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

Abstract

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 μM and 0.2 μM 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 μM 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 μM 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.