Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists.
OBJECTIVE
To investigate the retrovirus-mediated transfer and expression of multidrug resistance gene (mdr1) in hematopoietic cells and to develop a model for studying the possible reversal of the MDR-mediated phenotype.


METHODS
A retroviral vector HaMDR expressing the human mdr1 gene was packaged by PA317 cells with a titer of up to 8.5 x 10(5) CFU/ml. K562 leukemia cells were infected with MDR retrovirus, and transfectant K562/MDR cells were generated. The integration and expression of the exogenous mdr1 gene in K562/MDR cells were determined by polymerase chain reaction and flow cytometry. The reversal ability of P-glycoprotein (P-gp) antagonists was analyzed by in vitro drug sensitivity, accumulation and efflux of rhodamine 123 (Rh123) in this model.


RESULTS
Transduction with amphotropic MDR retrovirus resulted in integration and expression of the mdr1 gene in the resistant cells, where an aberrant splicing transcript of the mdr1 gene was found. The K562/MDR cells displayed a classic MDR phenotype with a 41-78 fold resistance to vincristine and colchicine in comparison with parental K562 cells. The drug sensitivity of K562/MDR cells to vincristine can be completely restored by cyclosporin A (CsA, 2 mg/L) and Cremophor EL (CRE 132 mg/L), either individually or in combination (P < 0.05). CsA (3 mg/L) can block the efflux pump function of P-gp shown by the significantly increased accumulation and efflux reduction of Rh123 in K562/MDR cells.


CONCLUSIONS
Retroviral vector HaMDR allows transfection with high-level expression of the mdr1 gene in human myeloid progenitor cells K562. The transfected K562/MDR provides a simple, sensitive model for developing antagonists of P-gp and studying their mechanism of action.
Fu
J
J
Jiangsu Institute of Hematology, First Affiliated Hospital, Suzhou Medical College, Suzhou 215006, China.
Chen
Z
Z
Cen
J
J
Ruan
C
C
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors*; D016572:Cyclosporine / Q000494:pharmacology; D019450:Genes, MDR; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D020112:Rhodamine 123 / Q000378:metabolism; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020112:Rhodamine 123; D014750:Vincristine; D016572:Cyclosporine