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Laboratoire de Physicochimie Biomoléculaire et Cellulaire, Université Paris Nord, Bobigny, France
Correspondence: Address reprint requests to Prof. A. Garnier-Suillerot, Laboratoire de Physicochimie Biomoléculaire et Cellulaire (LPBC-CSSB UMR CNRS 7033), Université Paris Nord, 74 rue Marcel Cachin, 93017 Bobigny, France. Tel.: 33-14-838-7748; Fax: 33-14-838-7777; E-mail: garnier{at}lpbc.jussieu.fr.
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ABSTRACT |
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10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes. |
INTRODUCTION |
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; Nielsen and Skovsgaard, 1992). Both proteins are members of the ATP-Binding-Cassette superfamily of transport proteins that lower the intracellular drug content through active drug efflux. Transfection experiments with the mdr-1 gene (Fojo et al., 1987) or the mrp-gene (Grant et al., 1994) have demonstrated that overexpression of these genes was sufficient to render otherwise drug-sensitive cells multidrug resistant.
Characterization of drug retention as functional assays for MDR has been primarily focused on P-gp-mediated MDR, and the recognition that Rh 123 is substrate for P-gp that can be applied at noncytotoxic concentrations has stimulated the use of this agent in flow cytometric drug retention studies. After the discovery of MRP1 in 1996, studies were undertaken to determine if agents used to evaluate P-gp function also can be used to evaluate MRP function. Thus, cellular retention of rhodamine 123 was studied in MRP1-expressing cell lines. Several studies have suggested that Rh123 is a substrate for MRP1 (Zaman et al., 1994; Twentyman et al., 1994; Minderman et al., 1996); however, no quantitative studies of the MRP1-mediated efflux of rhodamine have, up to now, been performed.
In this article, using two types of measurements, rate and steady-state, with the latter being performed under single-cell conditions, we have derived values for the internal concentration of free rhodamine, and hence values for the real rate of outward pumping of rhodamine. In addition, the use of potassium and a potassium carrier to eliminate the effect of membrane potential makes a very useful simplification. This was done using intact GLC4/ADR cells. We are therefore able to present data that quantitatively characterized the transport by MRP1 of four rhodamine analogs: rhodamine 6G (Rh 6G), tetramethylrosamine (TMR), tetramethylrhodamine ethyl ester or rhodamine I (Rh I), and tetramethylrhodamine methyl ester or rhodamine II (Rh II). The results are compared with those obtained in the same cell line with other substrates versus those obtained in cells having a P-gp-mediated MDR phenotype.
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MATERIALS AND METHODS |
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).
Cell lines and cultures
GLC4 and the MRP1-expressing GLC4/ADR cells (Zijlstra et al., 1987) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum in a humidified incubator with 5% CO2. The resistant GLC4/ADR cells were cultured with 1.2 µM doxorubicin until 1–4 weeks before experiments. Cell cultures used for experiments were split 1:2 one day before use to ensure logarithmic growth. Cell viability was assessed by Trypan blue exclusion and was >95% under the various experimental conditions used. Cell counting and cell diameter were determined using a Coulter Channelyzer 256 (Beckman, St. Louis, MO). The curve distribution of the cell number vs. cell radius had a Gaussian shape with a maximum corresponding to mean cell radius of 12.5 µm and 75% of the cells had a radius equal to 12.5 ± 1.5 µm, which yields a mean volume 1.0 ± 0.3 x 10-12 L. The cytotoxicity of one rhodamine derivative, RhII, was determined by incubating cells (105) with different concentrations of the compound for 72 h in standard 6-well plates. Three independent experiments were performed. Then the IC50 values (50% inhibitory drug concentrations) were determined by counting the cells using a Coulter counter. The resistance factor (RF) was defined as the IC50 for the resistant cells divided by the IC50 for the corresponding sensitive cells.
GSH depletion
To examine the effect of glutathione depletion by L-buthionine sulphoximine on rhodamine efflux, cells were cultured in the presence of 25 µM BSO for 24 h. Intracellular GSH was quantified using an enzymatic technique (Salerno and Garnier-Suillerot, 2001).
Real-time fluorescence measurement of drug transport in living cells
Intracellular rhodamine accumulation was measured with a flow cytometer (Becton-Dickinson, Beckman, St. Louis, MO). Cells, 106/ml, were put in the K+ buffer at 37°C and a small volume of the mother rhodamine solution was quickly added to get a CT rhodamine concentration. Fluorescence intensity was measured continuously until steady state was reached.
