Bogenhagen D, Clayton D A
J Biol Chem. 1976 May 25;251(10):2938-44.
The effects of 5-fluorodeoxyuridine and methotrexate on [3H]thymidine and 32P labeling of mtDNA were studied in two lines of mouse L-cells. LMTK- cells, which lack the major cellular thymidine kinase (EC 2.7.1.21) but contain a genetically distinct mitochondrial enzyme, were compared to LA9 cells, which contain both thymidine kinase activities. LMTK- cells were resistant to 5-flurodeoxyuridine by a factor of 200 in comparison to LA9 cells. In both cells lines appropriate drug treatment increased utilization of exogenous thymidine for mtDNA synthesis. The maximum enhancement was 10- to 12-fold for LA9 cells and approximately 20-fold for LMTK- cells when treated with 10 muM methotrexate. The rates of mtDNA and nuclear DNA synthesis during drug treatment were analyzed with 32P labeling and 5-bromo-2'-deoxyuridine density labeling experiments. Synthesis of both mtDNA and nuclear DNA were strongly inhibited by drug treatment of either LA9 or LMTK- cells in the absence of exogenous thymidine. The rate of mtDNA synthesis substantially exceeded that of nuclear DNA in LA9 cells treated with 4 muM 5-fluorodeoxyuridine and less than 5 muM thymidine. Both synthetic rates approached those of untreated LA9 control cultures if 20 muM thymidine was present during 5-fluorodeoxyuridine treatment. In contrast, in LMTK- cells treated with 10 muM methotrexate and 20 muM thymidine, mtDNA synthesis continued at 50 to 60% of the control rate for at least 10 hours while nuclear DNA synthesis was 96% inhibited. Synthesis of mtDNA mass-labeled in both strands with 5-bromouracil occurred when LMTK- cells were incubated for 30 hours with 10 muM methotrexate and 20 muM 5-bromodeoxyuridine. These results indicate that mtDNA synthesis is resistant to a limitation of the thymidine triphosphate supply and is not strictly dependent upon concomitant nuclear DNA synthesis in these cells.
在两株小鼠L细胞系中研究了5-氟脱氧尿苷和甲氨蝶呤对[3H]胸苷和32P标记线粒体DNA（mtDNA）的影响。将缺乏主要细胞胸苷激酶（EC 2.7.1.21）但含有一种遗传上不同的线粒体酶的LMTK-细胞与同时含有两种胸苷激酶活性的LA9细胞进行比较。与LA9细胞相比，LMTK-细胞对5-氟脱氧尿苷的抗性高200倍。在两种细胞系中，适当的药物处理均可增加外源性胸苷用于mtDNA合成的利用率。用10μM甲氨蝶呤处理时，LA9细胞的最大增强倍数为10至12倍，LMTK-细胞约为20倍。在药物处理期间，通过32P标记和5-溴-2'-脱氧尿苷密度标记实验分析了mtDNA和核DNA的合成速率。在没有外源性胸苷的情况下，对LA9或LMTK-细胞进行药物处理会强烈抑制mtDNA和核DNA的合成。在用4μM 5-氟脱氧尿苷和少于5μM胸苷处理的LA9细胞中，mtDNA的合成速率大大超过核DNA的合成速率。如果在5-氟脱氧尿苷处理期间存在20μM胸苷，则两种合成速率均接近未处理的LA9对照培养物的速率。相比之下，在用10μM甲氨蝶呤和20μM胸苷处理的LMTK-细胞中，mtDNA合成至少持续10小时，速率维持在对照速率的50%至60%，而核DNA合成受到96%的抑制。当LMTK-细胞与10μM甲氨蝶呤和20μM 5-溴脱氧尿苷孵育30小时时，会发生两条链均用5-溴尿嘧啶进行质量标记的mtDNA合成。这些结果表明，mtDNA合成对三磷酸胸苷供应的限制具有抗性，并且在这些细胞中并不严格依赖于伴随的核DNA合成。
