DNA-mediated Transfer and Cloning of a Human Multidrug-resistant Gene of Adriamycin-resistant Myelogenous Leukemia K562

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 X 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^-Adri-amycin-resistant Ltk^-and Adtiamycin-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.