Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection.

In 12 healthy male volunteers (27-53 yr), a placebo-controlled randomized double blind cross-over trial was performed to study the effect of the intravenous injection of 0.25, 0.5, 1, 2, 4, and 6 mg draflazine (a selective nucleoside transport inhibitor) on hemodynamic and neurohumoral parameters and ex vivo nucleoside transport inhibition. We hypothesized that an intravenous draflazine dosage without effect on hemodynamic and neurohumoral parameters would still be able to augment the forearm vasodilator response to intraarterially infused adenosine. Heart rate (electrocardiography), systolic blood pressure (Dinamap 1846 SX; Critikon, Portanje Electronica BV, Utrecht, The Netherlands) plasma norepinephrine and epinephrine increased dose-dependently and could almost totally be abolished by caffeine pretreatment indicating the involvement of adenosine receptors. Draflazine did not affect forearm blood flow (venous occlusion plethysmography). Intravenous injection of 0.5 mg draflazine did not affect any of the measured hemodynamic parameters but still induced a significant ex vivo nucleoside-transport inhibition of 31.5 +/- 4.1% (P < 0.05 vs placebo). In a subgroup of 10 subjects the brachial artery was cannulated to infuse adenosine (0.15, 0.5, 1.5, 5, 15, and 50 micrograms/100 ml forearm per min) before and after intravenous injection of 0.5 mg draflazine. Forearm blood flow amounted 1.9 +/- 0.3 ml/100 ml forearm per min for placebo and 1.8 +/- 0.2, 2.0 +/- 0.3, 3.8 +/- 0.9, 6.3 +/- 1.2, 11.3 +/- 2.2, and 19.3 +/- 3.9 ml/100 ml forearm per min for the six incremental adenosine dosages, respectively. After the intravenous draflazine infusion, these values were 1.6 +/- 0.2 ml/100 ml forearm per min for placebo and 2.1 +/- 0.3, 3.3 +/- 0.6, 5.8 +/- 1.1, 6.9 +/- 1.4, 14.4 +/- 2.9, and 23.5 +/- 4.0 ml/100 ml forearm per min, respectively (Friedman ANOVA: P < 0.05 before vs after draflazine infusion). In conclusion, a 30-50% inhibition of adenosine transport significantly augments the forearm vasodilator response to adenosine without significant systemic effects. These results suggest that draflazine is a feasible tool to potentiate adenosine-mediated cardioprotection in man.

Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes. Effects of adenosine and deoxyadenosine.

Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phytohemagglutinin the ATP concentrations decreased in these lymphocytes. The concentrations of TTP and dATP were measured with a DNA polymerase assay. Phytohemagglutinin-stimulation increased the TTP concentration in lymphocytes of all three species, the dATP concentration only in human lymphocytes. ATP, TTP and dATP concentrations and thymidine incorporation were measured in phytohemagglutinin-stimulated lymphocytes after 24 and 48 h culturing in the presence of adenosine or deoxyadenosine. Adenosine increased the ATP concentration in porcine and equine, but not in human lymphocytes. Deoxyadenosine and adenosine did not affect the TTP concentration. Deoxyadenosine decreased the ATP concentration only in the presence of EHNA in human lymphocytes, but increased it in other conditions and in equine and porcine lymphocytes. Deoxyadenosine in the presence of EHNA increased the dATP concentration in human, equine and porcine lymphocytes 3-, 10-, and 9-fold, respectively, and decreased considerably thymidine incorporation. Deoxyadenosine without EHNA increased the dATP concentration 2-5-fold, decreased the thymidine incorporation in lymphocytes of man and horse, but stimulated incorporation in porcine lymphocytes about 5-fold. The latter results indicate that accumulation of dATP is not always associated with inhibition of cell proliferation.

Deconjugation of glucuronides catalysed by UDPglucuronyltransferase.

Evidence was found for UDPglucuronyltransferase-catalysed deconjugation of p-nitrophenol-, 4-methylumbelliferone- and phenolphthalein-glucuronides. The evidence is based on the following observations: 1, deconjugation is UDP-dependent and the reactions show Michaels-Menten kinetics with respect to UDP and glucuronide saturability; 2, UDP-glucuronic acid was identified as reaction product; 3, all studies were done in the presence of a beta-glucuronidase inhibitor; 4, induction profiles, using 3-methylcholanthrene and phenobarbital as inducing agents, were identical for conjugation and deconjugation reactions. Optimal deconjugation rates for p-nitrophenol- and 4-methylumbelliferone-glucuronides were at pH 5.1 and for phenolphthalein-glucuronide at pH 6.5. Only conjugation reactions showed latency; the corresponding deconjugation reactions were not latent. UDPglucuronyltransferase is a group of oligomeric isoenzymes with different molecular masses. The molecular masses of the isoenzyme species catalysing the forward and reverse reactions were determined by radiation-inactivation analysis. The molecular masses of the isoenzyme species mediating the catalyses of deconjugation reactions were significantly smaller than those mediating catalyses of conjugation reactions: 66 +/- 4 kDa vs. 109 +/- 7 kDa for p-nitrophenol; 82 +/- 8 kDa vs. 105 +/- 6 kDa for 4-methylumbelliferone; and 74 +/- 8 kDa vs. 159 +/- 14 kDa for phenolphthalein. This suggests that for catalyses of deconjugation reactions only part of a UDPglucuronyltransferase isoenzyme is needed, whereas for forward reactions the complete isoenzymes are required.

