Trippett T, Schlemmer S, Elisseyeff Y, Goker E, Wachter M, Steinherz P, Tan C, Berman E, Wright J E, Rosowsky A
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Blood. 1992 Sep 1;80(5):1158-62.
Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4'-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.
虽然在对甲氨蝶呤（MTX）产生耐药性的实验肿瘤中，对该药物的耐药机制已为人所知，但关于患者对MTX获得性耐药的信息却很少。使用MTX的荧光赖氨酸类似物N-(4-氨基-4-脱氧-N10-甲基蝶酰基)-Nε-(4'-荧光素-硫代氨基甲酰基)-L-赖氨酸（PT430）开发了一种竞争性置换试验，作为检测细胞系以及白血病患者原始细胞对MTX转运耐药性的灵敏方法。白血病原始细胞在高浓度（20 μmol/L）下对PT430的快速摄取导致在2小时内达到稳态水平。随后与在载体转运模式上存在差异的叶酸拮抗剂MTX和三甲曲沙（TMTX）一起孵育，产生了PT430置换的特征模式。对人CCRF-CEM T细胞淋巴母细胞白血病细胞系及其MTX耐药亚系的平均荧光强度进行流式细胞术分析，清楚地确定了耐药亚系中存在转运缺陷。对17例白血病患者的原始细胞进行分析，其中9例未接受过化疗，8例曾接受过化疗，发现在4例接受MTX治疗且被认为对该药物临床耐药的患者中，有2例存在MTX转运耐药的证据。一些被认为对MTX临床耐药的白血病患者的原始细胞因MTX转运减少这一发现，对该药物的临床应用和新药开发具有重要意义。
