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Functional Modulation of ATPase of P-Glycoprotein by C219, a Monoclonal Antibody against P-Glycoprotein
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
230, 398–401 (1997)
RC965970
Functional Modulation of ATPase of P-Glycoprotein by C219, a Monoclonal Antibody against P-Glycoprotein Noriko Kokubu,* Dalia Cohen,† and Toru Watanabe*,1 *Oncology/Hematology Unit, Pharmacology Department, Sandoz Tsukuba Research Institute, Sandoz Pharmaceuticals, Ltd., Tsukuba-shi, Ibaraki 300-26, Japan; and †Oncology Research Group, Preclinical Research, Sandoz Research Institute, Sandoz Pharmaceutical Corporation, East Hanover, New Jersey 07936
Received November 29, 1996
P-glycoprotein functions as an ATP-driven efflux pump for antitumor agents. C219 is a monoclonal antibody which recognizes regions near both ATP binding domains in each half of P-glycoprotein. In this study, we have demonstrated that C219 inhibits the ATPase activity of P-glycoprotein based on the following findings: 1) the inhibition of total ATPase activity by C219 was selective to P-glycoprotein-positive membranes; 2) the C219-sensitive fraction of ATPase correlated the expression of P-glycoprotein; and 3) modulators of P-glycoprotein ATPase, verapamil and cyclosporin A, affected the C219-sensitive fraction of ATPase. The photolabeling of P-glycoprotein with 8-azido-[a-32P]ATP was inhibited by C219, suggesting that the inhibition of ATP binding by C219 reduced the activity. Since C219 interacts with P-glycoprotein ATPase, C219 might become a useful tool for studying the role of P-glycoprotein ATPase. q 1997 Academic Press
P-glycoprotein (P-gp) is a membrane ATPase that serves as efflux pump for multiple anticancer agents (1, 2). This protein has 12 transmembrane domains contained in two homologous halves and two ATP-binding cassette domains in each half that catalyze ATP hydrolysis. Its primary sequence and structure are highly conserved to members of the superfamily of ATP-binding cassette transmembrane transporters (1, 2). Monoclonal antibodies recognizing defined regions of P-gp could be useful for investigating conformational changes caused by the interaction of molecules with Pgp. Recently, Georges et al. determined the binding properties of an anti-P-gp monoclonal antibody C219 (3). C219 binds to regions near the ATP-binding cassette in the C- and N-terminal halves. The property of C219 binding suggests this monoclonal antibody is a useful tool for studying the interaction of molecules 1 Corresponding author. Fax: /81 298 65 2385. Abbreviation: P-gp, P-glycoprotein.
with P-gp ATP binding domains. Recently it was reported that C219 inhibits photolabeling of P-gp with 8azido-[a-32P]ATP and [3H]azidopine (4), and that cyclosporin A modulates the C219-P-gp binding (5). To further characterize the molecular interaction of C219 with P-gp, we examined the effect of C219 binding to the defined regions of the P-gp on its ATPase activity. MATERIALS AND METHODS Tumor cells. Human breast adenocarcinoma MCF7 (MCF7/WT) and its adriamycin-resistant subline (MCF7/ADR) were kindly supplied by Dr. Cowan (National Cancer Institute, NIH). The two sublines of MCF7 were maintained in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum in a 5% CO2/95% air atmosphere at 377C. The MCF7/ADR cells were maintained in the growth medium in the presence of 10 mM adriamycin. Preparation of plasma membrane-enriched fraction. Cells (3 1 109) of the sublines of MCF7 were detached by incubation with phosphate buffered saline containing 20 mM EDTA. The cell suspension was centrifuged and resultant pellet was resuspended in homogenate buffer (10 mM Tris-HCl (pH 8.0), 75 mM sucrose, 25 mM MgCl2 , 1.5 mM EDTA, 150 mM NaCl, 150 mM KCl, 5 mM DTT). The homogenate was disrupted by sonication for 30 s at level 2 (20% of maximum power in Sonifer 450 (Branson)), and unbroken cells and nuclei were removed by centrifugation. The supernatant was laid onto a discontinuous sucrose gradient consisting of 16%, 31% and 45% sucrose. Centrifugation was carried out using an SW41Ti rotor (Beckman) for 18 h at 76900 1 g, 47C. The opaque band at the 16/31% interface was collected, and then diluted with STM buffer (10 mM Tris-HCl (pH 7.5), 250 mM sucrose, 1.5 mM MgCl2 , 20 mg/ml aprotinin and 1 mM PMSF). The fraction was sedimented and the resultant pellet was resuspended in STM buffer. The protein concentration was determined by the Lowry method with bovine serum albumin as a standard. The expression levels of P-gp in the membrane fractions from MCF7/ADR and MCF7/WT cells were determined by immunoblotting using JSB-1 as a probe as previously described (6). Measurement of ATPase activity. The ATPase activity was estimated by a coupled enzyme assay as described previously (7). The membrane vesicles were diluted with distilled water and incubated for 30 min on ice to disrupt the vesicles osmotically. The disrupted membranes were directly subjected to the ATPase assay, in which the Na//K/-ATPase activity was inhibited by ouabain, the Ca2/ATPase was inhibited by EGTA, and the F1-F0-ATPase activity was
