Quantitative characterization of P-glycoprotein-mediated transport in mdr1-gene-transfected lymphoma cells.
We have established a quantitative flow cytometry system to elucidate the causal role of P-glycoprotein in the phenomenon of multidrug resistance. We have used this method to analyze the accumulation and release of adriamycin (ADM) in intact L5178Y and L5178Y/VMDR/C.06 (L5178Y/R) cells, by determining the effect of sodium orthovanadate (Na3VO4), verapamil, bovine serum albumin (BSA) and physiologically operative materials on the cells. Based on the experiments, we prepared a standard solution that contained NaCl, D-glucose, L-cysteine, HCO3- and BSA, which was sufficient to perform transport experiments. In particular, BSA caused a decrease in ADM accumulation and a facilitation of the rate of ADM release in both L5178Y and L5178Y/R cells, probably due to its relatively high affinity for ADM as compared to the cell membrane. In multidrug-resistant L5178Y/R cells, sodium orthovanadate, a strong ATP-binding inhibitor, caused a marked increase in the accumulation of ADM, whereas vanadate-treated drug-sensitive L5178Y cells showed little increase in ADM accumulation. In a release (0-trans exit) experiment, vanadate-treated L5178Y/R cells exhibited an apparent decrease in ADM release (increase in ADM retention), to a level which was almost the same as L5178Y cells. We thus confirmed that the P-glycoprotein-mediated efflux system is coupled with P-glycoprotein-associated ATP-hydrolysis. Further, verapamil, a potent inhibitor of P-glycoprotein-mediated transport, facilitated the ADM accumulation in L5178Y/R cells up to the level of L5178Y and vanadate-treated L5178Y/R cells. A more important finding is that, in the release experiment, verapamil-treated L5178Y/R cells exhibited a much greater ADM retention than drug-sensitive L5178Y and vanadate-treated L5178Y/R cells. These findings, in particular the potent effect of verapamil on drug-resistant cells, may afford new insight into the pathophysiology of the phenomenon of multidrug resistance and the mechanism of action of the multidrug transporter.
Oonishi
T
T
Medical Information Institute, Kanto Teishin Hospital, Tokyo, Japan.
Sakashita
K
K
Suematsu
N
N
Schechter
A N
AN
Uyesaka
N
N
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005434:Flow Cytometry; D006801:Humans; D008223:Lymphoma / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014700:Verapamil