Mathematical calculations
The mathematical symbols used are the following:
10-12 L cell-1). 
is the concentration of internal rhodamine bound to its receptors. i is the concentration of free internal rhodamine.
 | (1) |
/i x [receptors] or K = ß x [receptors] with ß = /i. 
is the molar fluorescence of rhodamine free in the cytosol. 
is the mean fluorescence quantum yield for rhodamine bound to its receptors. x (1 + ß x ) is the proportionality constant between the fluorescence intensity recorded via flow cytometry and i. i. i. We intend to derive, from the data ka, the rate constant for outward pumping at limiting low substrate concentration, and for this purpose we need to determine 1), the concentration of free internal rhodamine; 2), the mean binding constant for rhodamine to its receptors, whatever they are; 3), the mean fluorescence quantum yield for rhodamine bound to its receptors, whatever they are; 4), the passive permeability rate constant; and 5), the rate constant for outward pumping at limiting low substrate concentration.
The determination of the kinetic parameters, e.g., the maximum rate, VM, and the Michaelis constant, Km, characteristic of the transporter-mediated efflux of drugs, required the measurement of Va and i. When Va can be determined for various intracellular free drug concentrations i, the maximal efflux rate (VM) and the apparent Michaelis-Menten constant (Km) can be computed by nonlinear regression analysis of transport velocity Va vs. i, assuming that the transport follows the Michaelis equation
 | (2) |
i is much lower than Km, Eq. 2 becomes
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 | (3) |
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 | (4) |
 | (5) |
i.
As it will be demonstrated below, the determination of k requires the knowledge of (the molar fluorescence of rhodamine free in the cytosol), of (the mean fluorescence quantum yield for rhodamine bound to its receptors whatever they are), and of ß = /i. For this purpose, sensitive cells were incubated with rhodamine in K+ buffer. Under these experimental conditions, where 
= 0, the positively-charged rhodamines cannot accumulate inside mitochondria. However, they can interact with different receptors within the cell. The intracellular concentration of rhodamine bound to these receptors () is in thermodynamic equilibrium with the rhodamine free in the cytosol (i). A mean binding constant can be defined as K = /i x [receptors]. As we were working under experimental conditions where the receptors were in large excess compared to the intracellular rhodamine concentration, the concentration of free receptors could be considered as constant. It follows that the binding of the different rhodamine to the receptors can be characterized by ß = /i. The concentration of rhodamine inside the cells is therefore
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x i) and that of the rhodamine bound to its receptors ( x x ).
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Let us consider sensitive cells, N x 109 L, in K+ buffer, incubated with rhodamine at concentration CT. At steady state, Ce = i, and taking into account Eqs. 1 and 6, it becomes
 | (8) |
 | (9) |
, , and ß can then be computed by a nonlinear analysis of Fcyto, measured at fluorescence steady state, vs. N.
The parameter k was determined from the continuous monitoring of the fluorescence signal, Fcyto (flow cytometry), when sensitive cells in K+ buffer were incubated with rhodamine. Actually, when cells are incubated with rhodamine, before reaching the steady state, rhodamine enters continuously into the cells. According to Eq. 6, during dt, the increase of the intracellular rhodamine concentration is (1 + ß) x dCi and the increase of the number of moles per cell and per second is
 | (10) |
 | (11) |
 | (12) |
 | (13) |
 | (13a) |
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RESULTS |
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i) - 1]. This requires the measurement of 1), the coefficient of passive diffusion k; and 2), the gradient of concentration generated by the pump, e.g., the extracellular Ce and the cytosolic i free drug concentrations at steady state. The following experiments were designed to determine these three parameters.
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Comparison of the MRP1-mediated efflux of daunorubicin (DNR) in the presence and in the absence of membrane potential
First, we have checked that in K+ buffer plasma and mitochondrial potentials were dissipated. We have performed a continuous spectrofluorometric monitoring of the fluorescence signal of a cationic rhodamine (TMR 0.2 µM) during incubation with sensitive cells in a 1-cm quartz cuvette containing Na+ buffer on the one hand and K+ buffer on the other hand. In Na+ buffer a strong decrease of the fluorescence signal was observed due to the accumulation of the lipophilic cation mainly in the mitochondrial compartments, leading to a quenching of the fluorescence signal. However, when the same experiments was performed in K+ buffer, no quenching of the fluorescence was observed—from which we inferred that there was no accumulation of TMR inside the cells and therefore that the potentials were eliminated.
