Activation of silent MDR1 genes in revertant cells by fusion with multidrug-resistant cells.

We isolated revertant and resistant clones from multidrug-resistant K562/ADM cells and evaluated the expression of P-glycoprotein and the DNA copy number of MDR1. The 9 revertant clones contained 2- to 26-fold DNA copies of MDR1; however, they expressed an extensively decreased P-glycoprotein compared with K562/ADM, while the 10 multidrug-resistant clones contained 4- to 48-fold DNA copies, and the expression level of P-glycoprotein was dependent on the copy number of MDR1 DNA. The decreased expression of P-glycoprotein in the revertants was not due only to the loss of the copy number of MDR1 DNA. To elucidate the mechanism of P-glycoprotein expression decrease in the revertants, a revertant clone (R1-5) was fused with a multidrug-resistant clone (A2-1) or with a drug-sensitive clone isolated from K562. Compared with K562 clone, the A2-1 contained 32-fold MDR1 DNA copies and showed 131-fold resistance to Adriamycin. The revertant clone R1-5 contained 26-fold MDR1 DNA copies but expressed only 5% the P-glycoprotein of A2-1 cells and showed only 2-fold resistance to Adriamycin. For selection of intraspecific hybrids, a neomycin-resistant or a blasticidin S-resistant gene was introduced into clones by electroporation of pSV2neo or pSV2bsr. The introduction of these resistant genes did not alter the copy number or expression of MDR1 in the clones. Hybrid cells between R1-5bsr and A2-1neo were found to express 136 +/- 15% of the P-glycoprotein of A2-1 cells evaluated by quantitive flow cytometry. These hybrid cells contained 41- to 48-fold MDR1 copies and showed the multidrug-resistant phenotype, such as decrease of rhodamine123 accumulation and 120- to 210-fold resistance to Adriamycin (compared with K562), indicating that the 'silent' MDR1 genes in the revertant clone R1-5 were activated by cell fusion with an MDR clone. R1-5bsr x K562neo hybrids were found to contain 8- to 11-fold MDR1 copies and there was no increase in P-glycoprotein expression as compared with R1-5.