Cancer Letters, 66 (1992) 83 - 89 Elsevier Scientific Publishers Ireland Ltd.
Abnormal cells S. Gollapudi”, ‘Division
83
chloride conductance
T. McDonaldb,
P. Gardnerb,
of Basic and Clinical Immunology,
Medicine,
Falk Cardiooascular
(Received
28 April 1992)
Research
in multidrug resistant HLGO/AR
Unioersity
Center,
Stanford
N. Kanga of California,
and Irvine,
S. Gupta” California
Uniuersity School of Medicine,
92717
and bDepartment
Stanford,
California
of
(USA)
(Revision received 6 July 1992) (Accepted
8 July 1992)
Summary
Introduction
Chloride channel currents were measured in drug sensitive parental HL60 and multidrug resistant (MDR) subline HL60/AR cells, using a whole cell patch-clamp technique. In addition, the in uitro effects of 4,4’diisothiocyanatostilbene-2,2’ -disulfonic acid (DIDS), a Cl channel blocker, on intracellular accumulation and sensitivity to daunorubicin and intracellular pH [pHJ in HL60 cells were examined. Baseline DIDS blockable CI- currents were consistently lower in HL60/AR cells (0.9
The development of resistance by malignant cells to cytotoxic drugs poses a major obstacle to the chemotherapeutic cure of cancer. Acquired drug resistance occurs when drug sensitive malignant cells become resistant to antineoplastic agents after exposure to them. These malignant cells often exhibit cross resistance to a wide variety of structurally and functionally unrelated drugs. This pattern of resistance is seen in tumors that are initially responsive to chemotherapy that is followed by untreatable relapse. A large number of mechanisms have been proposed to explain this broad resistance, the multidrug resistance (MDR). These include, overexpression of Pglycoprotein, increased levels of glutathione-Stransferase, glutathione and topoisomerase II, etc. [l-4]. At the biochemical level, the role of plasma membrane potential [5,6] and protein kinase C (PKC) [7] has been suggested. The plasma membranes of all cells have transport systems that mediate the exchange of solutes between the cells and their environment. These include, ATPase or ion pumps, Cl -/HC03 - anion exchanger and ion channels that provide selective ion permeabilities in the cell membrane. The chloride channels are controlled by ligands, membrane potential, intracellular second messengers (i.e. PKC, PKA,
pA/pF) pA/pF).
as compared to HL60 cells (7.0 Similarly CAMP-activated CI- currents were minimal in HL60/AR cells (0.2 pA/pF) as compared to HL60 cells (8 pA/pF). In vitro treatment of drug sensitive HL60 cells with DIDS resulted in concentration-dependent decreased accumulation and increased resistance to daunorubicin and decreased pH,. These data show that altered Cl- permeability is associated with MDR and suggest that Clchannels may play a role in MDR.
Kegwords: multidrug resistance; pH; chloride current
intracellular
Correspondence to: S. Gupta, Medical Sciences I, Room 264A, University of California, Irvine, CA 92717, USA.
0304-3835/92/$05.00 Printed and Published
C-
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd
84
Ca + +), etc. and appear to play a role in establishing the membrane potential [8]. Because of the suggested role of plasma membrane potential, PKC and intracellular pH in MDR, we compared the properties of Cl currents, using patch-clamp technique, in MDR HL60/AR cells and parental drug sensitive HL60 cells. Our results show that baseline DIDS (Cl - channel blocker) sensitive and CAMP-activated Cl - channel currents are decreased in HL60/AR cells as compared to HL60 cells, suggesting decreased Cl - permeability in MDR cells. Furthermore, blocking of Cl - channel with DIDS in drug sensitive HL60 cells resulted in decreased intracellular drug accumulation and increased drug resistance. These data suggest a possible role of Cl - channels in MDR. Materials
and Methods
Cell lines The human promyelogenous leukemia Adriamycin sensitive parental cell line (HL60) and Adriamycin resistant (HL60/AR) subline were provided by Dr. Kapil Bhalla, Medical University of South Carolina. HL60/AR cells are cross-resistant to daunorubicin, vincristine and vinblastin. These cell lines are maintained in vitro as suspension culture. Both HL60 and HL60/AR cells lack mdr 1 [9]. Chemicals and reagents 4,4’ Diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) ,caged CAMP, 2 ’ ,7 ’ -his- (2-carboxyethyl) - 5 - (and - 6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM) and BAPTA were purchased from Molecular Probes, Eugene, OR. Daunorubicin was purchased from Sigma Chemicals, St. Louis, MO. Electrophysiological studies of HL60 and HL60/AR cells
Chloride currents of HL60 cells were studied with patch pipettes in a whole-cell voltage clamp configuration [lo]. Tip resistance of the pipettes was 2-4 MQ. The extracellular solution for electrophysiologic studies