398
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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12-26-96 15:42:01
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Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
ATPase Activities of Membrane Vesicles from MCF7/WT and MCF7/ADR ATPase activity (nmol/min/mg protein)a
ATPase activity / NaN3 (10 mM), ouabain (2 mM) and EGTA (4 mM) /C219 60 mg/ml C219-sensitive ATPase a
MCF7/WT
MCF7/ADR
22.1 { 1.0 (n Å 3) 21.6 { 2.0 (n Å 3) 0.87 { 0.52 (n Å 3)
72.3 { 3.6 (n Å 6) 35.4 { 3.2 (n Å 6) 35.5 { 4.7 (n Å 6)
Values are expressed as the mean { S.E.M. of indicated number of membrane preparations.
inhibited by NaN3 . One hundred mg of the membrane protein was added into buffer A (10 mM Tris-HCl (pH 8.0), 2 mM MgCl2 , 100 mM NaCl, 10 mM KCl and 1 mM DTT) supplemented with 5 mM Mg2//ATP, 4 mM EGTA, 2 mM ouabain, 10 mM NaN3 , 0.1 mg/ml pyruvate kinase, 1 mM phosphoenolpyruvate, 0.1 mg/ml lactate dehydrogenase and 1 mM NADH. The absorbance at 340 nm was followed and the degradation of NADH was determined by linear regression. Photoaffinity labeling and immunoprecipitation. Two hundred mg of the membrane were incubated in distilled water for 30 min, and then resuspended in reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NH4Cl, 2 mM MgCl2 , 2 mM PMSF, 2 mM leupeptin, 3 mM pepstatin and 50 mM DTT) and preincubated for 1 h at 47C in the presence of either non-specific mouse IgG2a or C219. 8-Azido-[a-32P]ATP (Amersham, 370 GBq/mmol) was added (final concentration of 20 mM). After incubation for 5 min, the mixtures were irradiated for 10 min at 47C with a UV lamp (254 nm, R-52G, UVP). The photolabeled membranes were immunoprecipitated with MRK16, and analyzed using 7.5% SDS-PAGE and detected by autoradiography.
1, FIG. 2A). The different activity between the C219sensitive fractions of MCF7/ADR and MCF7/WT was consistent with the difference in P-gp level detected by immunoblotting (FIG. 2B). From these results, it was suggested that the effect of C219 on total ATPase activity was due to inhibition of P-gp ATPase by blocking the binding of ATP to P-gp. Modulation of the C219-Sensitive ATPase by Verapamil and the Cyclosporin A To confirm that the ATPase activity of the C219sensitive fraction was produced by P-gp, the effects of
RESULTS Effect of C219 on the ATPase Activity of the Membrane Fraction Prepared from MCF7 The plasma membrane-enriched fraction contains not only P-gp but also other ATPases (e.g., F1-F0ATPase, Na//K/-ATPase and Ca2/-ATPase). To inhibit F1-F0-ATPase, Na//K/-ATPase and Ca2/-ATPase, the inhibitors NaN3 , ouabain and EGTA were added to the incubation mixture. In the presence of 10 mM NaN3 , 4 mM EGTA and 2 mM ouabain, the total ATPase activity of the MCF7/ADR membranes was 72.3 { 3.6 nmol/ min/mg protein, which was 3.3-fold higher than the ATPase actually found in P-gp-deficient MCF7/WT membranes (TABLE 1). Incubation of the membranes with increasing concentration of C219 demonstrated that C219 caused a dose-dependent inhibition of the ATPase and a steady-state inhibition at more than 60 mg/ml C219 (FIG. 1A). Evidence that C219 interacts with P-gp was observed by the inhibition of the photoaffinity labeling of P-gp with 8-azido-[a-32P]ATP by C219 (FIG. 1B). In contrast, C219 at 60 mg/ml reduced minimally the activity of MCF7/WT membranes (TABLE 1). The ATPase activity of the C219-sensitive fraction from MCF7/ADR and MCF7/WT was respectively 35.5 { 4.7 and 0.87 { 0.52 nmol/min/mg protein (TABLE
FIG. 1. Concentration-dependent inhibition of ATPase activity and 8-azido-[a-32P]ATP binding by C219 in membrane fractions from MCF7/ADR. (A) The effect of C219 on the ATPase activity in membrane fractions from MCF7/ADR. The data represent the mean { S.E.M. of three membrane preparations. (B) Autoradiogram of photolabeled P-Gp with 20 mM 8-azido-[a-32P]-ATP.
