Fabre G, Goldman I D
Cancer Res. 1985 Jan;45(1):80-5.
The rapid synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-MTX) and their selective intracellular retention are reported in human chronic myelogenous leukemia cells, K-562. After a 30-min exposure to 5 microM [3H]7-OH-MTX, three different polyglutamyl derivatives were detected by high-performance liquid chromatography. When extracellular 7-OH-MTX was removed, the 7-OH-MTX diglutamate level declined slowly in comparison to the monoglutamate, but the higher polyglutamyl derivative levels increased. Within 10 min after exposure of cells to 7-OH-MTX, the level of these polyglutamyl derivatives far exceeds the dihydrofolate reductase binding capacity. Gel filtration or charcoal binding analysis followed by high-performance liquid chromatography analysis of the bound component showed intracellular binding of virtually all 7-OH-MTX tetraglutamate at a level 4-fold higher than that of the dihydrofolate reductase binding capacity. No bound 7-OH-MTX diglutamate or triglutamate could be detected. Treatment of the 7-OH-MTX tetraglutamate: protein complex with 100 microM unlabeled methotrexate (MTX) for 15 min resulted in only a partial dissociation of this complex to an extent compatible with the dihydrofolate reductase level. The residual 7-OH-MTX tetraglutamate remained bound to a site with a molecular weight of approximately 25,000 to 35,000 as assessed by Bio-Gel P-60 analysis and could not be displaced by folic acid, 5-formyltetrahydrofolate, 7-OH-MTX, or the tetraglutamate of MTX. 7-OH-MTX and MTX cytotoxicities were compared by clonogenic assay in agar and by their effects on cell growth. After a 2-hr exposure, the 50% inhibitory concentrations for 7-OH-MTX and MTX in cells growing in agar were 10(-5) and 10(-6) M, respectively. A 10-fold difference in cytotoxicity was also observed in cells growing in suspension. Continuous exposure to glycine: adenosine: thymidine completely protects cells from a sustained exposure to 7-OH-MTX over the entire period of clonal growth. However, even a brief exposure to 7-OH-MTX also requires continuous exposure to glycine: adenosine: thymidine for protection. This suggests that, as observed for MTX, the 7-OH-MTX polyglutamyl derivatives that are retained within the cells have a sustained cytotoxic effect after the monoglutamate is removed.
据报道，在人慢性髓性白血病细胞K-562中，7-羟基甲氨蝶呤（7-OH-MTX）的聚γ-谷氨酰衍生物能够快速合成，且具有选择性细胞内滞留特性。在5微摩尔[3H]7-OH-MTX作用30分钟后，通过高效液相色谱法检测到三种不同的聚谷氨酸衍生物。当去除细胞外的7-OH-MTX后，与单谷氨酸相比，7-OH-MTX二谷氨酸水平下降缓慢，但更高的聚谷氨酸衍生物水平有所增加。在细胞暴露于7-OH-MTX后10分钟内，这些聚谷氨酸衍生物的水平远远超过二氢叶酸还原酶的结合能力。凝胶过滤或活性炭结合分析，随后对结合成分进行高效液相色谱分析表明，几乎所有的7-OH-MTX四谷氨酸在细胞内的结合水平比二氢叶酸还原酶的结合能力高4倍。未检测到结合的7-OH-MTX二谷氨酸或三谷氨酸。用100微摩尔未标记的甲氨蝶呤（MTX）处理7-OH-MTX四谷氨酸：蛋白质复合物15分钟，结果该复合物仅部分解离，解离程度与二氢叶酸还原酶水平相符。通过Bio-Gel P-60分析评估，残留的7-OH-MTX四谷氨酸仍与分子量约为25,000至35,000的位点结合，且不能被叶酸、5-甲酰四氢叶酸、7-OH-MTX或MTX的四谷氨酸取代。通过琼脂克隆形成试验及其对细胞生长的影响，比较了7-OH-MTX和MTX的细胞毒性。暴露2小时后，在琼脂中生长的细胞对7-OH-MTX和MTX的50%抑制浓度分别为10^(-5)和10^(-6) M。在悬浮生长的细胞中也观察到细胞毒性有10倍差异。持续暴露于甘氨酸：腺苷：胸苷可在整个克隆生长期间完全保护细胞免受7-OH-MTX的持续暴露。然而，即使短暂暴露于7-OH-MTX也需要持续暴露于甘氨酸：腺苷：胸苷来提供保护。这表明，正如对MTX所观察到的那样，细胞内滞留的7-OH-MTX聚谷氨酸衍生物在单谷氨酸被去除后具有持续的细胞毒性作用。