consisted of 150 mM Tris-Cl, 2.5 mM CaQ., 1 mM MgQ, 5 mM glucose (pH 7.4). Intracellular pipette solution consisted of 140 mM CsCI, 5.5 mM BAPTA, 0.5 mM CaCl*, 4 mM ATP-Mg, 20 mM HEPES (pH 7.2). The calculated chloride current reversal potential for these solutions.was 0 mV. The osmolarity of the extracellular solution was maintained at 25 mM hypertonic relative to the intracellular solution and was monitored by a vapor pressure osmometer (Wescor; Logan, UT). The external hypertonicity was necessary to prevent volume-induced chloride currents [ll]. Studies were conducted at 29 -3OOC. The cell membrane was voltage clamped and whole-cell currents were obtained with an Axopatch amplifier (Axon Instruments; Burlingame, CA) which was controlled by an 80386-based PC and one 16-bit AD/DA convertor. Current signals were recorded on FM tape and digitized for storage on floppy diskettes. The holding membrane potential was maintained at - 50 mV and series of voltage steps from - 100 to + 100 mV were performed for construction of current voltage (I-V) curves. The current was measured as the average current during each voltage pulse and normalized to the measured cell capacitance expressed as picoamps/picofarad (pA/pF) . 4,4’ Diisothiocyanatostilbene-2,2’-disulfonit acid (DIDS) was introduced by means of a rapid superinfusion chamber resulting in an extracellular concentration of 500 PM. CAMP effects were obtained by either superinfusion of chlorophenylthiol-CAMP (cpt-CAMP, 400 PM) to the extracellular solution or by flash photolysis of 4,5-dimethoxy-2-nitrobenzyl-CAMP (Caged-CAMP). Photolysis was performed by brief (1 ms) flashes from a high pressure xenon arc lamp focused onto a 2 - 3-mm area of the cell chamber with quartz optics. Wave lengths below 305 nM were eliminated with a WG305 filter. This system resulted in a 6% release of caged-CAMP per flash as measured by HPLC. intracellular drug accumulation HL60 and HL60/AR cells were resuspen-
ded at 1
x
lo6 cells/ml in 12
x
75-mm
85
culture tubes in the presence or absence of various concentrations of DIDS. Daunorubicin (DNR, 4 ,ug/ml) was added to the cells, gently mixed, and incubated at 37*C for 45 min and DNR accumulation was analyzed by FACScan (Becton-Dickinson, San Jose, CA) at a flow rate of 500 events per second [12]. Mean fluorescence was recorded from histogram and expressed as mean fluorescence linear channel numbers. Drug sensitivity
assay
Drug sensitivity of HL60 and HL60/AR cells was done by MTT assay [13]. Cells (1 x lo6 cells/ml) were incubated with various concentrations of DNR, in the presence or absence of various concentrations of DIDS, for 1 h at 37*C. Cells were washed twice with prewarmed medium RPMI-1640, resuspended in drug free medium, distributed in triplicate ( lo5 cells/well) .into 96-well tissue culture plates and incubated for 3 days at 37*C in a humified CO:! incubator. Three hours prior to termination of the culture, 25 ~1 of MTT (5 mg/ml) was added to each well and the plates incubated at 37*C for an additional 3 h. The formazon crystals formed by the viable cells were dissolved by the addition of 100 ~1 of 1 N HC1:isopropanol (1:24) and the absorbance was measured at 570 nm, using automated microplate reader (UVMax, Molecular Devices, Menlo Park, CA). Cells not exposed to drugs were used as controls (baseline). Results are calculated as percent of the baseline absorbance, ED50 is defined as the concentration of DNR which induced the 50 % cytotoxicity . Measurement of intracellular pH Intracellular pH was measured with an intracellularly trapped and pH-sensitive dye, BCECF-AM by a technique previously described [14]. Cells (3 x 106) were loaded with 2 PM of BCECF-AM for 30 min at 37*C. Cells were washed and resuspended in fresh medium RPMI-1640 at 1 x 107/ml. Intracellular pH was measured using FACScan. Calibration of fluorescence intensity of
BCECF-AM was accomplished by measuring fluorescence intensity after equilibration of the internal and external pH with nigericin. Calibration of pHi versus fluorescence intensity was carried out with buffers of different pH values ranging from 6.5 to 7.9. Results
channel currents Baseline and DlDS sensitive whole-cell currents. Cl - currents at baseline were measured 3 - 5 min after establishing the whole-cell configuration which allowed for thorough dialysis of the cytoplasm with the pipette solution. Baseline Cl- currents were consistently lower in HL60/AR drug resistant (3.2 pA/pF at + 100 mV; n = 47) as compared to drug sensitive HL60 cells (5.2 pA/pF at + 100 mV; n = 42). Representative examples are shown in Fig. 1A. DIDS sensitive current was determined as the baseline current minus that current remaining after the addition of 500 PM DIDS. Drug resistant HLGO/AR cells had much less DIDS blockable baseline current (0.9 pA/pF at + 100 mV; n = 18) than did the drug sensitive HL60 cells (7.2 pA/pF at + 100 mV; n = 16). The DIDS sensitive current had an Erev of 0 mV consistent with a Cl - current and a current-voltage relationship that was usually outward in rectification (Fig. 1B). Chloride
CAMP-actiuated Cl- currents Currents activated by CAMP were determined by measuring maximal current after the addition of CAMP and subtracting the baseline current. The addition of cpt-CAMP or flash photolysis of caged-CAMP was performed after a stable baseline current was obtained. Drug sensitive HL60 cells responded to the addition of cpt-CAMP with an average maximal current of 8 pA/pF at + 100 mV (n = 14)) while MDR HL60/AR cells showed minimal response (0.2 pA/pF at + 100 mV; n = 11). Representative examples are shown in Fig. 2A. Flash photolysis of caged-CAMP resulted in a similar response from HL60 (9.7 pA/pF at
Baseline
DIDS
HLBO/AR
Fig. 1. DIDS blockable Cl- current tracings and I-V relation. (A), examples of whole cell Cl - currents in response to the voltage pulse protocol demonstrated in the left lower inset. Upward deflections represent chloride movement into the cell. The left upper and lower sets of tracing are baseline currents in HL60 and HMO/AR cells, respectively. Currents tracing on the right from the same cells after the addition of DIDS 500 PM to the extracellular solution. (B) , the average I-V relation of the DIDS-sensitive currents in HL60 (open circles; n = 18) and HL60/AR (closed circles; n = 16). Currents were calculated by substracting the residual post-DIDS currents from the baseline current and normalized to cell capacitance (pA/pF) .