399
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6916$$$702
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modulators of P-gp ATPase, verapamil and cyclosporin A, were examined. Verapamil stimulated ATPase activity of the C219-sensitive fraction in the MCF7/ADR. The maximum stimulation by verapamil was approximately 2-fold at 60 mM (FIG. 3A). In contrast, verapamil did not stimulate the ATPase activity in the MCF7/WT (data not shown). Cyclosporin A inhibited the 60 mM verapamil-stimulated C219-sensitive fraction of ATPase activity in the MCF7/ADR in a concentration-dependent manner (FIG. 3B). The concentration of cyclosporin A required to inhibit the ATPase activity by 50% was 2.36 { 0.99 mM. DISCUSSION Previously (4), it was found that the binding of C219 to regions near both of the ATP binding cassettes of P-gp perturbed the binding of 8-azido-[a-32P]ATP and [3H]azidopine, suggesting functional modulation of Pgp by C219. The observation of this report indicated that C219 inhibited the ATPase activity of P-gp; 1) the inhibition of total ATPase activity by C219 was selective to P-gp-positive membranes (TABLE 1 and FIG. 2A), 2) the C219-sensitive fraction ATPase correlated the expression of P-gp (FIG. 2A and B), and 3) modulators of P-gp ATPase, verapamil and cyclosporin A, modulated the C219-sensitive ATPase. The inhibition of Pgp ATPase by C219 is suggested to result from the
FIG. 2. C219-sensitive ATPase activity and P-Gp expression in MCF7/WT and MCF7/ADR. (A) The ATPase activity of the 60 mg/ml C219-sensitive plasma membrane fraction of MCF7/ADR and MCF7/ WT is given as mean { S.E.M. of at least three independent preparations (data from Table 1). (B) After the fractionation of the plasma membrane by SDS-PAGE and transfer to imobiron membrane, P-Gp was probed by 1 mg/ml JSB-1. The arrow indicates the immunoreactive P-Gp.
FIG. 3. Modulation of the C219-sensitive ATPase activity by verapamil and cyclosporin A. (A) The concentration-dependent stimulation by verapamil of 60 mg/ml C219-sensitive ATPase activity in the membrane fraction from MCF7/ADR. (B) The effect of cyclosporin A on the MCF7/ADR membrane fraction containing C219-sensitive ATPase activity activated by 60 mM verapamil. All data represent the mean { S.E.M. of three membrane preparations.