Kinetics of adenylate metabolism in human and rat myocardium.

Pathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP- and IMP-specific 5'-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was not detectable in human heart. The Km-values of the AMP-catabolizing enzymes were 2-5 times higher in human heart; the substrate affinity of the other enzymes was in the same order of magnitude in both species. The maximal activity (Vmax) of adenosine kinase was the same in both species, but HGPRT in man was only 12% of that in the rat. For human heart the Vmax-values of adenosine deaminase, nucleoside phosphorylase, AMP- and IMP-specific 5'-nucleotidases, and AMP deaminase were 25-50% of those for rat heart. We conclude that human heart is less geared to purine catabolism than rat heart as is evident from the lower activities of the catabolic enzymes. Maintenance of the nucleotide pool may thus play a more important role in human heart.

A biochemical basis for synergism of 6-mercaptopurine and mycophenolic acid in Molt F4, a human malignant T-lymphoblastic cell line.

6-Mercaptopurine (6MP) cytotoxicity was studied in Molt F4 cells, a T-cell acute lymphoblastic leukemia (ALL) cell line. The effects on cytotoxicity were concentration-dependent. Measurements of intracellular thionucleotide intermediates of 6MP demonstrated a rapid rise of thio-IMP (tIMP) levels, and subsequently a rapid decrease. Thio-GMP (tGMP) and methyl-thio-IMP (Me-tIMP) appeared later in time, and persisted longer. Mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase (IMPDH), was used to inhibit the conversion of tIMP into tGMP, thereby decreasing the incorporation of 6MP into DNA. A synergistic effect on cell viability and cell growth was observed when cells were treated with a combination of 2 microM 6MP and 0.5 microM MPA. Also, intracellular Me-tIMP increased 5 times with the combination. Based on the increase of Me-tIMP concentration and the observed synergism between 6MP and MPA, we conclude that methylation of tIMP into Me-tIMP is an important alternative route for 6MP cytotoxicity.

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.

Inhibition of lymphoid cell growth by adenine ribonucleotide accumulation. The role of phosphoribosylpyrophosphate-depletion induced pyrimidine starvation.

The exact role of adenosine in the adenosine deaminase (EC 3.5.4.4) deficiency-related severe combined immunodeficiency disease has not been ascertained. We analysed the effects of adenosine, in the presence of the adenosine deaminase inhibitor, deoxycoformycin, on cell growth, cell phase distributions and intracellular nucleotide concentrations of cultured human lymphoblasts. Adenosine had a biphasic effect on cell growth and cell cycle distribution of a partial hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) deficient MOLT-HPRT cell line. After 24 h of incubation, 60 microM adenosine inhibited cell growth more extensively than did 100 and 200 microM adenosine. The distribution of the MOLT-HPRT cells in the various phases of the cell cycle showed a similar biphasic pattern. Adenosine concentrations in the medium below 10 microM caused accumulation of adenine ribonucleotides and depletion of phosphoribosylpyrophosphate, UTP and CTP in the cells. This was associated with inhibition of cell growth. Medium adenosine concentrations above 10 microM neither resulted in accumulation of adenine ribonucleotides nor in inhibition of cell growth.

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 (P < 0.01). 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.

Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk for developing a severe 5FU-associated toxicity. To evaluate the importance of this specific type of inborn error of pyrimidine metabolism in the etiology of 5FU toxicity, an analysis of the DPD activity, the DPD gene, and the clinical presentation of patients suffering from severe toxicity after the administration of 5FU was performed. Our study demonstrated that in 59% of the cases, a decreased DPD activity could be detected in peripheral blood mononuclear cells. It was observed that 55% of patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of patients with a normal DPD activity (P = 0.01). Furthermore, the onset of toxicity occurred, on average, twice as fast in patients with low DPD activity as compared with patients with a normal DPD activity (10.0 +/- 7.6 versus 19.1 +/- 15.3 days; P < 0.05). Analysis of the DPD gene of 14 patients with a reduced DPD activity revealed the presence of mutations in 11 of 14 patients, with the splice site mutation IVS14+1G-->A being the most abundant one (6 of 14 patients; 43%). Two novel missense mutations 496A-->G (M166V) and 2846A-->T (D949V) were detected in exon 6 and exon 22, respectively. Our results demonstrated that at least 57% (8 of 14) of the patients with a reduced DPD activity have a molecular basis for their deficient phenotype.

