John M. van Baal

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps. The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD+, cyclic AMP, NADP+ and uridinediphosphoglucose (UDPG). In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.

High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides in biological fluids

A method is presented for the separation and quantitative determination of compounds normally related to purine and pyrimidine metabolism in biological material. The retention behaviour of nucleobases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides has been systematically investigated by reversed-phase high-performance liquid chromatography using a non-linear gradient. Ultimately a separation of the purine and pyrimidine compounds was achieved in a 35-min run with an average detection limit of 5-10 pmol per injection. Recoveries of standards added to urine, plasma or serum were 96 +/- 5%.

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.

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.

The effect of methotrexate on purine and pyrimidine deoxyribonucleoside triphosphate pools and on cell viability and cell-phase distribution in malignant human T- and B-lymphoblasts

Methotrexate (MTX) and 6-mercaptopurine (6-MP) are widely used in the maintenance treatment of acute lymphoblastic leukemia (ALL) in children. Based on their biochemical interactions there are reasons to support a combination therapy of both drugs. MTX binds tightly to dihydrofolate reductase, the enzyme that catalyses the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate coenzymes are required for one-carbon transfer reactions in purine de novo synthesis and thymidylate biosynthesis (Figure 1). As a consequence a purine-less and a thymidylate-less state will occur, ultimately resulting in inhibition of DNA biosynthesis [1–10].

Influence of methotrexate on purine and pyrimidine pools and on cell phase distribution of cultured human lymphoblasts.

Methotrexate (MTX) and 6-mercaptopurine (6MP) are among the most commonly used agents in the maintenance treatment of acute lymphoblastic leukemia (ALL) in children.1-3 There are biochemical considerations for an increased efficacy of a combination of both drugs in maintenance therapy of ALL. MTX is a strong inhibitor of dihydrofolate reductase. As a result tetrahydrofolates are reduced. Tetrahydrofolate coenzymes are required for one-carbon transfer reactions in purine de novo synthesis and thymidine biosynthesis. As a consequence a purine-less and a thymidy-late-less state will occur, ultimately resulting in inhibition of DNA biosynthesis.4–9

INFLUENCE OF METHOTREXATE ON PURINE AND PYRIMIDINI POOLS AND ON CELL PHASE DISTRIBUTION OF CULTURED HUMAN LYMPHOBLASTS: 50

Human leukemic lymphoblastic cell lines (Molt-4, Raji) were treated for 24 hrs with 0.2 μM Methotrexate (MTX). After 12 hrs, intracellular dCTP and dTTP concentrations decreased to 20% of the values of untreated cells. We noted a significant increase of UMP, UTP and CTP concentrations under these conditions. At the same time dATP and dGTP levels dropped to 30% and 50%, respectively. Accumulation of cells in G1 phase and decrease of cells in G2+M phase (to almost zero) was observed, after treatment with 0.2 μMMTX. The effect of 0.02 μM MTX on intracellular purine and pyrimidine concentrations was less pronounced, than that of 0.2 μM MTX, except for dCTP. In contrast to a decrease of dCTP, observed in experiments with 0.2 μM MTX, an increase of dCTP was measured in experiments with 0.02 μM MTX. Cell cycle phase distribution studies with 0.02 μM MTX, revealed an accumulation of cells in early S phase and decrease of cells in G1 phase and G2+M phase.

6-Thioguanine: High-dose 2-H infusions in goats

Summary6-Thioguanine (6TG) is poorly absorbed after oral administration. Bolus injections of 6TG result in high peak concentrations with relatively shortlived plasma concentrations. In vitro studies have shown the importance of prolonged exposure to 6TG. Therefore we administered 6TG by infusion at a dose rate of 2 mg/h over 2 h. In three goats we determined the plasma concentration-time curves of 6TG and its riboside (6TGR).A steady state was reached for 6TG and was almost reached for 6TGR within the 2 h of infusion. In one experiment we obtained several samples of CSF and observed good penetration of 6TG and 6TGR into CSF. Urinary excretion of 6TG and 6TGR was also quantitated. The amount of drug and metabolite excreted later than 4 h after the end of the infusion was negligible. By infusing 6TG, the problems of both erratic absorption after oral administration and acute renal toxicity after bolus injection, can be averted. In our opinion prolonged infusions of 6TG may be of advantage in humans suffering from actively proliferating malignant diseases, and thus should be studied.