We have measured the accumulation of DNR (cells, 106/ml, and 1 µM DNR) in GLC4 and GLC4/ADR cells in Na+ and K+ buffers ( = 0) respectively, using a method previously described (Marbeuf-Gueye et al., 1998). We observed that the accumulation of DNR in sensitive cells was much lower than in sensitive cells and that, in both cases, the accumulation did not depend on the membrane potentials.
Uptake of rhodamine by GLC4 cells in the absence of membrane potential
A continuous cytofluorometric monitoring (Facscan) of the fluorescence signal of sensitive cells incubated in K+ buffer with various rhodamine concentrations CT (0.02–0.2 µM) was performed. Fig. 2 shows such a record for Rh6G. Data points of Fcyto vs. time (or the experimental records) were fitted to Eq. 13a) and the a- and b-parameters determined (see Table 1) with b = k/Vcell x (1 + ß).
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= 0, there is a transmembrane equilibrium—and the free rhodamine activity should be the same on both sides of the plasma membrane. Here, the concentrations being low, we have made the reasonable assumption that concentration could be used in place of activity, e.g., Ce = i, therefore CT = i. As can be seen, there is a linear dependency of Fcyto as a function of CT = i:Fcyto = A x i, where A is a constant which depends on the nature of the rhodamine. However, the fluorescence signal recorded from the cells is due not only to the rhodamine free (i) in the cytosol but also to the rhodamine bound () to intracellular sites and the ratio of these two concentrations is a constant ß = /i. As we have shown in the experimental section, Fcyto = x (1 + ß x ) x i, and therefore A = x (1 + ß x ).
The following experiments were designed to determine ß. In this set of experiments cells were incubated with always the same rhodamine concentration CT = 0.2 µM but the number of cells used during the incubation in K+ buffer was varied from 105 to 5 x 107 per ml. The flow cytometry signal was measured at steady state. Fig. 4 shows typical records of Fcyto as a function of the cells' number for TMR, Rh 6G, and RhII. As can be seen, the intensity of the signal decreased when the number of cells increased. Data points of Fcyto vs. number of cells were fitted to Eq. 9 and the values of , , and ß were estimated. To check if ß-values in resistant cells were similar to those observed in sensitive cells, experiments were performed with energy-depleted resistant cells. The values of the three parameters , , and ß were the same respectively as those determined for sensitive cells. They are reported in Table 2. The ß-values for RhI and RhII rhodamines were comparable; however, the ß-value for TMR was 15-fold higher. It was then possible to calculate the k-value (Table 1).
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i, the concentration of free rhodamine in the cytosol of GLC4/ADRi value and then the gradient of concentration i/Ce generated by the pump. This calculation was performed for the various concentrations of rhodamines used (0.02–0.2 µM), and we did not observe significant variation of the gradient value, indicating that we were working under conditions where the MRP1 transporter was far from being saturated. The values of the gradient generated by the pump are reported in Table 1; they are within the range of 0.35–0.78.
Determination of the active efflux coefficient ka
Once the parameters k, Ce, and i were measured, it was easy to calculate ka according to Eq. 5. The values are reported in Table 1. For the sake of comparison, the values for DNR are also shown.
Validation of the method
The method was validated in two different ways.
Using Eq. 13a to fit the data in Fig. 2, we have determined a and b. On the other hand, using the data of a completely different set of experiments, and fitting them with Eq. 9, we have determined -, ß-, and -values. These values were then used to recalculate a = (1 + ß x ) x x Ce. As can be seen in Tables 1 and 2, the values of a obtained either from the data Fcyto = f(t) or from the data Fcyto = f(N) (at steady state) are in good agreement.
We have further validated the method using a totally different substrate for which the k+/- and ka parameters were previously determined using a totally different methodology. For this purpose we have used daunorubicin. We have recorded Fcyto as a function of time when cells (106/mL) were incubated with 1 µM DNR to avoid accumulation of the drug (which is a weak base in acidic compartments; Loetchutinat et al., 2001); the experiments were performed in the presence of 20 nM concanamycin A. DNR is well-known to accumulate in the nucleus and therefore the extracellular concentration cannot be consider as constant. When taken into account as a function of time, the modification of the extracellular drug concentration is always
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| (13a) |
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Of course this complete expression can be used for rhodamines, but in this case, the D-value is close to 1 (because of the relatively low value of ß).