Effects of 8-aminoguanosine on the toxicity of guanosine and deoxyguanosine for malignant and normal lymphoid cells.

The toxicity of guanosine and deoxyguanosine in the presence or absence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine, for malignant lymphoid cell lines and mitogen-stimulated peripheral blood lymphocytes has been studied. Deoxyguanosine inhibited the proliferation of lymphoid cells more strongly than guanosine. Addition of 100 microM 8-aminoguanosine neither enhanced nor diminished the toxicity of guanosine to the lymphoid cells. Only the toxicity of deoxyguanosine for the leukemic T cell line, MOLT 4, and the leukemic nonBnonT cell line, KM-3, was enhanced by the addition of 100 microM 8-aminoguanosine. These data suggest a possible role of purine nucleoside phosphorylase inhibitors in the treatment of lymphoproliferative disorders of the T-acute lymphoblastic leukemia (ALL) as well as the nonBnonT-ALL subclass.

Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity.

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5-FU), thereby limiting the efficacy of the therapy. It has been suggested that patients suffering from 5-FU toxicities due to a low activity of DPD are genotypically heterozygous for a mutant allele of the gene encoding DPD. In this study we investigated the cDNA and a genomic region of the DPD gene of a cancer patient experiencing severe toxicity following 5-FU treatment for the presence of mutations. Although normal activity of DPD was observed in fibroblasts, the DPD activity in leucocytes of the cancer patient proved to be in the heterozygous range. Analysis of the DPD cDNA showed heterozygosity for a 165bp deletion that results from exon skipping. Sequence analysis of the genomic region encompassing the skipped exon showed that the tumour patient was heterozygous for a G-->A point mutation in the invariant GT splice donor sequence in the intron downstream of the skipped exon. So far, the G-->A point mutation has also been found in 8 out of 11 patients suffering from a complete deficiency of DPD. Considering the frequent use of 5-FU in the treatment of cancer patients, the severe 5-FU-related toxicities in patients with a low activity of DPD and the high frequency of the G-->A mutation in DPD deficient patients, analysis of the DPD activity and screening for the G-->A mutation should be routinely carried out prior to the start of the treatment with 5-FU.

Analysis of DNA methylation of the 5' region of the deoxycytidine kinase gene in CCRF-CEM-sensitive and cladribine (CdA)- and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)-resistant cells.

DNA methylation of the CpG-rich 5' region of the deoxycytidine kinase (dCK) gene is potentially involved in the suppression of the gene and the resistance of tumour cells to arabinosylcytosine (ara-C). 2-Chlorodeoxyadenosine (cladribine, CdA) and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA) are purine nucleoside analogues which are also phosphorylated by dCK. We observed a reduction in dCK activity in a number of CCRF-CEM-derived cell lines that are resistant to these drugs and hypothesized that this reduction is due to DNA methylation of the 5' region of the dCK gene. The DNA methylation state was analyzed at the DNA sequence level after bisulfite modification of genomic DNA. The investigated region included 0.3 kb of DNA upstream to the start site of transcription, exon 1 and part of intron 1. Sensitive cells (CCRF-CEM/0) and three resistant cell lines (CCRF-CEM/CdA4000, CCRF-CEM/CAFdA100 and CCRF-CEM/CAFdA4000) were investigated. The region that was analyzed contained no methylated cytosine residues in the parental cell line CCRF-CEM/0 or in the resistant cell lines. Therefore, it is highly unlikely that DNA methylation plays a role in the suppression of dCK gene expression in these cell lines.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages.

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-related effects of methotrexate on purine and pyrimidine nucleotides and on cell-kinetic parameters in MOLT-4 malignant human T-lymphoblasts.

