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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.

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.

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.

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.

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)

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.

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.

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.

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)