inhibition of ATP binding to P-gp by C219, which is indicated by the previous (4) and present studies. It remains unclear how C219 blocks the 8-azido-[a-32P]ATP binding to P-gp. It is speculated that steric hindrance by the C219 binding might result in limiting the amount of ATP able to access the ATP-binding pockets. Alternatively, the C219 binding may cause some conformational change in P-gp that could induce allosteric inhibition of the ATP binding. In a number of studies it has been assumed that the vanadate-sensitive ATPase in the presence of EGTA, NaN3 and ouabain was due to P-gp (10). Sarkadi reported that 100 mM vanadate inhibited the ATPase activity by approximately 30% in membrane vesicles from P-gp-negative cells even in the presence of EGTA, NaN3 and ouabain (11). This suggests that the inhibition of ATPase by vanadate even in the presence of the inhibitors was not specific to P-gp ATPase. In the present study, C219 was demonstrated to be a specific inhibitor of P-gp ATPase, since C219 minimally reduced the ATPase activity of P-gp-negative membranes. MRK16 is reported to be another antibody able to modulate the functions of P-gp. Hamada and Tsuruo (8, 9) reported that MRK16 modulates the transport of anticancer agents by P-gp but did not modulate P-gp ATPase. Perturbation of P-gp functions by MRK16 and C219 is a useful tool for studying P-gp functions. Recently (5), cyclosporin A was shown to increase the binding of C219 to P-gp, which was detected by using a surface plasmon resonance method, suggesting that binding of C219 to P-gp is a useful indicator for studying molecular interactions with P-gp. REFERENCES 1. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137– 171.
400
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6916$$$702
12-26-96 15:42:01
bbrcg
AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385–427. 3. Georges, E., Bradley, G., Gariepy, J., and Ling, V. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 152–156. 4. Georges, E., Zhang, J. T., and Ling, V. (1991) J. Cell. Physiol. 148, 479–484. 5. Demeule, M., Vachon, V., Delisle, M.-C., Beaulieu, E., AverillBates, D., Murphy, G. F., and Beliveau, R. (1995) Anal. Biochem. 230, 239–247.
6. Dong, J., Naito, M., Tatsuta, T., Seimiya, H., Johdo, O., Tsuruo, T. (1995) Oncol. Res. 7, 245–252. 7. Garrigos, M., Belehradek, Jr., J., Mir, L. M., and Orlowski, S. (1993) Biochem. Biophys. Res. Commun. 196, 1034–1041. 8. Hamada, H., and Tsuruo, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7785–7789. 9. Hamada, H., and Tsuruo, T. (1988) Cancer Res. 48, 4926–4932. 10. Scarborough, G. A. (1995) J. Bioener. Biomem. 27, 37–41. 11. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992) J. Biol. Chem. 267, 4854–4858.
401
AID
BBRC 5970
/
6916$$$702
12-26-96 15:42:01
bbrcg
AP: BBRC
230, 398–401 (1997)
RC965970
Functional Modulation of ATPase of P-Glycoprotein by C219, a Monoclonal Antibody against P-Glycoprotein Noriko Kokubu,* Dalia Cohen,† and Toru Watanabe*,1 *Oncology/Hematology Unit, Pharmacology Department, Sandoz Tsukuba Research Institute, Sandoz Pharmaceuticals, Ltd., Tsukuba-shi, Ibaraki 300-26, Japan; and †Oncology Research Group, Preclinical Research, Sandoz Research Institute, Sandoz Pharmaceutical Corporation, East Hanover, New Jersey 07936
Received November 29, 1996
P-glycoprotein functions as an ATP-driven efflux pump for antitumor agents. C219 is a monoclonal antibody which recognizes regions near both ATP binding domains in each half of P-glycoprotein. In this study, we have demonstrated that C219 inhibits the ATPase activity of P-glycoprotein based on the following findings: 1) the inhibition of total ATPase activity by C219 was selective to P-glycoprotein-positive membranes; 2) the C219-sensitive fraction of ATPase correlated the expression of P-glycoprotein; and 3) modulators of P-glycoprotein ATPase, verapamil and cyclosporin A, affected the C219-sensitive fraction of ATPase. The photolabeling of P-glycoprotein with 8-azido-[a-32P]ATP was inhibited by C219, suggesting that the inhibition of ATP binding by C219 reduced the activity. Since C219 interacts with P-glycoprotein ATPase, C219 might become a useful tool for studying the role of P-glycoprotein ATPase. q 1997 Academic Press
P-glycoprotein (P-gp) is a membrane ATPase that serves as efflux pump for multiple anticancer agents (1, 2). This protein has 12 transmembrane domains contained in two homologous halves and two ATP-binding cassette domains in each half that catalyze ATP hydrolysis. Its primary sequence and structure are highly conserved to members of the superfamily of ATP-binding cassette transmembrane transporters (1, 2). Monoclonal antibodies recognizing defined regions of P-gp could be useful for investigating conformational changes caused by the interaction of molecules with Pgp. Recently, Georges et al. determined the binding properties of an anti-P-gp monoclonal antibody C219 (3). C219 binds to regions near the ATP-binding cassette in the C- and N-terminal halves. The property of C219 binding suggests this monoclonal antibody is a useful tool for studying the interaction of molecules 1 Corresponding author. Fax: /81 298 65 2385. Abbreviation: P-gp, P-glycoprotein.
