Buchholz B, Frei E, Eisenbarth J, Weigand M, Ludwig R
Children's Hospital, Klinikum, Mannheim, Germany.
Eur J Cancer. 1996 Nov;32A(12):2101-7. doi: 10.1016/s0959-8049(96)00230-4.
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.
为了研究白血病治疗难治性儿童对甲氨蝶呤（MTX）耐药的机制，我们建立了一种分析Nalm6细胞（人前B细胞）中MTX代谢的方法。在细胞密度为5×10⁶个细胞/毫升时，MTX摄取和MTX多聚谷氨酸（MTXPG2 - 6）形成的最佳细胞外浓度为1微摩尔³H - MTX。在此浓度下孵育15小时后，以等量的多聚谷氨酸3、4和5以及少量的MTX和多聚谷氨酸2和6形式积累的总MTX达到5皮摩尔/10⁶个细胞的平台期。预先加载MTX的细胞在无MTX培养基中迅速丢失MTX和MTXPG2，而MTXPG5仍在形成，然后降解非常缓慢。在无MTX培养基中培养8小时后，总MTXPG的40%丢失，24小时后，70%丢失。该方法对患者的原始细胞是可行的。诊断后从骨髓中分离出的原始细胞数量足以进行小型动力学研究。MTX进入患者原始细胞的摄取量约为Nalm6细胞的1/10。
