endonuclease digestion protocol. PCR products were desalted by microdialysis on Millipore VS type filters (Pall Corporation, Ann Arbor, MI, USA) prior to an extended overnight digestion. In order to detect

Purine enzymes in rheumatoid arthritis: possible association with response to azathioprine. A pilot study.

OBJECTIVE--To study the possible association of purine enzyme activities with response to azathioprine (AZA) treatment in rheumatoid arthritis (RA) and their correlation with parameters of disease activity. PATIENTS AND METHODS--Lymphocyte activities of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), adenine phosphoribosyltransferase (APRT), purine nucleoside phosphorylase (PNP) and 5-nucleotidase (5NT), and erythrocyte activities of thiopurine methyltransferase (TPMT) were measured in 14 healthy controls and 36 patients with RA. Eight patients had not previously been treated with AZA. Response to AZA therapy in 28 patients, determined in a prospective trial, was considered good in nine (group 1), insufficient in seven (group 2). In 12 patients AZA was withdrawn because of adverse reactions (group 3). Disease activity parameters were obtained simultaneously with purine enzyme measurements. Purine enzyme levels in the different groups were compared. RESULTS--Levels of 5NT activity were significantly lower in patients with RA than in healthy controls. PNP activity was higher in patients with RA not using prednisone compared with those who did and healthy controls. No clear correlation between purine enzyme levels and disease activity parameters was found. 5NT activities were significantly higher in group one than in group three (p = 0.012; alpha = 0.017), and almost significantly higher than in group two (p = 0.03; alpha = 0.017). CONCLUSIONS--The results indicate that purine enzyme activities in patients with RA differ from healthy controls, are associated with the outcome of AZA treatment and seem not to be associated with disease activity. Our findings may offer a clue to predict the response to AZA therapy in RA.

Inhibition of IMP Dehydrogenase by Mycophenolic Acid in Molt F4 Human Malignant Lymphoblasts

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 0–5 μM MPA. After a 2 h exposure of the cells to 0–5 μM MPA, guanine nucleotides were depleted to approximately 50% of control values, whereas 5-phosphoribosyl-1-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 MPA 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.

Reversal of 6-mercaptopurine and 6-methylmercaptopurine ribonucleoside cytotoxicity by amidoimidazole carboxamide ribonucleoside in Molt F4 human malignant T-lymphoblasts

Cytotoxicity of 6-mercaptopurine (6MP) and 6-methylmercaptopurine ribonucleoside (Me-MPR) was studied in Molt F4 human malignant lymphoblasts. Both drugs are converted into methylthioIMP (Me-tIMP), which inhibits purine de novo synthesis. Addition of amidoimidazole carboxamide ribonucleoside (AICAR) circumvented inhibition of purine de novo synthesis, and thus partly prevented 6MP and Me-MPR cytotoxicity. Purine nucleotides, and especially adenine nucleotides, were recovered by addition of AICAR. Under these conditions, Me-tIMP formation decreased. The results of this study indicate that formation of Me-tIMP may be important for 6MP cytotoxicity in Molt F4 cells. These data suggest that depletion of adenine nucleotides is the main cause for Me-tIMP cytotoxicity.

Synergistic interaction of methotrexate and 6-mercaptopurine in human derived malignant T-ALL and CALLA+ cell lines.

Methotrexate (MTX and 6-mercaptopurine (6MP) are antimetabolites which are commonly used in the treatment of acute lymphoblastic leukemia (ALL). Synergistic cytotoxic effects can be expected from combinations of MTX and 6MP, based on the biochemical pharmacology of both drugs.

Action and Sequence Dependent Interaction of Acivicin and 6-Thioguanine in Human Derived Malignant T-All and Calla+ Cell Lines

Acivicin is an irreversible inhibitor of L-glutamine amino transferases and therefore effects several enzymatic steps in purine de novo sythesis (PDNS) including phosphoribosyl pyrophosphate (PRPP) amidotransferase and enzymes which are specifically rate limiting for GTP, for example, GMP synthetase. This drug biochemically targets GTP concentration in leukemic blast cells (fig. 1)1. Acivicin may also be effective as a modulator of other anti-leukemic agents such as AraC and 6-thioguanine (6TG).2 Acivicin interferes with PDNS resulting in PRPP elevations which may allow for synergistic action with antipurines.3 In response to inhibition of (PDNS), an increase in the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) occurs. As a result of this increased availability of PRPP potentiation of 6TG incorporation can be expected in cells with an active PDNS.