+ 100 mV; n = 7) and HL60/AR (0.3 pA/pF at + 100 mV; n = 8). The CAMPactivated current had an Erev of 0 mV and had a milder degree of outward rectification as compared to the DIDS blockable baseline currents (Fig. ZB). Effect of DZDS on intracellular drug accumulation HL60 cells were incubated with DNR (4
pg/mI) for 1 h at 37OC in the presence or absence of various concentrations of DIDS and intracellular accumulation of DNR was measured using FACScan. Data are expressed
as mean fluorescence channel numbers (MFC). Data in Table I show that DIDS in a concentration-dependent manner reduced intracellular accumulation of DNR in HL60 cells. As expected, no significant effect of DIDS was observed on DNR accumulation in HLGO/AR cells (data not shown). Drug sensitioity
The effect of DIDS on the sensitivity of HL60 cells to DNR was assessed by MTT cytotoxicity assay. Table I shows that DIDS, in a concentration-dependent manner, increased ED/50 for DNR in drug sensitive HL60 to a
87
CAMP
Baseline
Fi2. 2. Cyclic AMP-dependent activation of Cl- current tracings and I-V relation. (A), examples of whole-cell Cl current tracings on the upper and lower left are examples of baseline currents from HL60 and HL60/AR cells, respectively. Tracing on the right show resulting currents from the same cells shown on the left after the addition of CAMP. Voltage pulse protocol is demonstrated in the left lower inset. (B), the average I-V relation of currents induced by either cpt-CAMP or caged-CAMP in HL60 (open circles; n = 21) and HL60/AR (closed circles; n = 19) cells. Current values were calculated as the current after addition of CAMP minus the baseline current and normalized to cell capacitance.
Table 1. Effect of DIDS on daunorubicin accumulation and sensitivity in HL60. DIDS (PM)
HL60/AR HL60 HL60 HL60 HL60
None None 125 250 500
MFC no.
Daunorubicin EDs.s (rM)
878 2226 1718 1725 1328
17.3 4.1 6.8 10.5 15.0
zt zt zt zt f
81 65 0 165 43
l
f f f f
5.4 0.9 1.0 2.1 2.8
HL60 cells were incubated with DNR (4c(Q/ml) in the presence or absence of DIDS. DNR accumulation was measured using FACScan. Data are presented as mean fluorescence channel number (MFC). EDss is defined as the concentration of DNR required for 50% cytotoxicity of cells as determined by MTT assay. Data represent mean f S.D. of 3 separate experiments.
level comparable to drug resistant HLGO/AR cells, indicating development of resistance in HL60 cells to DNR. No significant effect on drug sensitivity of HL60/AR cells was observed (data not shown). This would be consistant with the presence of few DIDS-sensitive Cl channels in HLGO/AR cells. These data would indicate that decreased Cl - permeability is associated with MDR. Intracellular pH The data for the effect of DIDS on pH, of HL60 cells is shown in Table II. In a dosedependent manner, DIDS reduced the pH, in HL60 cells. This would be consistent with the observation of the amplification of vacuolar
88
Table Il. In vitro effect of DIDS on intracellular pH of HL60 cells. DIDS (PM)
None 125 250 500
Intracellular
pH
Exp. 1
Exp. 2
7.82 7.57 7.38 7.17
7.73 7.40 7.30 7.16
HL60 drug-sensitive cells were incubated with various concentrations of DIDS and pH, was measured with BCECF dye, using FACScan (see Materials and Methods).
