Stegmann A P, Honders W H, Willemze R, Ruiz van Haperen V W, Landegent J E
Department of Hematology, University Hospital Leiden, The Netherlands.
Blood. 1995 Mar 1;85(5):1188-94.
The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro-induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1-ADCK) that were stably transfected and monitored over a period of 14 weeks. Northern and Western blot analysis showed restoration of dck mRNA and protein expression. Initial rate measurements of DCK activity showed that Km values for dck were only slightly altered as a result of transfection, whereas strongly increased Vmax values were observed, resulting in a 12-fold increased phosphorylation efficiency for both dC and AraC, compared with the AraC-sensitive parental cell line RO/1 from which the RO/1-A was originally derived. In vitro sensitivity to AraC- and DAC-mediated cytotoxicity was fully restored in RO/1-ADCK. The data pinpoint acquired DCK deficiency caused by mutations of the dck gene as the major cause of AraC resistance in this model.
抗阿糖胞苷（AraC）的大鼠白血病细胞系RO/1-A已被证明具有典型的脱氧胞苷激酶（DCK）缺陷表型，且无法代谢抗白血病药物阿糖胞苷（AraC）和地西他滨（DAC）。为了研究dck基因突变对体外诱导的AraC耐药性形成的相对贡献，将携带野生型dck编码区的新霉素可选择质粒构建体转染到RO/1-A中。聚合酶链反应分析证实了在稳定转染并持续监测14周的靶细胞（RO/1-ADCK）中存在载体DNA。Northern和Western印迹分析显示dck mRNA和蛋白表达得以恢复。DCK活性的初始速率测量表明，转染后dck的Km值仅略有改变，而Vmax值显著增加，导致与最初衍生出RO/1-A的AraC敏感亲本细胞系RO/1相比，dC和AraC的磷酸化效率提高了12倍。RO/1-ADCK对AraC和DAC介导的细胞毒性体外敏感性完全恢复。数据表明，在该模型中，dck基因突变导致的获得性DCK缺陷是AraC耐药的主要原因。
