ISOLATION AND CHARACTERIZATION OF DAUNORUBICIN-RESISTANT AML-2 SUBLINES

An attempt was made to isolate resistant sublines of acute myelogenous leukemia (OCI/AML-2) cells by chronic exposure to gradually increasin g concentrations of daunorubicin in order to determine the mechanism o f its resistance to this drug, Four daunorubicin-resistant sublines, A ML-2/D100, /D250, /D500, and /D1,000 were isolated. The values of rela tive resistance of each daunorubicin-resistant AML subline were about 3, 6, 18, and 23-fold, respectively, as compared to the AML-2 line wit h an IC50 of 5 nM. The daunorubicin-resistant AML-2 sublines also show ed cross resistance to various anticancer drugs including another anth racycline doxorubicin, a Vinca alkaloid vincristine, and an epipodophy llotoxin etoposide. A functional assay using flow cytometry showed dec reased accumulation of daunorubicin in these sublines as compared to t hat of AML-2, which was reversed by cyclosporin A or cyanide. The deve lopment of the ATP-dependent multidrug resistant phenotype was due to low to high levels of expression of P-glycoprotein (PGP). The major me chanism of increased PGP appears to be associated with gene amplificat ion. In addition, other mechanisms such as increased stability of prot ein or mRNA might be involved depending on the concentration of daunor ubicin used for selection, However, a multidrug resistance-associated protein (MRP) was not involved in these resistant sublines. These daun orubicin-resistant AML-2 sublines could provide a useful model for the study of multidrug resistance mediated by PGP.