with P-gp ATP binding domains. Recently it was reported that C219 inhibits photolabeling of P-gp with 8azido-[a-32P]ATP and [3H]azidopine (4), and that cyclosporin A modulates the C219-P-gp binding (5). To further characterize the molecular interaction of C219 with P-gp, we examined the effect of C219 binding to the defined regions of the P-gp on its ATPase activity. MATERIALS AND METHODS Tumor cells. Human breast adenocarcinoma MCF7 (MCF7/WT) and its adriamycin-resistant subline (MCF7/ADR) were kindly supplied by Dr. Cowan (National Cancer Institute, NIH). The two sublines of MCF7 were maintained in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum in a 5% CO2/95% air atmosphere at 377C. The MCF7/ADR cells were maintained in the growth medium in the presence of 10 mM adriamycin. Preparation of plasma membrane-enriched fraction. Cells (3 1 109) of the sublines of MCF7 were detached by incubation with phosphate buffered saline containing 20 mM EDTA. The cell suspension was centrifuged and resultant pellet was resuspended in homogenate buffer (10 mM Tris-HCl (pH 8.0), 75 mM sucrose, 25 mM MgCl2 , 1.5 mM EDTA, 150 mM NaCl, 150 mM KCl, 5 mM DTT). The homogenate was disrupted by sonication for 30 s at level 2 (20% of maximum power in Sonifer 450 (Branson)), and unbroken cells and nuclei were removed by centrifugation. The supernatant was laid onto a discontinuous sucrose gradient consisting of 16%, 31% and 45% sucrose. Centrifugation was carried out using an SW41Ti rotor (Beckman) for 18 h at 76900 1 g, 47C. The opaque band at the 16/31% interface was collected, and then diluted with STM buffer (10 mM Tris-HCl (pH 7.5), 250 mM sucrose, 1.5 mM MgCl2 , 20 mg/ml aprotinin and 1 mM PMSF). The fraction was sedimented and the resultant pellet was resuspended in STM buffer. The protein concentration was determined by the Lowry method with bovine serum albumin as a standard. The expression levels of P-gp in the membrane fractions from MCF7/ADR and MCF7/WT cells were determined by immunoblotting using JSB-1 as a probe as previously described (6). Measurement of ATPase activity. The ATPase activity was estimated by a coupled enzyme assay as described previously (7). The membrane vesicles were diluted with distilled water and incubated for 30 min on ice to disrupt the vesicles osmotically. The disrupted membranes were directly subjected to the ATPase assay, in which the Na//K/-ATPase activity was inhibited by ouabain, the Ca2/ATPase was inhibited by EGTA, and the F1-F0-ATPase activity was
398
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BBRC 5970
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6916$$$701
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
ATPase Activities of Membrane Vesicles from MCF7/WT and MCF7/ADR ATPase activity (nmol/min/mg protein)a
ATPase activity / NaN3 (10 mM), ouabain (2 mM) and EGTA (4 mM) /C219 60 mg/ml C219-sensitive ATPase a
MCF7/WT
MCF7/ADR
22.1 { 1.0 (n Å 3) 21.6 { 2.0 (n Å 3) 0.87 { 0.52 (n Å 3)
72.3 { 3.6 (n Å 6) 35.4 { 3.2 (n Å 6) 35.5 { 4.7 (n Å 6)
Values are expressed as the mean { S.E.M. of indicated number of membrane preparations.
