TY - JOUR
T1 - pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines
AU - Martínez-Zaguilán, Raul
AU - Raghunand, Natarajan
AU - Lynch, Ronald M.
AU - Bellamy, William
AU - Martinez, Gloria M.
AU - Rojas, Bertha
AU - Smith, Douglas
AU - Dalton, William S.
AU - Gillies, Robert J.
N1 - Funding Information:
This work was supported by US Army Breast Cancer Initiative Grant DAMD17–94-J-4368.
PY - 1999/5/1
Y1 - 1999/5/1
N2 - 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 (pH(i)) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pH(i) 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 pH(i) was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pH(i) and increased the rates of pH(i) 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 pH(i) 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. Copyright (C) 1999 Elsevier Science Inc.
AB - 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 (pH(i)) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pH(i) 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 pH(i) was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pH(i) and increased the rates of pH(i) 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 pH(i) 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. Copyright (C) 1999 Elsevier Science Inc.
KW - Cancer cells
KW - H-ATPase
KW - Intracellular pH
KW - MCF-7
KW - MDR
KW - P-glycoprotein
KW - SNARF-1
KW - Vacuolar pH
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U2 - 10.1016/S0006-2952(99)00022-2
DO - 10.1016/S0006-2952(99)00022-2
M3 - Article
C2 - 10796074
AN - SCOPUS:0345040164
SN - 0006-2952
VL - 57
SP - 1037
EP - 1046
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 9
ER -