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 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.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003078:Colchicine / Q000627:therapeutic use; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D004317:Doxorubicin / Q000627:therapeutic use*; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D007941:Leukemia P388 / Q000188:drug therapy / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000188:drug therapy; D014162:Transfection; D014750:Vincristine / Q000627:therapeutic use
D014750:Vincristine; D004317:Doxorubicin; D004247:DNA; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D003078:Colchicine