A study on the molecular diagnosis of antifolate resistance in leukemia cells : Establishment of anti-folate-resistant cell line and elucidation of molecular mechanisms of resistance

建立抗叶酸抗性细胞系，以阐明抗叶酸的分子机制。研究了还原或氧化叶酸对甲氨蝶呤（MTX）或N^<10>-丙炔-5,8-二氮杂酚（CB3717）耐酸性的影响。在翼酰基谷氨酸（PGA）中开发的大约60倍MTX耐药CCRF-CEM由于药物的膜转运减少而对MTX耐药。MTX抗性的进一步增强导致选择了大约5000倍的MTX抗性，由于基因扩增而增加了二氢叶酸还原酶（DHFR）。由于基因扩增导致DHFR增加，亚叶酸蛋白中产生了大约140倍和随后1500倍的MTX耐药亚系。大约200倍的cb3717耐药MOLT-3亚系在PGA或亚叶酸钙中开发，减少了膜运输。由于基因扩增，PGA中只有cb3717耐药亚系胸腺苷酸合成酶升高。膜运输缺陷的抗叶酸抗性亚群对三甲氧蝶呤表现出侧枝敏感性，在亚叶酸钙存在下，三甲氧蝶呤敏感性降低，表明对TMQ的侧枝敏感性是由细胞内叶酸缺乏引起的。

Antifolate resistant cell lines were established to elucidate molecular mechanisms of resistance. Effects of reduced or oxidized folates on the development of methotrexate (MTX) or N^<10>-propargyl-5,8-dideazafolic (CB3717) acid resistance were examined. An approximately 60-fold MTX-resistant CCRF-CEM developed in pteroylglutamic acid (PGA) was resistant to MTX by virtue of decreased membrance transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX resistance as noted by increased dihydrofolate reductase (DHFR) due to gene amplification. An approximately 140-fold and subsequently 1500-fold MTX-resistant subline developed in leucovorin were resistant to MTX by virtue of increased DHFR due to gene amplification. Approximately 200-fold CB3717-resistant MOLT-3 sublines developed in PGA or leucovorin had decreased membrance transport. Only CB3717-resistant subline developed in PGA had increased thymidylate synthase due to gene amplification. Membrane transport defective anti-folate-resistant sublines showed collateral sensitivity to trimeterxate, which was reduced in the presence of leucovorin, suggesting collateral sensitivity to TMQ was caused by intracellular folate defficiency.