Miyachi H, Takemura Y, Kobayashi H, Ando Y
Department of Clinical Pathology, Tokai University School of Medicine, Isehara.
Jpn J Cancer Res. 1997 Sep;88(9):900-6. doi: 10.1111/j.1349-7006.1997.tb00467.x.
Cytotoxicity of trimetrexate (TMQ), a lipophilic dihydrofolate reductase inhibitor, was examined in antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. An approximately 60-fold methotrexate (MTX)-resistant subline was developed in oxidized folate (pteroylglutamic acid: PGA) (CCRF-CEM/MTX60-PGA) from human T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX5000-PGA), which showed increased dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced folate (10 nM leucovorin) (CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV); they exhibited increased dihydrofolate reductase due to gene amplification accompanied by increased intracellular drug accumulation of MTX. While CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX60-PGA and CCRF-CEM/MTX5000-PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N10-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human T-cell leukemia MOLT-3 sublines developed in PGA (MOLT-3/CB3717(200)-PGA) or leucovorin (MOLT-3/CB3717(200)-LV), as compared to the parent cells; MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells were resistant to CB3717 by virtue of impaired transport, only the former possessing gene amplification of thymidylate synthase. The cytotoxicity of TMQ in both MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells was reduced by addition of leucovorin in a dose-dependent manner, suggesting intracellular folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired antifolate resistance, irrespective of gene amplification of dihydrofolate reductase or thymidylate synthase. Types of folate used during the development of antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of antifolate resistance.
亲脂性二氢叶酸还原酶抑制剂三甲曲沙（TMQ）对在氧化型或还原型叶酸中培养出的抗叶酸人T细胞白血病细胞系的细胞毒性进行了检测。从人T细胞白血病细胞系CCRF-CEM中，在氧化型叶酸（蝶酰谷氨酸：PGA）中培养出了一个对甲氨蝶呤（MTX）耐药约60倍的亚系（CCRF-CEM/MTX60-PGA）；该细胞系对药物的膜转运受损。MTX耐药性的进一步增强导致选择出了一个对MTX耐药约5000倍的亚系（CCRF-CEM/MTX5000-PGA），除了MTX转运进一步受损外，该亚系还因基因扩增而表现出二氢叶酸还原酶活性增加。在还原型叶酸（10 nM亚叶酸）中培养出了一个对MTX耐药约140倍的亚系，随后又培养出了一个对MTX耐药1500倍的亚系（CCRF-CEM/MTX140-LV和CCRF-CEM/MTX1500-LV）；它们因基因扩增而表现出二氢叶酸还原酶增加，同时MTX的细胞内药物积累也增加。虽然CCRF-CEM/MTX140-LV和CCRF-CEM/MTX1500-LV细胞对TMQ表现出交叉耐药，但CCRF-CEM/MTX60-PGA和CCRF-CEM/MTX5000-PGA细胞对TMQ的敏感性至少与亲本细胞相同。TMQ对在PGA（MOLT-3/CB3717(200)-PGA）或亚叶酸（MOLT-3/CB3717(200)-LV）中培养出的对N10-炔丙基-5,8-二去氮叶酸（CB3717）耐药约200倍的人T细胞白血病MOLT-3亚系的作用比亲本细胞更强；MOLT-3/CB3717(200)-PGA和MOLT-3/CB3717(200)-LV细胞因转运受损而对CB3717耐药，只有前者胸苷酸合成酶有基因扩增。在MOLT-3/CB3717(200)-PGA和MOLT-3/CB3717(200)-LV细胞中，加入亚叶酸后TMQ的细胞毒性呈剂量依赖性降低，提示细胞内叶酸缺乏是TMQ敏感性的原因。这些结果表明，TMQ能克服转运受损的抗叶酸耐药性，而与二氢叶酸还原酶或胸苷酸合成酶的基因扩增无关。在抗叶酸耐药性形成过程中使用的叶酸类型似乎与TMQ反应性机制以及抗叶酸耐药性机制都很重要。