H +-ATPase activity and mRNA, that maintain an acidic intravasicular environment, in multidrug resistant HL60 cells [ 15,161. Discussion Accumulating evidence indicates that MDR is accompanied by several distinct types of cellular alterations in the cell drug transport process [l - 41. A common finding in most of these studies is that the MDR cells accumulate less drug as compared to sensitive parental cell lines. A number of mechanisms have been proposed for MDR. At the biochemical levels, increased levels of PKC and altered membrane potentials appear to play a role in MDR [6,7,17]. Recent studies of Cl- channels have shown that they regulate various cellular functions, including maintenance of plasma membrane potential [8,17]. In the present study we observed decreased DIDS-blockable baseline Cl - currents, as well as CAMP-activated Cl channel currents in MDR HLGO/AR cells as compared to drug sensitive HL60 cells. This would indicate decreased Cl - permeability in MDR cells. The homology of cystic fibrosis transmembrane regulator (CFTR) to P-glycoprotein [l&19] raised the question of whether these transport proteins might be bifunctional and by, extension, whether or not mdr 1 might encode for Cl - conductance. This issue has been directly addressed by Valverde et al. [20], who suggests that mdr 1 not only en-
codes for a drug transport protein but also the hypotonicity induced Cl - current. However, we did not observe any difference in hypotonicity Cl - currents between HL60 and HLGO/AR (data not shown). The difference in our observations from those of Valverde et al. [20] are not surprising because our multidrug resistant cell line lacks mdr 1 mRNA and Pglycoprotein. Furthermore, we did not find any difference in ionomycin-induced Cl - currents between HL60 and HL60/AR cells (data not shown). The linkage of MDR and decreased drug transport to altered Cl - permeabilities in our present study appears to be a second example of aberrant transport and altered Cl permeability mechanisms. A similar defect in Cl - permeability has been observed in cystic fibrosis [21,22]. Since the precise molecular mechanism for multidrug resistance in HLGO/AR is unknown, we cannot further speculate on the molecular basis for the linkage. But the linkage itself is intriguing. Because of decreased Cl - currents in HLGO/AR cells, we reasoned that if the Cl channel plays a role in MDR, then blocking the Cl - channels with DIDS in drug sensitive cells should render them resistant. In this study we show that in vitro treatment of drug sensitive HL60 cells with DIDS resulted, in a concentration-dependent manner, in decreased intracellular accumulation of DNR and increased resistance to DNR. Although DIDS is known to block Cl - channels, the possibility of its effect on other Cl - transport mechanisms and on proton transport cannot be excluded. Furthermore, DIDS is known to alter pHi via Cl -/HCO< transport. In this study, we show that DIDS in a dose-dependent manner, decreased pHi of HL60 drug sensitive cells. Recent evidence suggests that vacuolar H +ATPase activity, which maintains an acidic vasicular microenviroment [23], may be involved in multidrug resistance in HL60 1151. Marquardt and Center [ 151, demonstrated that bafilomycin Al, a specific inhibitor of H +ATPase, was capable of blocking efflux and inducing an increase in drug accumulation. More recently, Ma and Center [16] have demonstrated increased levels of SC-H +-ATPase
89
(a subunit C of H +-ATPase) mRNA in multidrug resistant HL60 cells. Although the precise mechanism(s) by which DIDS reduces intracellular drug accumulation and confers resistance to HL60 sensitive cells remains unclear, it is possible that DIDS might alter the activity of H +-ATPase. Additionally, the data of the Cl - current and dose-dependent effects of DIDS suggest a possible role for Cl - channels in MDR. The relationship between the Cl - channel and H +-ATPase activity remains to be evaluated. Acknowledgments
This work was supported in part by grants from USPHS Al 26 456 (SG) and DK 41324 (PG) .P. Gardner is a recipient of the Borrough Wellcome Faculty Scholar Award in Clinical Pharmacology. T. McDonald is supported by NIH grant KllHL 02 397.
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11
12
13
14
15
16
References Gottesman, M.M. and Pastan, I. (1988). Resistance to multiple chemotherapeutic agents in human cancer cells. Trends Pharmacol. Sci., 9, 54 - 58. Juranka, P.F., Zastawny, R.L. and Ling, V. (1989). Pglycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins. Fed. Am. Sot. Exp. Biol. J., 3, 2583-2592. Lazo, J.S. and Bahnson, R.R. (1989). Pharmacological modulators of DNA-interactive antitumor drugs. Trends Pharmacol. Sci., 10, 369- 373. Moscow, LA. and Cowan, K.H. (1989). Multidrug resistance. J. Natl. Cancer Inst., 80, 14- 20. Hasmann, M., Vallet, G.K., Tapeiro, H., Trevorrow, K. and Lampidis, T. (1989). Membrane potential differences between Adriamycin-sensitive and -resistant cells as measured by flow cytometry. Biochem. Pharmcol., 38, 305-312. Vayuvegula, B., Slater, L., Meador, J. and Gupta, S. (1988). Correction of altered plasma membrane potentials. A possible mechanism of cyclosporin A and verapamil reversal of pleiotropic drug resistance in neoplasia. Cancer Chemother. Pharmacol., 22, 163- 168. O’Brian, C.A., Fan, D., Ward, N.E., Seid, C. and Fidler, I.J. (1989). Levels of protein kinase C activity correlates directly with resistance to Adriamycin in murine fibrosarcoma cells. Fed. Eur. Biochem. Sot. Lett., 246, 78-89. Welsh, M. J. (1990). Abnormal regulation of ion channels in cystic fibrosis. Fed. Am. Sot. Exp. Biol. J. 4, 2718 - 2725. McGrath, T., Latoud, C., Arnold, S.T., Safa, A.R., Felsted, R.L. and Center, M.S. (1989). Mechanisms of
17
18
multidrug resistance in HL60 cells. Biochem. Pharmacol.. 38, 3611-3619. Hamill, O.P., Marty, A.J., Neher, E., Sakmann, B., Sigworth, F.T. (1981). Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch., 391, 85- 100. Worrell, R.T., Butt, A.G., Cliff, W.H. and Frizell, R.A. (1989). A volume-sensitive chloride conductance in human colonic cell line T84. Am. Inst. Physiol., 256, Cllll-c1119. Gupta, S., Kim, J. and Gollapudi, S. (1991). ltraconazole reverses daunorubicine resistance in P388/ADR cells. J. Clin. Invest., 87, 1467- 1469. Mossman, T. (1983). Rapid calorimetric assay of cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63. Tron, L., Siegel, J.P., and Aszalos, A. (1989). Effect of cyclosporin A and ionophores on the intracellular pH of lymphocytes as measured by flow cytometry. Biochem. Med. Metab. Biol.. 41, 164-170. Marquart. D. and Center, M.S. (1991). Involvement of vacuolar H( + )-adenosine triphosphatase activity in multidrug resistance in HL60 cells. J. Natl. Cancer Inst., 83, 1098 - 1102. Ma, 1. and Center, M.S. (1992). The gene encoding vacuolar H( +)-ATPase subunit C is overexpressed in multidrug resistant HL60 cells. Biochem. Biophys. Res. Commun. 182, 675-681. Gollapudi, S., Patel. K., Jain, V. and Gupta, S. (1992) Protein kinase C isoforms in multidrug resistant P388/ADR cells: a possible role in daunorubicin transport. Cancer Lett., 62, 69 - 75. Riordan. J.R., Rommens, J.M., Kerem B.S., Alon, N., Rosmachel, R., Grzelenski, J., Lok, S., Plavsic, N., Chou, J.L., Iannuzsi, M.C., Collins, F.C. and Tsui L.-C. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 245, 1066 - 1073.
