Are altered pHi 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 protein (P-glycoprotein) has been associated with intracellular alkalinization, 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 alter cells in many ways. Thus it is not clear which phenotypic alterations are explicitly due to MDR protein overexpression alone. To more precisely define the MDR phenotype mediated by hu MDR 1 protein, we co-transfected hu MDR 1 cDNA and a neomycin resistance marker into LR73 Chinese hamster ovary fibroblasts and selected stable G418 (geneticin) resistant transfectants. Several clones expressing different levels of hu MDR 1 protein were isolated. Unlike previous work with hu MDR 1 transfectants, the clones were not further selected with, or maintained on, chemotherapeutic drugs. These clones were analyzed for chemotherapeutic drug resistance, intracellular pH (pHi), membrane electrical potential (Vm), and stability of MDR 1 protein overexpression. LR73/hu MDR 1 clones exhibit elevated pHi and are depolarized, consistent with 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 that is expressed. Cytotoxicity experiments with untransfected LR73 cells with elevated pHi due to manipulating percent CO2 show that the pHi perturbations in the MDR 1 clones can account for much of the measured drug resistance. Membrane depolarization in the absence of MDR protein expression is also found to confer mild drug resistance, and we find that the pHi and Vm changes can conceivably account for the altered drug accumulation measured for representative clones. These data indicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in Vm and pHi that accompany this overexpression. In addition, MDR mediated by MDR protein overexpression alone differs significantly from that observed for MDR cell lines expressing similar levels of MDR protein but also exposed to chemotherapeutic drugs.
Program in Molecular Pharmacology and Therapeutics, Raymond and Beverly Sackler Foundation Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
M M
MM
Hoffman
L Y
LY
Wei
P D
PD
Roepe
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D018432:Drug Resistance, Multiple; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D001639:Bicarbonates; D006859:Hydrogen