IDENTIFICATION OF METHOTREXATE TRANSPORT DEFICIENCY IN MAMMALIAN-CELLS USING FLUORESCEINATED METHOTREXATE AND FLOW-CYTOMETRY
IDENTIFICATION OF METHOTREXATE TRANSPORT DEFICIENCY IN MAMMALIAN-CELLS USING FLUORESCEINATED METHOTREXATE AND FLOW-CYTOMETRY
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DOI：
10.1073/pnas.84.20.7154
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发表时间：
1987-10-01
影响因子：
11.1
通讯作者：
SCHIMKE, RT
中科院分区：
文献类型：
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作者：
ASSARAF, YG;SCHIMKE, RT
我们用快速流式细胞仪技术研究了耐甲氨蝶呤的中国仓鼠卵巢细胞的转运突变频率。在用荧光甲氨蝶呤浸泡细胞后，我们检测了亲水性和亲脂性抗叶酸取代荧光甲氨蝶呤与二氢叶酸还原酶结合的能力。甲氨蝶呤转运缺陷的细胞不能摄取甲氨蝶呤，从而保留荧光，而在敏感和耐药的细胞系中，亲脂性的抗叶酸取代荧光的甲氨蝶呤效果相同。这些抗性克隆不能吸收甲氨蝶呤，并且在甲氨蝶呤浓度为.apprxeq的单步选择中出现频率很高。是50%杀灭浓度的7倍。大多数这样的第一步抗性克隆似乎完全是由于运输不足而产生的抗性；它们没有表现出二氢叶酸还原酶的过量生产，也没有表现出稳定的mRNA水平或基因拷贝数的增加。讨论了使用荧光甲氨蝶呤来表征混合细胞群体中甲氨蝶呤耐药性的各种机制的可能应用。
We have studied the frequency of transport mutations in methotrexate-resistant Chinese hamster ovary cells using a rapid-flow cytometric technique. After saturating cells with fluoresceinated methotrexate, we examined the ability of hydrophilic and lipophilic antifolates to displace fluoresceinated methotrexate binding to dihydrofolate reductase. Cells with methotrexate transport deficiency are unable to take up methotrexate and thus retain the fluorescence, whereas the lipophilic antifolates displace fluoresceinated methotrexate equally well in sensitive and resistant cell lines. These resistant clones fail to take up methotrexate and occur with high frequencies upon single-step selections at methotrexate concentrations .apprxeq. 7-fold the 50% killing concentration. The majority of such first-step resistant clones appear to derive their resistance solely from transport deficiency; they exhibit no overproduction of dihydrofolate reductase and no increase in either steady-state mRNA levels or gene copy number. Possible applications of the use of fluoresceinated methotrexate to the characterization of various mechanisms of methotrexate resistance in mixed cell populations are discussed.