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Hyde, S.C.. Emsley, P., Hartshorn, M.J., Mimmack, M.M.. Gileadi, U., Pearce, S.R., Gallagher, M.P., Gill, D.R.. Hubbard, R.E. and Higgins, C.F. (1990). Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature, 346, 362 - 365. Valverde, M.A., Diaz, M., Sepulveda, F.V., Gill, D.R., Hyde, S.C. and Higgins, C.F. (1992). Volume-regulated chloride channels associated with the human multidrug resistance P-glycoprotein. Nature, 355, 830- 833. Hwang T.-C., Lu. L., Zeitlin, P.L., Gruenert, D.C., Huganir, R. and Guggino, W.B. (1989). Membrane potential differences between adriamycin-sensitive and resistant cells as measured by flow cytometry. Science, 244. 1351- 1353. Chen, J.H., Schulman, H. and Gardner, P. (1989). A CAMP-regulated chloride channel in lymphocytes that is affected by cystic fibrosis. Science, 244, 657 - 660. Njus, D., Kelley, P.M. and Harnadek. G.J. (1986). Bioenergetics of secretory vessels. Biochim. Biophys. Acta, 853, 237-265.
Abnormal cells S. Gollapudi”, ‘Division
83
chloride conductance
T. McDonaldb,
P. Gardnerb,
of Basic and Clinical Immunology,
Medicine,
Falk Cardiooascular
(Received
28 April 1992)
Research
in multidrug resistant HLGO/AR
Unioersity
Center,
Stanford
N. Kanga of California,
and Irvine,
S. Gupta” California
Uniuersity School of Medicine,
92717
and bDepartment
Stanford,
California
of
(USA)
(Revision received 6 July 1992) (Accepted
8 July 1992)
Summary
Introduction
Chloride channel currents were measured in drug sensitive parental HL60 and multidrug resistant (MDR) subline HL60/AR cells, using a whole cell patch-clamp technique. In addition, the in uitro effects of 4,4’diisothiocyanatostilbene-2,2’ -disulfonic acid (DIDS), a Cl channel blocker, on intracellular accumulation and sensitivity to daunorubicin and intracellular pH [pHJ in HL60 cells were examined. Baseline DIDS blockable CI- currents were consistently lower in HL60/AR cells (0.9
The development of resistance by malignant cells to cytotoxic drugs poses a major obstacle to the chemotherapeutic cure of cancer. Acquired drug resistance occurs when drug sensitive malignant cells become resistant to antineoplastic agents after exposure to them. These malignant cells often exhibit cross resistance to a wide variety of structurally and functionally unrelated drugs. This pattern of resistance is seen in tumors that are initially responsive to chemotherapy that is followed by untreatable relapse. A large number of mechanisms have been proposed to explain this broad resistance, the multidrug resistance (MDR). These include, overexpression of Pglycoprotein, increased levels of glutathione-Stransferase, glutathione and topoisomerase II, etc. [l-4]. At the biochemical level, the role of plasma membrane potential [5,6] and protein kinase C (PKC) [7] has been suggested. The plasma membranes of all cells have transport systems that mediate the exchange of solutes between the cells and their environment. These include, ATPase or ion pumps, Cl -/HC03 - anion exchanger and ion channels that provide selective ion permeabilities in the cell membrane. The chloride channels are controlled by ligands, membrane potential, intracellular second messengers (i.e. PKC, PKA,
pA/pF) pA/pF).
as compared to HL60 cells (7.0 Similarly CAMP-activated CI- currents were minimal in HL60/AR cells (0.2 pA/pF) as compared to HL60 cells (8 pA/pF). In vitro treatment of drug sensitive HL60 cells with DIDS resulted in concentration-dependent decreased accumulation and increased resistance to daunorubicin and decreased pH,. These data show that altered Cl- permeability is associated with MDR and suggest that Clchannels may play a role in MDR.
