Drake J C, Allegra C J, Johnston P G
NCI-Navy Medical Oncology Branch, National Cancer Institute, NIH, Naval Medical Hospital, Bethesda, MD 20889-5105.
Anticancer Drugs. 1993 Aug;4(4):431-5. doi: 10.1097/00001813-199308000-00002.
Thymidylate synthase (TS) is responsible for the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. One of the principal mechanisms of action of 5-fluorouracil (5-FU) is the inhibition of TS by formation of a ternary covalent complex consisting of TS-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate. We have developed a Western immunoblot assay using the monoclonal antibody TS 106 to measure ternary complex and free TS in intact human carcinoma cells following exposure to either 5-FU alone or 5-FU plus leucovorin. Lysates from cells treated with either 5-FU or 5-FU/leucovorin were resolved in 15% polyacrylamide gel, transferred onto nitrocellulose and immunoblotted using TS 106 antibody. Detection of positive bands was by a chromogenic substrate strain. Immunoblotting detected free TS at 36 kDa and TS in ternary complex at 38.5 kDa which were quantitated by densitometric scanning. This assay was able to detect a ternary complex from intact cells treated with 5-FU or 5-FU/leucovorin up to 96 h after drug removal. The ratio of complex to free TS was up to 2-fold greater in 5-FU/leucovorin-treated cells compared to those treated with 5-FU alone. This assay may be applied to measuring the formation and stability of ternary complex and free TS in patient tissue samples.
胸苷酸合成酶（TS）负责将脱氧尿苷一磷酸转化为脱氧胸苷一磷酸。5-氟尿嘧啶（5-FU）的主要作用机制之一是通过形成由TS-5-氟脱氧尿苷酸-5,10-亚甲基四氢叶酸组成的三元共价复合物来抑制TS。我们开发了一种使用单克隆抗体TS 106的蛋白质免疫印迹分析方法，以测量完整的人癌细胞在单独暴露于5-FU或5-FU加亚叶酸后三元复合物和游离TS的情况。用5-FU或5-FU/亚叶酸处理的细胞裂解物在15%聚丙烯酰胺凝胶中进行分离，转移到硝酸纤维素膜上，并用TS 106抗体进行免疫印迹。通过显色底物菌株检测阳性条带。免疫印迹检测到36 kDa的游离TS和38.5 kDa的三元复合物中的TS，通过光密度扫描进行定量。该分析方法能够检测在用5-FU或5-FU/亚叶酸处理的完整细胞中直至药物去除后96小时的三元复合物。与单独用5-FU处理的细胞相比，5-FU/亚叶酸处理的细胞中复合物与游离TS的比例高达2倍。该分析方法可用于测量患者组织样本中三元复合物和游离TS的形成和稳定性。
