Houghton P J, Rahman A, Will C L, Dolnick B J, Houghton J A
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Cancer Res. 1992 Feb 1;52(3):558-65.
Biological characterization of a human colon adenocarcinoma cell line deficient in thymidylate synthase (TS-) is described. The clone, designated TS-C1/C1, was derived from the parental line GC3/C1 by selection in medium containing aminopterin, thymidine (dThd), and low concentrations of 5-formyltetrahydrofolate (5-CHO-H4PteGlu), and was subsequently reselected by single-step cloning in 500 microM methotrexate in the presence of dThd. This clone retained its TS- phenotype, was highly resistant to methotrexate (greater than 100,000-fold), and remained tumorigenic in mice (P.J. Houghton, et al., Proc. Natl. Acad. Sci. USA, 86: 1377-1381, 1989). In studies reported, it is shown that high levels of exogenous folate can support the growth of the TS- C1/C1 clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 microM [6R,S]5-CHO-H4PteGlu, and greater than or equal to 80% of activity was lost within 24 h of removing this folate from the medium. The labeling index was determined by autoradiographic techniques using [6-3H]2'-deoxyuridine. None of the greater than 6,000 cells radiolabeled in the absence of [6R,S]5-CHO-H4PteGlu, whereas 33.5% labeled in the presence of 20 microM exogenous folate. Relative to the parental (TS+) clone, there was a greater than 87,500-, an 8,182-, and a 425-fold higher requirement for 5-methyltetrahydrofolate ([6R,S]5-CH3-H4PteGlu), PteGlu, and [6R,S]5-CH3-H4PteGlu to support 50% maximal colony formation in the absence of dThd. Quantitative analysis of the combined pools of 5,10-methylenetetrahydrofolate (CH2-H4PteGlun) and H4PteGlun showed that parental GC3/C1 cells had higher endogenous folate pools compared to TS-C1/C1 cells [168 +/- 40 (SD) and 10.9 +/- 0.3 fmol/10(6) cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 microM [6R,S]5-CHO-H4PteGlu were similar in the two lines. Analysis of pools in a second, independently derived, TS- clone (TS-C3/C3, a transcription-negative mutant) demonstrated undetectable levels of CH2-H4PteGlun and H4PteGlun. This line cannot be rescued by exogenous folate. The data thus suggest that deletion of dTMP synthase activity may cause redistribution of reduced folate pools. In cytosolic extracts from parental GC3/C1 (TS+) cells, [6R]CH2-H4PteGlu1 acted as a cofactor in the release of 3H2O from [5-3H]dUMP, whereas no activity was detected in cytosols from TS-C1/C1. In contrast dTMP synthase activity was detected in cytosols from TS- C1/C1 cells in the presence of [6R]CH2-H4PteGlu5.(ABSTRACT TRUNCATED AT 400 WORDS)
本文描述了一种胸苷酸合成酶缺陷型（TS-）人结肠腺癌细胞系的生物学特性。该克隆株命名为TS-C1/C1，是通过在含有氨基蝶呤、胸腺嘧啶核苷（dThd）和低浓度5-甲酰四氢叶酸（5-CHO-H4PteGlu）的培养基中筛选，从亲代细胞系GC3/C1获得的，随后在dThd存在的情况下，通过在500微摩尔甲氨蝶呤中进行单步克隆进行重新筛选。该克隆株保留了其TS-表型，对甲氨蝶呤具有高度抗性（大于100,000倍），并且在小鼠中仍具有致瘤性（P.J.霍顿等人，《美国国家科学院院刊》，86: 1377 - 1381, 1989）。在本报告的研究中，结果表明，在没有dThd的情况下，高水平的外源性叶酸可以支持TS-C1/C1克隆株的生长。在将细胞暴露于20微摩尔[6R,S]5-CHO-H4PteGlu后的6小时内，从头合成dTMP的过程被激活，而在从培养基中去除这种叶酸后的24小时内，大于或等于80%的活性丧失。使用[6-3H]2'-脱氧尿苷通过放射自显影技术测定标记指数。在没有[6R,S]5-CHO-H4PteGlu的情况下，超过6000个细胞均未被放射性标记，而在存在20微摩尔外源性叶酸的情况下，33.5%的细胞被标记。相对于亲代（TS+）克隆株，在没有dThd的情况下，支持50%最大集落形成所需的5-甲基四氢叶酸（[6R,S]5-CH3-H4PteGlu）、蝶酰谷氨酸（PteGlu）和[6R,S]5-CH3-H4PteGlu的需求量分别高出87,500倍以上、8182倍和425倍。对5,10-亚甲基四氢叶酸（CH2-H4PteGlun）和H4PteGlun的联合池进行定量分析表明，亲代GC3/C1细胞比TS-C1/C1细胞具有更高的内源性叶酸池[分别为168±40（标准差）和10.9±0.3飞摩尔/10(6)个细胞]。定性分析表明，在两条细胞系中，多聚谷氨酸物种的分布及其在暴露于20微摩尔[6R,S]5-CHO-H4PteGlu的细胞中的重新分布是相似的。对第二个独立获得的TS-克隆株（TS-C3/C3，一种转录阴性突变体）的池分析表明，未检测到CH2-H4PteGlun和H4PteGlun的水平。该细胞系不能被外源性叶酸挽救。因此，数据表明dTMP合成酶活性的缺失可能导致还原型叶酸池的重新分布。在亲代GC3/C1（TS+）细胞的胞质提取物中，[6R]CH2-H4PteGlu1在[5-3H]dUMP释放3H2O的过程中作为辅因子起作用，而在TS-C1/C1细胞的胞质溶胶中未检测到活性。相反，在存在[6R]CH2-H4PteGlu5的情况下，在TS-C1/C1细胞的胞质溶胶中检测到了dTMP合成酶活性。（摘要截短于400字）