Kegwords: multidrug resistance; pH; chloride current
intracellular
Correspondence to: S. Gupta, Medical Sciences I, Room 264A, University of California, Irvine, CA 92717, USA.
0304-3835/92/$05.00 Printed and Published
C-
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd
84
Ca + +), etc. and appear to play a role in establishing the membrane potential [8]. Because of the suggested role of plasma membrane potential, PKC and intracellular pH in MDR, we compared the properties of Cl currents, using patch-clamp technique, in MDR HL60/AR cells and parental drug sensitive HL60 cells. Our results show that baseline DIDS (Cl - channel blocker) sensitive and CAMP-activated Cl - channel currents are decreased in HL60/AR cells as compared to HL60 cells, suggesting decreased Cl - permeability in MDR cells. Furthermore, blocking of Cl - channel with DIDS in drug sensitive HL60 cells resulted in decreased intracellular drug accumulation and increased drug resistance. These data suggest a possible role of Cl - channels in MDR. Materials
and Methods
Cell lines The human promyelogenous leukemia Adriamycin sensitive parental cell line (HL60) and Adriamycin resistant (HL60/AR) subline were provided by Dr. Kapil Bhalla, Medical University of South Carolina. HL60/AR cells are cross-resistant to daunorubicin, vincristine and vinblastin. These cell lines are maintained in vitro as suspension culture. Both HL60 and HL60/AR cells lack mdr 1 [9]. Chemicals and reagents 4,4’ Diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) ,caged CAMP, 2 ’ ,7 ’ -his- (2-carboxyethyl) - 5 - (and - 6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM) and BAPTA were purchased from Molecular Probes, Eugene, OR. Daunorubicin was purchased from Sigma Chemicals, St. Louis, MO. Electrophysiological studies of HL60 and HL60/AR cells
Chloride currents of HL60 cells were studied with patch pipettes in a whole-cell voltage clamp configuration [lo]. Tip resistance of the pipettes was 2-4 MQ. The extracellular solution for electrophysiologic studies
consisted of 150 mM Tris-Cl, 2.5 mM CaQ., 1 mM MgQ, 5 mM glucose (pH 7.4). Intracellular pipette solution consisted of 140 mM CsCI, 5.5 mM BAPTA, 0.5 mM CaCl*, 4 mM ATP-Mg, 20 mM HEPES (pH 7.2). The calculated chloride current reversal potential for these solutions.was 0 mV. The osmolarity of the extracellular solution was maintained at 25 mM hypertonic relative to the intracellular solution and was monitored by a vapor pressure osmometer (Wescor; Logan, UT). The external hypertonicity was necessary to prevent volume-induced chloride currents [ll]. Studies were conducted at 29 -3OOC. The cell membrane was voltage clamped and whole-cell currents were obtained with an Axopatch amplifier (Axon Instruments; Burlingame, CA) which was controlled by an 80386-based PC and one 16-bit AD/DA convertor. Current signals were recorded on FM tape and digitized for storage on floppy diskettes. The holding membrane potential was maintained at - 50 mV and series of voltage steps from - 100 to + 100 mV were performed for construction of current voltage (I-V) curves. The current was measured as the average current during each voltage pulse and normalized to the measured cell capacitance expressed as picoamps/picofarad (pA/pF) . 4,4’ Diisothiocyanatostilbene-2,2’-disulfonit acid (DIDS) was introduced by means of a rapid superinfusion chamber resulting in an extracellular concentration of 500 PM. CAMP effects were obtained by either superinfusion of chlorophenylthiol-CAMP (cpt-CAMP, 400 PM) to the extracellular solution or by flash photolysis of 4,5-dimethoxy-2-nitrobenzyl-CAMP (Caged-CAMP). Photolysis was performed by brief (1 ms) flashes from a high pressure xenon arc lamp focused onto a 2 - 3-mm area of the cell chamber with quartz optics. Wave lengths below 305 nM were eliminated with a WG305 filter. This system resulted in a 6% release of caged-CAMP per flash as measured by HPLC. intracellular drug accumulation HL60 and HL60/AR cells were resuspen-
ded at 1
x
lo6 cells/ml in 12
x
75-mm
85
culture tubes in the presence or absence of various concentrations of DIDS. Daunorubicin (DNR, 4 ,ug/ml) was added to the cells, gently mixed, and incubated at 37*C for 45 min and DNR accumulation was analyzed by FACScan (Becton-Dickinson, San Jose, CA) at a flow rate of 500 events per second [12]. Mean fluorescence was recorded from histogram and expressed as mean fluorescence linear channel numbers. Drug sensitivity
assay