The effects of methotrexate (MTX) on cytotoxicity (trypan blue exclusion and soft agar clonal growth), cell cycle perturbation, and purine and pyrimidine ribonucleotide and deoxyribonucleotide pools have been studied in MOLT-4 malignant T-lymphoblasts. Two concentrations of MTX, 0.02 microM and 0.2 microM have been utilized, which can be maintained in vivo during many hours in the maintenance therapy of acute lymphoblastic leukemia (ALL). The results are correlated with the effects of MTX on the inhibition of purine de novo synthesis. Treatment with 0.02 microM MTX results in an accumulation of cells in early S phase after 20 hr, as measured by DNA flow cytometry and by a significant increase of dCTP levels, followed by a slow progression of a cohort of cells through the cell cycle. Cytotoxicity also becomes evident starting from this point of time. The effects on deoxyribonucleotide pools are discussed in correlation with the inhibition of DNA synthesis. The changes in ribonucleotide pools are associated with the partial inhibition of purine de novo synthesis at 20-28 hr and suggest an inhibition of RNA synthesis. After 48 hr a reutilization of nucleotide precursors due to nucleic acid breakdown and a recovery of purine de novo synthesis is shown, associated with a recovery of RNA synthesis, whereas cytotoxicity increases. Treatment of MOLT-4 cells with 0.2 microM MTX results in a rapid complete cessation of cell progression through all parts of the cell cycle after 8 hr, associated with a depletion of all deoxyribonucleotide pools, complete inhibition of purine de novo synthesis, inhibition of RNA synthesis and a marked cytotoxicity. Ribonucleotide pools demonstrate a reutilization of nucleotide precursors after 12 hr of incubation without a recovery of purine de novo synthesis and RNA synthesis. These data show a close dose- and time-dependent correlation of the effects of MTX on purine de novo synthesis, UMP levels and other (deoxy)ribonucleotide pools, and on RNA and DNA synthesis in MOLT-4 cells having an active purine de novo synthesis. This correlation is absent in normal bone marrow cells and peripheral blood lymphocytes. These data can be used in order to elucidate the synergistic effects of sequential administration of MTX and 6-mercaptopurine.

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.

6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts.

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro inhibition of cell growth of MOLT-4 malignant human T-lymphoblasts by coenzyme F420.

The inhibitory effect of methanogenic coenzymes on the proliferation of MOLT-4 human malignant T-lymphoblasts was tested. Furthermore the effects of methanogenic coenzymes on dihydrofolate reductase activity (DHFR) from chicken liver have been examined. The results showed that heat-stable extracts of the hydrogenotrophs Methanobacterium thermoautotrophicum, Methanoculleus thermophilicum and Methanogenium tationis inhibit both proliferation of human T-lymphoblasts and DHFR activity. Heat-stable extract of the methylotroph Methanosarcina barkeri showed neither inhibitory nor stimulatory effects in both test systems. The present study proves coenzyme F420 to be the active, inhibitory component in methanogenic extracts.

The IMP dehydrogenase inhibitor mycophenolic acid antagonizes the CTP synthetase inhibitor 3-deazauridine in MOLT-3 human leukemia cells: a central role for phosphoribosyl pyrophosphate.

Mycophenolic acid, an inhibitor of the enzyme IMP dehydrogenase, antagonizes the CTP synthetase inhibitor 3-deazauridine in its anti-proliferative effects on MOLT-3 human T leukemia cells. No depletion of CTP occurred, and decreased amounts of 3-deazuridine-triphosphate were measured in cells incubated with mycophenolic acid and 3-deazuridine. Most probably, these phenomena are related to the increased amounts of PRPP observed, which can result in an increased pyrimidine biosynthesis de novo and, as a consequence, a decreased metabolism of 3-deazauridine via the salvage pathway.

Effects on transmethylation by high-dose 6-mercaptopurine and methotrexate infusions during consolidation treatment of acute lymphoblastic leukemia.

6-mercaptopurine (6MP) cytotoxicity is caused by thioguanine and methylthioinosine nucleotides. Thiopurine methylation occurs to a large extent in vivo and in vitro. In this reaction, S-adenosyl-L-methionine (AdoMet), produced from methionine and ATP, is converted into S-adenosyl-L-homocysteine (AdoHcy) which, in turn, is hydrolyzed into homocysteine. Remethylation of homocysteine into methionine is inhibited by methotrexate (MTX). In cultured lymphoblasts, AdoMet: AdoHcy ratio and DNA methylation decrease after incubation with 6MP. The aim of the present study was to investigate the influence of high-dose 6MP on the methylation capacity in children with acute lymphoblastic leukemia. Five patients received 4 courses with high-dose intravenous MTX (5' g.m-2 in 24 hr) immediately followed by high-dose 6MP (1300 mg.m-2 in 24 hr). Five control patients received high-dose MTX and oral 6MP (25 mg.m -2 daily for 8 weeks). Leucovorin rescue was started at 36 hr in both groups. In the intravenous 6MP group, 6-methylmercaptopurine, its riboside, and 6-methylmercapto-8-hydroxypurine were detectable in plasma in concentrations of 0.3-2.6 muM (6MP steady state levels: 11.6 muM). In red blood cells, mean methylthioinosine nucleotide levels were one third of those of ATP (13.1 nmol/10(8)). AdoHcy levels (10 pmol/10(8)) remained constant in both groups and AdoMet was not detectable ( < 20 pmol/10(8)). In both groups, plasma homocysteine increased and methionine decreased following administration of MTX. The delay in the recovery of methionine in the intravenous 6MP group after MTX infusion is probably the result of an increased demand on methyl groups during 6MP infusion.