DNA-mediated transfer and cloning of a human multidrug-resistant gene of adriamycin-resistant myelogenous leukemia K562.
DNA-mediated transfer and cloning of a human multidrug-resistant gene of adriamycin-resistant myelogenous leukemia K562.
复制标题
DOI：
--
复制
发表时间：
1987-05
期刊：
影响因子：
11.2
通讯作者：
Y. Sugimoto;T. Tsuruo
中科院分区：
文献类型：
--
作者：
Y. Sugimoto;T. Tsuruo
我们利用DNA介导的基因转移技术成功地转移并克隆了一个人多药耐药基因片段。将人多药耐药K562细胞的大分子DNA转染到药物敏感的小鼠Ltk-细胞中，以获得具有人耐药基因的耐药转染子。原代和二代转染子对阿霉素和长春新碱的交叉耐药模式相似。转染子的耐药机制归因于药物滞留减少。获得的三个二级转染子独立地含有15、6.5、3.7、2.6和1.9个内切酶长的共同的含EcoRI片段。从由第二转染子的DNA制备的λ噬菌体基因组文库中克隆2.6-内切酶EcoRI片段。在原代和二代转染子中检测到2.6-腺苷酸酶片段，但在亲本Ltk-、阿霉素抗性Ltk-和阿霉素抗性P388细胞中未检测到。发现该序列在几种多药耐药细胞系如阿霉素耐药卵巢癌A2780和秋水仙素耐药KB癌细胞中扩增。2.6-腺苷酸酶片段与4.5-腺苷酸酶mRNA杂交，4.5-腺苷酸酶mRNA在阿霉素抗性K562细胞和阿霉素抗性A2780细胞中过表达，但在亲本K562细胞中未检测到。从mRNA的大小及其在某些P-糖蛋白高表达的多药耐药细胞系中的过表达情况判断，本研究中转移和克隆的基因似乎与P-糖蛋白基因有关。
We have successfully transferred and cloned a fragment of a human multidrug-resistant gene by using DNA-mediated gene transfer. Macromolecular DNA of human multidrug-resistant K562 cells was transfected to drug-sensitive mouse Ltk- cells to obtain a drug-resistant transfectant with a human resistant gene. Both primary and secondary transfectants showed similar patterns of cross-resistance to Adriamycin and vincristine. The mechanism of drug resistance of the transfectants was attributed to decreased retention of the drug. Three secondary transfectants obtained independently contained common Alu-containing EcoRI fragments 15, 6.5, 3.7, 2.6, and 1.9 kilobases long. The 2.6-kilobase EcoRI fragment was cloned from a lambda phage genomic library made from DNA of a secondary transfectant. The 2.6-kilobase fragment was detected in the primary and secondary transfectants but not in the parental Ltk-, Adriamycin-resistant Ltk-, and Adriamycin-resistant P388 cells. This sequence was found to be amplified in several multidrug-resistant cell lines such as Adriamycin-resistant ovarian carcinoma A2780 and colchicine-resistant KB carcinoma cells. The 2.6-kilobase fragment hybridized with a 4.5-kilobase mRNA which is overexpressed in the Adriamycin-resistant K562 cells and the Adriamycin-resistant A2780 cells but not detected in the parental K562 cells. The gene transferred and cloned in this study seems to be related to the P-glycoprotein gene as judged from the size of mRNA and its overexpression in some of the multidrug-resistant cell lines where P-glycoprotein was found to be highly expressed.