inhibited by NaN3 . One hundred mg of the membrane protein was added into buffer A (10 mM Tris-HCl (pH 8.0), 2 mM MgCl2 , 100 mM NaCl, 10 mM KCl and 1 mM DTT) supplemented with 5 mM Mg2//ATP, 4 mM EGTA, 2 mM ouabain, 10 mM NaN3 , 0.1 mg/ml pyruvate kinase, 1 mM phosphoenolpyruvate, 0.1 mg/ml lactate dehydrogenase and 1 mM NADH. The absorbance at 340 nm was followed and the degradation of NADH was determined by linear regression. Photoaffinity labeling and immunoprecipitation. Two hundred mg of the membrane were incubated in distilled water for 30 min, and then resuspended in reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NH4Cl, 2 mM MgCl2 , 2 mM PMSF, 2 mM leupeptin, 3 mM pepstatin and 50 mM DTT) and preincubated for 1 h at 47C in the presence of either non-specific mouse IgG2a or C219. 8-Azido-[a-32P]ATP (Amersham, 370 GBq/mmol) was added (final concentration of 20 mM). After incubation for 5 min, the mixtures were irradiated for 10 min at 47C with a UV lamp (254 nm, R-52G, UVP). The photolabeled membranes were immunoprecipitated with MRK16, and analyzed using 7.5% SDS-PAGE and detected by autoradiography.
1, FIG. 2A). The different activity between the C219sensitive fractions of MCF7/ADR and MCF7/WT was consistent with the difference in P-gp level detected by immunoblotting (FIG. 2B). From these results, it was suggested that the effect of C219 on total ATPase activity was due to inhibition of P-gp ATPase by blocking the binding of ATP to P-gp. Modulation of the C219-Sensitive ATPase by Verapamil and the Cyclosporin A To confirm that the ATPase activity of the C219sensitive fraction was produced by P-gp, the effects of
RESULTS Effect of C219 on the ATPase Activity of the Membrane Fraction Prepared from MCF7 The plasma membrane-enriched fraction contains not only P-gp but also other ATPases (e.g., F1-F0ATPase, Na//K/-ATPase and Ca2/-ATPase). To inhibit F1-F0-ATPase, Na//K/-ATPase and Ca2/-ATPase, the inhibitors NaN3 , ouabain and EGTA were added to the incubation mixture. In the presence of 10 mM NaN3 , 4 mM EGTA and 2 mM ouabain, the total ATPase activity of the MCF7/ADR membranes was 72.3 { 3.6 nmol/ min/mg protein, which was 3.3-fold higher than the ATPase actually found in P-gp-deficient MCF7/WT membranes (TABLE 1). Incubation of the membranes with increasing concentration of C219 demonstrated that C219 caused a dose-dependent inhibition of the ATPase and a steady-state inhibition at more than 60 mg/ml C219 (FIG. 1A). Evidence that C219 interacts with P-gp was observed by the inhibition of the photoaffinity labeling of P-gp with 8-azido-[a-32P]ATP by C219 (FIG. 1B). In contrast, C219 at 60 mg/ml reduced minimally the activity of MCF7/WT membranes (TABLE 1). The ATPase activity of the C219-sensitive fraction from MCF7/ADR and MCF7/WT was respectively 35.5 { 4.7 and 0.87 { 0.52 nmol/min/mg protein (TABLE
FIG. 1. Concentration-dependent inhibition of ATPase activity and 8-azido-[a-32P]ATP binding by C219 in membrane fractions from MCF7/ADR. (A) The effect of C219 on the ATPase activity in membrane fractions from MCF7/ADR. The data represent the mean { S.E.M. of three membrane preparations. (B) Autoradiogram of photolabeled P-Gp with 20 mM 8-azido-[a-32P]-ATP.
399
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6916$$$702
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modulators of P-gp ATPase, verapamil and cyclosporin A, were examined. Verapamil stimulated ATPase activity of the C219-sensitive fraction in the MCF7/ADR. The maximum stimulation by verapamil was approximately 2-fold at 60 mM (FIG. 3A). In contrast, verapamil did not stimulate the ATPase activity in the MCF7/WT (data not shown). Cyclosporin A inhibited the 60 mM verapamil-stimulated C219-sensitive fraction of ATPase activity in the MCF7/ADR in a concentration-dependent manner (FIG. 3B). The concentration of cyclosporin A required to inhibit the ATPase activity by 50% was 2.36 { 0.99 mM. DISCUSSION Previously (4), it was found that the binding of C219 to regions near both of the ATP binding cassettes of P-gp perturbed the binding of 8-azido-[a-32P]ATP and [3H]azidopine, suggesting functional modulation of Pgp by C219. The observation of this report indicated that C219 inhibited the ATPase activity of P-gp; 1) the inhibition of total ATPase activity by C219 was selective to P-gp-positive membranes (TABLE 1 and FIG. 2A), 2) the C219-sensitive fraction ATPase correlated the expression of P-gp (FIG. 2A and B), and 3) modulators of P-gp ATPase, verapamil and cyclosporin A, modulated the C219-sensitive ATPase. The inhibition of Pgp ATPase by C219 is suggested to result from the
FIG. 2. C219-sensitive ATPase activity and P-Gp expression in MCF7/WT and MCF7/ADR. (A) The ATPase activity of the 60 mg/ml C219-sensitive plasma membrane fraction of MCF7/ADR and MCF7/ WT is given as mean { S.E.M. of at least three independent preparations (data from Table 1). (B) After the fractionation of the plasma membrane by SDS-PAGE and transfer to imobiron membrane, P-Gp was probed by 1 mg/ml JSB-1. The arrow indicates the immunoreactive P-Gp.
