Membrane potential differences between adriamycin-sensitive and -resistant cells as measured by flow cytometry.
Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3], we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K+), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positively-charged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux "pump" system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR "pump." How, or whether, plasma membrane potentials and the MDR efflux "pump" are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here.
Hasmann
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Max-Planck Institut fur Biochemie, Mildred-Scheel-Labor fur Krebszellforschung, Martinsried, F.R.G.
Valet
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GK
Tapiero
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Trevorrow
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Lampidis
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D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance; D005434:Flow Cytometry; D007211:Indoles / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D011188:Potassium / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D002232:Carbocyanines; D007211:Indoles; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D004317:Doxorubicin; D014700:Verapamil; C048960:indo-1; D011188:Potassium