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

Abstract

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 μM), 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, depencling 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 μM 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 μM 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 μM 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 μM 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. Increased levels of ribonucleotides after prolonged exposure to 0.2 μM MTX were due to reutilization of purine and pyrimidine precursors from nucleic acid breakdown consuming PRPP, whereas consumption of ribo-nucleotides for RNA synthesis was absent. The perturbations of deoxyribonucleotide pools after exposure to 0.02 and 0.2 μM MTX were in agreement with the changes in DNA-flow cytometry. Increased levels of dCTP after exposure to 0.02 μM MTX correlated with accumulation of cells in early S phase. A consplcuous phenomenon was the absence of decrease in dGTP pools in RAJI cells, in which cytotoxicity was low. Cytotoxicity was correlated with the degree of PDNS inhibition and a severe decrease of both dGTP and dTTP pools. The differences between the T−, B−, and non-B-non-T-, common ALL cell lines with respect to the parameters studied may explain the different responses to treatment of patients with corresponding lymphoid leukemias and lymphomas and suggest possibilities for different treatment strategies.