Time course of methotrexate polyglutamate formation and degradation in the pre-B-leukaemia cell line Nalm6 and in lymphoblasts from children with leukaemia

With the aim of investigation, the mechanisms of resistance to methotrexate (MTX) in children refractory to leukaemia-treatment, we established a method of analysing MTX metabolism in Nalm6 cells (human pre-B). The optimal extracellular concentration for MTX uptake and MTX polyglutamate (MTXPG2-6) formation at a density of 5 x 10(6) cells/ml was 1 microM 3H-MTX. After 15 h incubation at this concentration, a plateau of 5 pmol/10(6) cells of total MTX accumulated in the form of equal amounts of polyglutamates 3, 4 and 5 and low amounts of MTX and polyglutamates 2 and 6. MTX preloaded cells rapidly lost MTX and MTXPG2 in MTX-free medium, while MTXPG5 was still formed and then degraded very slowly. After 8 h in medium without MTX, 40% of total MTXPG was lost, after 24 h, 70%. The method is feasible for patient blasts. The number of blasts isolated from bone marrow after diagnosis is enough to perform small kinetic studies. The uptake of MTX into patient blasts is about 1/10 of that in Nalm6 cells.