Houghton P J, Germain G S, Hazelton B J, Pennington J W, Houghton J A
Department of Biochemical and Clinical Pharmacology, Saint Jude Children's Research Hospital, Memphis, TN 38101.
Proc Natl Acad Sci U S A. 1989 Feb;86(4):1377-81. doi: 10.1073/pnas.86.4.1377.
GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.
用诱变剂甲磺酸乙酯处理GC3/c1人结肠腺癌细胞，筛选并鉴定出三个胸苷酸合成酶（5,10-亚甲基四氢叶酸：dUMP C-甲基转移酶，EC 2.1.1.45）活性缺陷的克隆。在缺乏胸苷的培养基中生长导致两个克隆（TS-c1和TS-c3）细胞死亡，而一个克隆（TS-c2）生长受限。生长与胸苷合成酶活性、5-氟-2'-脱氧尿苷5'-单磷酸结合能力以及2'-脱氧[6-³H]尿苷掺入DNA相关。在最佳胸苷存在的情况下，生长速率仅为亲本克隆（GC3/c1）的5%-18%，亲本克隆在缺乏胸苷或富含胸苷的培养基中生长情况相同。对多聚腺苷酸加尾RNA（poly(A)+ RNA）的分析表明，TS-c1和TS-c2中1.6千碱基转录本水平正常，但TS-c3中水平降低（约为对照的6%）。克隆TS-c3对5-氟尿嘧啶、5-氟-2'-脱氧尿苷和甲氨蝶呤的抗性分别比亲本克隆高32倍、750倍和超过100,000倍。当接种到无胸腺裸鼠体内时，每个TS克隆都产生了肿瘤，表明其在体内仍具有增殖能力。