Drug sensitivity of HL60 and HL60/AR cells was done by MTT assay [13]. Cells (1 x lo6 cells/ml) were incubated with various concentrations of DNR, in the presence or absence of various concentrations of DIDS, for 1 h at 37*C. Cells were washed twice with prewarmed medium RPMI-1640, resuspended in drug free medium, distributed in triplicate ( lo5 cells/well) .into 96-well tissue culture plates and incubated for 3 days at 37*C in a humified CO:! incubator. Three hours prior to termination of the culture, 25 ~1 of MTT (5 mg/ml) was added to each well and the plates incubated at 37*C for an additional 3 h. The formazon crystals formed by the viable cells were dissolved by the addition of 100 ~1 of 1 N HC1:isopropanol (1:24) and the absorbance was measured at 570 nm, using automated microplate reader (UVMax, Molecular Devices, Menlo Park, CA). Cells not exposed to drugs were used as controls (baseline). Results are calculated as percent of the baseline absorbance, ED50 is defined as the concentration of DNR which induced the 50 % cytotoxicity . Measurement of intracellular pH Intracellular pH was measured with an intracellularly trapped and pH-sensitive dye, BCECF-AM by a technique previously described [14]. Cells (3 x 106) were loaded with 2 PM of BCECF-AM for 30 min at 37*C. Cells were washed and resuspended in fresh medium RPMI-1640 at 1 x 107/ml. Intracellular pH was measured using FACScan. Calibration of fluorescence intensity of
BCECF-AM was accomplished by measuring fluorescence intensity after equilibration of the internal and external pH with nigericin. Calibration of pHi versus fluorescence intensity was carried out with buffers of different pH values ranging from 6.5 to 7.9. Results
channel currents Baseline and DlDS sensitive whole-cell currents. Cl - currents at baseline were measured 3 - 5 min after establishing the whole-cell configuration which allowed for thorough dialysis of the cytoplasm with the pipette solution. Baseline Cl- currents were consistently lower in HL60/AR drug resistant (3.2 pA/pF at + 100 mV; n = 47) as compared to drug sensitive HL60 cells (5.2 pA/pF at + 100 mV; n = 42). Representative examples are shown in Fig. 1A. DIDS sensitive current was determined as the baseline current minus that current remaining after the addition of 500 PM DIDS. Drug resistant HLGO/AR cells had much less DIDS blockable baseline current (0.9 pA/pF at + 100 mV; n = 18) than did the drug sensitive HL60 cells (7.2 pA/pF at + 100 mV; n = 16). The DIDS sensitive current had an Erev of 0 mV consistent with a Cl - current and a current-voltage relationship that was usually outward in rectification (Fig. 1B). Chloride
CAMP-actiuated Cl- currents Currents activated by CAMP were determined by measuring maximal current after the addition of CAMP and subtracting the baseline current. The addition of cpt-CAMP or flash photolysis of caged-CAMP was performed after a stable baseline current was obtained. Drug sensitive HL60 cells responded to the addition of cpt-CAMP with an average maximal current of 8 pA/pF at + 100 mV (n = 14)) while MDR HL60/AR cells showed minimal response (0.2 pA/pF at + 100 mV; n = 11). Representative examples are shown in Fig. 2A. Flash photolysis of caged-CAMP resulted in a similar response from HL60 (9.7 pA/pF at
Baseline
DIDS
HLBO/AR
Fig. 1. DIDS blockable Cl- current tracings and I-V relation. (A), examples of whole cell Cl - currents in response to the voltage pulse protocol demonstrated in the left lower inset. Upward deflections represent chloride movement into the cell. The left upper and lower sets of tracing are baseline currents in HL60 and HMO/AR cells, respectively. Currents tracing on the right from the same cells after the addition of DIDS 500 PM to the extracellular solution. (B) , the average I-V relation of the DIDS-sensitive currents in HL60 (open circles; n = 18) and HL60/AR (closed circles; n = 16). Currents were calculated by substracting the residual post-DIDS currents from the baseline current and normalized to cell capacitance (pA/pF) .
+ 100 mV; n = 7) and HL60/AR (0.3 pA/pF at + 100 mV; n = 8). The CAMPactivated current had an Erev of 0 mV and had a milder degree of outward rectification as compared to the DIDS blockable baseline currents (Fig. ZB). Effect of DZDS on intracellular drug accumulation HL60 cells were incubated with DNR (4
pg/mI) for 1 h at 37OC in the presence or absence of various concentrations of DIDS and intracellular accumulation of DNR was measured using FACScan. Data are expressed
as mean fluorescence channel numbers (MFC). Data in Table I show that DIDS in a concentration-dependent manner reduced intracellular accumulation of DNR in HL60 cells. As expected, no significant effect of DIDS was observed on DNR accumulation in HLGO/AR cells (data not shown). Drug sensitioity
The effect of DIDS on the sensitivity of HL60 cells to DNR was assessed by MTT cytotoxicity assay. Table I shows that DIDS, in a concentration-dependent manner, increased ED/50 for DNR in drug sensitive HL60 to a
87
CAMP
Baseline
Fi2. 2. Cyclic AMP-dependent activation of Cl- current tracings and I-V relation. (A), examples of whole-cell Cl current tracings on the upper and lower left are examples of baseline currents from HL60 and HL60/AR cells, respectively. Tracing on the right show resulting currents from the same cells shown on the left after the addition of CAMP. Voltage pulse protocol is demonstrated in the left lower inset. (B), the average I-V relation of currents induced by either cpt-CAMP or caged-CAMP in HL60 (open circles; n = 21) and HL60/AR (closed circles; n = 19) cells. Current values were calculated as the current after addition of CAMP minus the baseline current and normalized to cell capacitance.
Table 1. Effect of DIDS on daunorubicin accumulation and sensitivity in HL60. DIDS (PM)
HL60/AR HL60 HL60 HL60 HL60
None None 125 250 500
MFC no.
