pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.
A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.
Martínez-Zaguilán
R
R
Department of Biochemistry, University of Arizona Health Sciences Center, Tucson 85724-5042, USA.
Raghunand
N
N
Lynch
R M
RM
Bellamy
W
W
Martinez
G M
GM
Rojas
B
B
Smith
D
D
Dalton
W S
WS
Gillies
R J
RJ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001578:Benzopyrans; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D002451:Cell Compartmentation; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D011992:Endosomes / Q000502:physiology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009284:Naphthols / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D014407:Tumor Cells, Cultured; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology
D000970:Antineoplastic Agents; D001578:Benzopyrans; D001639:Bicarbonates; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C091666:seminaphthorhodaminefluoride; D004317:Doxorubicin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases