Washtien W L, Santi D V
Cancer Res. 1979 Sep;39(9):3397-404.
Methods have been developed to assay several aspects of 5-fluoro-2'-deoxyuridine and 5-fluorouracil metabolism in tissue culture cells. These methods allow measurement of (a) intracellular levels of the covalent complex formed between 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase; (b) incorporation of drug into RNA; and (c) analysis of drug metabolites. The methods were developed using radioactively labeled drugs, but they should be adaptable to studies using nonlabeled compounds. Sephadex G-25 chromatography or trichloroacetic acid precipitation were utilized for isolation of the macromolecular cell fraction; prior treatment of this fraction with RNase or heating at 65 degrees for 15 min resulted in selective removal of RNA or the thymidylate synthetase complex, respectively, from the precipitable fraction. Free intracellular drug metabolites present in the acid-soluble fraction were analyzed by high-pressure liquid chromatography. Following incubation of HTC cells with [6-3H]-5-fluoro-2'-deoxyuridine, a radioactive macromolecule was isolated and identified as a FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex. Intracellular formation of this complex was shown to be dependent on the presence of the enzyme thymidine kinase. Dissociation of the complex in vivo was first order with a t1/2 of 6.2 hr; in contrast, a t1/2 of 2 hr was determined for dissociation of the complex in cytosol. Incubation of L1210 cells with [6-3H]-5-fluorouracil for 22 hr resulted in formation of 80 fmol of FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex per 10(6) cells, as compared with 400 fmol of drug incorporated into RNA per 10(6) cells. Intracellular FdUMP could not be detected in the acid-soluble fraction of these cells unless the cells were first heated to dissociate the complex.
已开发出多种方法来检测组织培养细胞中5-氟-2'-脱氧尿苷和5-氟尿嘧啶代谢的多个方面。这些方法可用于测量：(a) 5-氟-2'-脱氧尿苷5'-单磷酸（FdUMP）、5,10-亚甲基四氢叶酸和胸苷酸合成酶之间形成的共价复合物的细胞内水平；(b) 药物掺入RNA的情况；以及(c) 药物代谢产物的分析。这些方法是使用放射性标记药物开发的，但它们应该适用于使用未标记化合物的研究。使用Sephadex G-25柱色谱法或三氯乙酸沉淀法分离大分子细胞组分；用核糖核酸酶预先处理该组分或在65℃加热15分钟，分别导致RNA或胸苷酸合成酶复合物从可沉淀组分中被选择性去除。通过高压液相色谱法分析酸溶性组分中存在的游离细胞内药物代谢产物。用[6-³H]-5-氟-2'-脱氧尿苷孵育HTC细胞后，分离出一种放射性大分子，并鉴定为FdUMP-5,10-亚甲基四氢叶酸-胸苷酸合成酶复合物。该复合物的细胞内形成显示依赖于胸苷激酶的存在。该复合物在体内的解离呈一级动力学，半衰期为6.2小时；相比之下，该复合物在细胞溶质中的解离半衰期为2小时。用[6-³H]-5-氟尿嘧啶孵育L1210细胞22小时后，每10⁶个细胞形成80 fmol的FdUMP-5,10-亚甲基四氢叶酸-胸苷酸合成酶复合物，而每10⁶个细胞中有400 fmol的药物掺入RNA。除非细胞首先被加热以解离复合物，否则在这些细胞的酸溶性组分中无法检测到细胞内FdUMP。