Daunorubicin EDs.s (rM)
878 2226 1718 1725 1328
17.3 4.1 6.8 10.5 15.0
zt zt zt zt f
81 65 0 165 43
l
f f f f
5.4 0.9 1.0 2.1 2.8
HL60 cells were incubated with DNR (4c(Q/ml) in the presence or absence of DIDS. DNR accumulation was measured using FACScan. Data are presented as mean fluorescence channel number (MFC). EDss is defined as the concentration of DNR required for 50% cytotoxicity of cells as determined by MTT assay. Data represent mean f S.D. of 3 separate experiments.
level comparable to drug resistant HLGO/AR cells, indicating development of resistance in HL60 cells to DNR. No significant effect on drug sensitivity of HL60/AR cells was observed (data not shown). This would be consistant with the presence of few DIDS-sensitive Cl channels in HLGO/AR cells. These data would indicate that decreased Cl - permeability is associated with MDR. Intracellular pH The data for the effect of DIDS on pH, of HL60 cells is shown in Table II. In a dosedependent manner, DIDS reduced the pH, in HL60 cells. This would be consistent with the observation of the amplification of vacuolar
88
Table Il. In vitro effect of DIDS on intracellular pH of HL60 cells. DIDS (PM)
None 125 250 500
Intracellular
pH
Exp. 1
Exp. 2
7.82 7.57 7.38 7.17
7.73 7.40 7.30 7.16
HL60 drug-sensitive cells were incubated with various concentrations of DIDS and pH, was measured with BCECF dye, using FACScan (see Materials and Methods).
H +-ATPase activity and mRNA, that maintain an acidic intravasicular environment, in multidrug resistant HL60 cells [ 15,161. Discussion Accumulating evidence indicates that MDR is accompanied by several distinct types of cellular alterations in the cell drug transport process [l - 41. A common finding in most of these studies is that the MDR cells accumulate less drug as compared to sensitive parental cell lines. A number of mechanisms have been proposed for MDR. At the biochemical levels, increased levels of PKC and altered membrane potentials appear to play a role in MDR [6,7,17]. Recent studies of Cl- channels have shown that they regulate various cellular functions, including maintenance of plasma membrane potential [8,17]. In the present study we observed decreased DIDS-blockable baseline Cl - currents, as well as CAMP-activated Cl channel currents in MDR HLGO/AR cells as compared to drug sensitive HL60 cells. This would indicate decreased Cl - permeability in MDR cells. The homology of cystic fibrosis transmembrane regulator (CFTR) to P-glycoprotein [l&19] raised the question of whether these transport proteins might be bifunctional and by, extension, whether or not mdr 1 might encode for Cl - conductance. This issue has been directly addressed by Valverde et al. [20], who suggests that mdr 1 not only en-
codes for a drug transport protein but also the hypotonicity induced Cl - current. However, we did not observe any difference in hypotonicity Cl - currents between HL60 and HLGO/AR (data not shown). The difference in our observations from those of Valverde et al. [20] are not surprising because our multidrug resistant cell line lacks mdr 1 mRNA and Pglycoprotein. Furthermore, we did not find any difference in ionomycin-induced Cl - currents between HL60 and HL60/AR cells (data not shown). The linkage of MDR and decreased drug transport to altered Cl - permeabilities in our present study appears to be a second example of aberrant transport and altered Cl permeability mechanisms. A similar defect in Cl - permeability has been observed in cystic fibrosis [21,22]. Since the precise molecular mechanism for multidrug resistance in HLGO/AR is unknown, we cannot further speculate on the molecular basis for the linkage. But the linkage itself is intriguing. Because of decreased Cl - currents in HLGO/AR cells, we reasoned that if the Cl channel plays a role in MDR, then blocking the Cl - channels with DIDS in drug sensitive cells should render them resistant. In this study we show that in vitro treatment of drug sensitive HL60 cells with DIDS resulted, in a concentration-dependent manner, in decreased intracellular accumulation of DNR and increased resistance to DNR. Although DIDS is known to block Cl - channels, the possibility of its effect on other Cl - transport mechanisms and on proton transport cannot be excluded. Furthermore, DIDS is known to alter pHi via Cl -/HCO< transport. In this study, we show that DIDS in a dose-dependent manner, decreased pHi of HL60 drug sensitive cells. Recent evidence suggests that vacuolar H +ATPase activity, which maintains an acidic vasicular microenviroment [23], may be involved in multidrug resistance in HL60 1151. Marquardt and Center [ 151, demonstrated that bafilomycin Al, a specific inhibitor of H +ATPase, was capable of blocking efflux and inducing an increase in drug accumulation. More recently, Ma and Center [16] have demonstrated increased levels of SC-H +-ATPase
89
(a subunit C of H +-ATPase) mRNA in multidrug resistant HL60 cells. Although the precise mechanism(s) by which DIDS reduces intracellular drug accumulation and confers resistance to HL60 sensitive cells remains unclear, it is possible that DIDS might alter the activity of H +-ATPase. Additionally, the data of the Cl - current and dose-dependent effects of DIDS suggest a possible role for Cl - channels in MDR. The relationship between the Cl - channel and H +-ATPase activity remains to be evaluated. Acknowledgments
This work was supported in part by grants from USPHS Al 26 456 (SG) and DK 41324 (PG) .P. Gardner is a recipient of the Borrough Wellcome Faculty Scholar Award in Clinical Pharmacology. T. McDonald is supported by NIH grant KllHL 02 397.
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