Cowan K H, Jolivet J
J Biol Chem. 1984 Sep 10;259(17):10793-800.
Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.
通过在甲氨蝶呤（MTX）中连续传代野生型ZR - 75 - 1细胞（野生型ZR - 75）获得了耐MTX的人乳腺癌细胞（MTXR ZR - 75）。与亲代细胞系相比，耐药细胞系中二氢叶酸还原酶在数量和质量上均无变化。耐药性与MTX转运至MTXR ZR - 75细胞的能力降低3倍以及耐药亚系中胸苷酸合成酶活性降低3倍有关。此外，在将MTX转化为其聚谷氨酸衍生物的能力方面，野生型和MTXR ZR - 75细胞之间观察到显著差异。野生型ZR - 75细胞在长时间（24小时）暴露于2 microM MTX期间会积累大量细胞内抗叶酸药物，这是由于MTX聚谷氨酸的形成。相比之下，即使在导致这些细胞内游离药物大量过剩的条件下，MTXR细胞中也基本不形成聚谷氨酸。这种缺陷与叶酸聚谷氨酸合成酶的活性没有任何明显变化无关，耐药细胞中该酶对MTX的表观Km也没有任何改变。进一步的研究表明，MTXR ZR - 75细胞对可转化为聚谷氨酸衍生物的抗叶酸类似物（氨基蝶呤和二氯甲氨蝶呤）具有交叉耐药性，但它们对不能转化为聚谷氨酸形式的抗叶酸类似物如2,4 - 二氨基 - 5 -（3'，4' - 二氯苯基）- 6 - 甲基嘧啶、三嗪酸盐和三甲氧苄氨嘧啶相对敏感。这些研究确定了一种与MTX耐药性相关的新机制（MTX聚谷氨酸积累减少），并为这些衍生物的形成是MTX细胞毒性的重要决定因素这一假说提供了额外支持。