FIG. 3. Modulation of the C219-sensitive ATPase activity by verapamil and cyclosporin A. (A) The concentration-dependent stimulation by verapamil of 60 mg/ml C219-sensitive ATPase activity in the membrane fraction from MCF7/ADR. (B) The effect of cyclosporin A on the MCF7/ADR membrane fraction containing C219-sensitive ATPase activity activated by 60 mM verapamil. All data represent the mean { S.E.M. of three membrane preparations.
inhibition of ATP binding to P-gp by C219, which is indicated by the previous (4) and present studies. It remains unclear how C219 blocks the 8-azido-[a-32P]ATP binding to P-gp. It is speculated that steric hindrance by the C219 binding might result in limiting the amount of ATP able to access the ATP-binding pockets. Alternatively, the C219 binding may cause some conformational change in P-gp that could induce allosteric inhibition of the ATP binding. In a number of studies it has been assumed that the vanadate-sensitive ATPase in the presence of EGTA, NaN3 and ouabain was due to P-gp (10). Sarkadi reported that 100 mM vanadate inhibited the ATPase activity by approximately 30% in membrane vesicles from P-gp-negative cells even in the presence of EGTA, NaN3 and ouabain (11). This suggests that the inhibition of ATPase by vanadate even in the presence of the inhibitors was not specific to P-gp ATPase. In the present study, C219 was demonstrated to be a specific inhibitor of P-gp ATPase, since C219 minimally reduced the ATPase activity of P-gp-negative membranes. MRK16 is reported to be another antibody able to modulate the functions of P-gp. Hamada and Tsuruo (8, 9) reported that MRK16 modulates the transport of anticancer agents by P-gp but did not modulate P-gp ATPase. Perturbation of P-gp functions by MRK16 and C219 is a useful tool for studying P-gp functions. Recently (5), cyclosporin A was shown to increase the binding of C219 to P-gp, which was detected by using a surface plasmon resonance method, suggesting that binding of C219 to P-gp is a useful indicator for studying molecular interactions with P-gp. REFERENCES 1. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137– 171.
400
AID
BBRC 5970
/
6916$$$702
12-26-96 15:42:01
bbrcg
AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385–427. 3. Georges, E., Bradley, G., Gariepy, J., and Ling, V. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 152–156. 4. Georges, E., Zhang, J. T., and Ling, V. (1991) J. Cell. Physiol. 148, 479–484. 5. Demeule, M., Vachon, V., Delisle, M.-C., Beaulieu, E., AverillBates, D., Murphy, G. F., and Beliveau, R. (1995) Anal. Biochem. 230, 239–247.
6. Dong, J., Naito, M., Tatsuta, T., Seimiya, H., Johdo, O., Tsuruo, T. (1995) Oncol. Res. 7, 245–252. 7. Garrigos, M., Belehradek, Jr., J., Mir, L. M., and Orlowski, S. (1993) Biochem. Biophys. Res. Commun. 196, 1034–1041. 8. Hamada, H., and Tsuruo, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7785–7789. 9. Hamada, H., and Tsuruo, T. (1988) Cancer Res. 48, 4926–4932. 10. Scarborough, G. A. (1995) J. Bioener. Biomem. 27, 37–41. 11. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992) J. Biol. Chem. 267, 4854–4858.
401
AID
BBRC 5970
/
6916$$$702
12-26-96 15:42:01
bbrcg
AP: BBRC