Dolnick B J, Pink J J
J Biol Chem. 1985 Mar 10;260(5):3006-14.
Growth of methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 5-fluorouracil results in an increase in dihydrofolate reductase mRNA. This increase can be solely attributed to a species of RNA of approximately 3.5 kilobase pairs in size. Although dihydrofolate reductase enzyme activity increases per cell with increasing 5-fluorouracil, there is a decrease of enzyme activity per mg of protein (Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299-13306). The rate of in vivo enzyme synthesis, as assayed by immunoprecipitation and supported by gel electrophoresis, does not decrease and may in fact increase with increasing 5-fluorouracil. Translation of purified dihydrofolate reductase mRNA in vitro shows that the rate of translation is unaffected by 5-fluorouracil incorporation into mRNA. The inhibition of dihydrofolate reductase by a monospecific polyclonal antiserum is reduced with extracts from 5-fluorouracil-treated cells. Inhibition of dihydrofolate reductase by methotrexate is significantly reduced in extracts from 5-fluorouracil-treated cells compared to control extracts. Tight binding of [3H]methotrexate is also different in extracts from 5-fluorouracil-treated cells. This data supports the hypothesis of translational miscoding during protein synthesis as a major mechanism of 5-fluorouracil-mediated cytotoxicity and suggests a new mechanism of 5-fluorouracil-methotrexate antagonism.
在5-氟尿嘧啶存在的情况下，耐甲氨蝶呤的二氢叶酸还原酶基因扩增的KB细胞生长会导致二氢叶酸还原酶mRNA增加。这种增加完全可归因于一种大小约为3.5千碱基对的RNA。尽管随着5-氟尿嘧啶浓度增加，每个细胞中二氢叶酸还原酶的酶活性增加，但每毫克蛋白质的酶活性却下降了（Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299 - 13306）。通过免疫沉淀测定并经凝胶电泳证实，体内酶合成速率并未下降，实际上可能随着5-氟尿嘧啶浓度增加而增加。体外纯化的二氢叶酸还原酶mRNA的翻译表明，翻译速率不受5-氟尿嘧啶掺入mRNA的影响。用5-氟尿嘧啶处理过的细胞提取物中，单特异性多克隆抗血清对二氢叶酸还原酶的抑制作用降低。与对照提取物相比，5-氟尿嘧啶处理过的细胞提取物中，甲氨蝶呤对二氢叶酸还原酶的抑制作用显著降低。在5-氟尿嘧啶处理过的细胞提取物中，[3H]甲氨蝶呤的紧密结合也有所不同。这些数据支持了蛋白质合成过程中翻译错义编码是5-氟尿嘧啶介导的细胞毒性主要机制的假说，并提示了5-氟尿嘧啶-甲氨蝶呤拮抗作用的新机制。
