ARE ALTERED PH(I) AND MEMBRANE-POTENTIAL IN HU MDR-1 TRANSFECTANTS SUFFICIENT TO CAUSE MDR PROTEIN-MEDIATED MULTIDRUG-RESISTANCE

Multidrug resistance (MDR) mediated by overexpression of the MDR prote in (P-glycoprotein) has been associated with intracellular alkalinizat ion, membrane depolarization, and other cellular alterations. However, virtually all MDR cell lines studied in detail have been created via protocols that involve growth on chemotherapeutic drugs, which can alt er cells in many ways. Thus it is not clear which phenotypic alteratio ns are explicitly due to MDR protein overexpression alone. To more pre cisely define the MDR phenotype mediated by hu MDR 1 protein, we co-tr ansfected hu MDR 1 cDNA and a neomycin resistance marker Into LR73 Chi nese hamster ovary fibroblasts and selected stable G418 (geneticin) re sistant transfectants. Several clones expressing different levels of h u MDR 1 protein were isolated. Unlike previous work with hu MDR 1 tran sfectants, the clones were not further selected with, or maintained on , chemotherapeutic drugs. These clones were analyzed for chemotherapeu tic drug resistance, intracellular pH (pH(i)), membrane electrical pot ential (V-m), and stability of MDR 1 protein overexpression. LR73/hu M DR 1 clones exhibit elevated pH(i) and are depolarized, consistent wit h previous work with LR73/mu MDR 1 transfectants (Luz, J.G. L.Y. Wei, S. Basu, and P.D, Roepe. 1994. Biochemistry. 33:7239-7249): The extent of these perturbations is related to the level of hu MDR 1 protein th at is expressed. Cytotoxicity experiments with untransfected LR73 cell s with elevated pH; due to manipulating percent CO, show that the pH(i ) perturbations in the MDR 1 clones can account for much of the measur ed drug resistance, Membrane depolarization in the absence of MDR prot ein expression is also found to confer mild drug resistance, and we fi nd that the pH(i) and V-m changes can conceivably account for the alte red drug accumulation measured for representative clones. These data i ndicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in V -m and pH(i) that accompany this overexpression. In addition, MDR medi ated by MDR protein overexpression alone differs significantly from th at observed for MDR cell lines expressing similar levels of MDR protei n but also exposed to chemotherapeutic drugs.