Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for the determination of 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthetase.
Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for the determination of 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthetase.
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肿瘤对 5-氟尿嘧啶敏感性的生化决定因素：测定 5-氟-2-脱氧尿苷酸、2-脱氧尿苷酸和胸苷酸合成酶的超灵敏方法。
DOI：
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发表时间：
1979
影响因子：
11.1
通讯作者：
C. Heidelberger
中科院分区：
文献类型：
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作者：
R. Moran;C. P. Spears;C. Heidelberger
已经开发了测量FdUMP（5-FUra的活性代谢物）、胸苷酸合成酶（TMP合成酶; 5，10-亚甲基四氢叶酸：dUMP C-甲基转移酶，EC 2.1.1.45）（该抗代谢物的靶酶）和dUMP（与FdUMP竞争结合TMP合成酶的底物）的技术。通过使用均质干酪乳杆菌/MTX TMP合成酶作为结合蛋白，可以用竞争性配体结合测定定量低至0.02 pmol的FdUMP。TMP合成酶的新结合测定允许检测0.005 pmol的酶。用5，10-亚甲基[（14）C]四氢叶酸盐将dUMP定量酶促转化为[甲基-（14）C]-TMP。干酪TMP合成酶用于dUMP测定，灵敏度为10 pmol。培养的CCRF-CEM人淋巴母细胞白血病细胞在暴露于30 μ M 5-FUra后11小时内形成高水平的FdUMP（2.6nmol/10（9）个细胞）。肿瘤细胞TMP合成酶水平下降，然后出现游离FdUMP。细胞内dUMP池在几种肿瘤细胞系的生长培养物中较低（2-5 nmol/10（9）个细胞），但在酶水平降低后暴露于5-FUra后，CCRF-CEM细胞中迅速扩增。所描述的方法足够灵敏，可以在细胞培养物或人肿瘤的穿刺活检样品中测量5-FUra作用的这些生物化学参数。
Techniques have been developed to measure FdUMP, the active metabolite of 5-FUra; thymidylate synthetase (TMP synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45), the target enzyme for this antimetabolite; and dUMP, the substrate that competes with FdUMP for binding to TMP synthetase. As little as 0.02 pmol of FdUMP can be quantitated with a competitive ligand binding assay by using homogeneous Lactobacillus casei/MTX TMP synthetase as a binding protein. A new binding assay for TMP synthetase allows detection of 0.005 pmol of enzyme. The quantitative enzymatic conversion of dUMP to [methyl-(14)C]-TMP using 5,10-methylene[(14)C]tetrahydrofolate by pure L. casei TMP synthetase is used as an assay for dUMP with a sensitivity of 10 pmol. Cultured CCRF-CEM human lymphoblastic leukemia cells formed high levels of FdUMP (2.6 nmol per 10(9) cells) within 11 hr after exposure to 30 muM 5-FUra. Tumor cell TMP synthetase levels dropped, and then free FdUMP appeared. The intracellular dUMP pool was low (2-5 nmol per 10(9) cells) in logarithmically growing cultures of several tumor cell lines but expanded rapidly in CCRF-CEM cells on exposure to 5-FUra after enzyme levels decreased. The levels of dUMP found after exposure to 5-FUra are sufficient to severely retard inhibition of TMP synthetase by FdUMP.The methods described are sufficiently sensitive to allow these biochemical parameters of 5-FUra action to be measured in cell culture or in needle biopsy samples of human tumors.