The mechanism of action of methotrexate in cultured L5178Y leukemia cells.
This study investigates the relationships between the methotrexate (MTX)-induced purineless state and thymineless state and between the thymineless state and the kill of L5178Y cells. As an index of the thymineless state, we measured the effect of MTX on conversion of deoxyuridylate to thymidylate. This was measured as the rate of incorporation of tritiated deoxyuridine into DNA, but it was corrected for changes in incorporation of tritiated thymidine. Thus we derived the "calculated tritiated deoxyuridine rate." During the MTX treatment, the calculated tritiated deoxyuridine rate decreased rapidly at first and then more slowly. The slow 2nd-phase block was not reversed by hypoxanthine. As the 2nd-phase block deepened, the lymphoblasts continued to die (loss of cloning ability) but recovered the ability to incorporate tritiated thymidine into DNA. After 7 hr of MTX treatment, the kinetics of the 2nd-phase block in calculated tritiated deoxyuridine rate correlated closely with the kinetics of cell kill. Thus, MTX may inhibit dihydrofolate reductase enzyme, rapidly deplete S-phase L5178Y of reduced folates, and thus produce a purineless and thymineless state. As treatment continues, MTX intensifies the thymineless state, possibly by direct inhibition of thymidylate synthetase enzyme, and the cells die predominantly a thymineless death. The purineless state initially contributes to cell kill but later does not, possibly because it partially reverses spontaneously.
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis; D003857:Deoxyuridine / Q000378:metabolism; D007042:Hypoxanthines / Q000494:pharmacology; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
D004273:DNA, Neoplasm; D007042:Hypoxanthines; D013936:Thymidine; D003857:Deoxyuridine; D008727:Methotrexate