The a-, b-, D- and ß-values obtained are the following:
On the other hand,
i/Ce = 0.30 ± 0.04, which yields ka = (0.8 ± 0.4) x 10-12 L cell–1 s–1.
These values are in good agreement with those previously obtained with a methodology that we have largely developed and used for the determination of the rate of uptake and pump-mediated efflux of anthracycline in living cells (Mankhetkorn et al., 1996; Marbeuf-Gueye et al., 1998). These values are k = (3.1 ± 0.5) x 10-13 L cell-1 s–1 and ka = (1.3 ± 0.3) x 10-12 L cell-1 s-1.
Rhodamine uptake in GSH-depleted GLC4/ADR cells
Cells were incubated in the culture medium for 24 h in the presence of 25 µM BSO. Under these conditions the concentration of GSH remaining inside the cells was <0.2 mM (Salerno and Garnier-Suillerot, 2001). Cells were then incubated with 0.2 µM rhodamine. For both cell lines the uptake was comparable to that observed for sensitive cells. These results clearly indicate that GSH is involved in the decrease of rhodamine retention in resistant cells.
Cell-growth inhibition
Cell lines that we used were made resistant by co-culture with doxorubicin. The IC50 value obtained with RhII for sensitive and resistant GLC4 cells are 0.09 ± 0.02 µM and 0.75 ± 0.15 µM, respectively (Fig. 5). These values represent means ± SD of triplicate determinations. The resistance factor was equal to 8. For comparison, the IC50 obtained with doxorubicin under similar experimental conditions were (9 ± 2) x 10 -3 µM and 0. 670 ± 0.070 µM for sensitive and resistant cells respectively, with RF = 74.
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DISCUSSION |
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; Eytan et al., 1997) and the kinetics parameters characteristic of their transport have been measured (Lu et al., 2001; Loetchutinat et al., 2003). In the case of MRP1, these things are far from clear. Zaman et al. (1994) have demonstrated cross-resistance to Rh123 in MRP1 transfected SW 1573 cells. Daoud et al. (2000) have shown that the IC50 of HeLa-MRP1 is 14- and fivefold higher than HeLa cells for VP16 and Rh123 respectively. Minderman et al. (1996) have demonstrated that in addition to being a substrate for P-gp, Rh123 is also a substrate for MRP-mediated drug efflux, but that this efflux takes place at a slower rate compared to P-gp. However, the comparison of the uptake of Rh123 by GLC4/ADR and GLC4 using flow cytometry has shown that the fluorescence signal recorded from GLC4 was less than that observed with GLC4/ADR cells (Feller et al., 1995; Garnier-Suillerot, unpublished data). More recently, Daoud et al. (2000) have reported on the characterization of MRP1 interactions with Rh123 using the photoactive analog iodoaryl azido Rh123, and shown that it interacts directly and specifically with MRP1. To our knowledge, up to now the MRP1-mediated efflux of one rhodamine only, Rh123, has been explored and no study was designed to determine the kinetics parameters of this transport.
Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In this article, we present data that characterized the transport of several rhodamine analogs which are positively charged. We took advantage of the intrinsic fluorescence of rhodamines, and performed a flow-cytometric analysis of dye accumulation in the wild-type, drug-sensitive GLC4 cells that do not express MRP1. For its MDR subline, which displays high level of MRP1, the resistance factor for daunorubicin was equal to 23 (Marbeuf-Gueye et al., 1999). The measurements were made in real-time using intact GLC4/ADR and GLC4 cells. We clearly show that GLC4/ADR cells are cross-resistant to rhodamines, that there is an energy-dependent efflux of rhodamines in GLC4/ADR cells, and that this efflux is GSH-dependent inasmuch as no efflux was observed after GSH depletion with BSO. Rh123 is widely used as a structural marker for mitochondria as an indicator of mitochondrial activity and, therefore, the relative accumulation of positively charged rhodamine in parental and resistant cells is likely to be influenced by any differences in mitochondrial number or membrane potential between the cell types in addition to effects of active transporters. For this reason we have studied the MRP1-mediated efflux of rhodamine in the absence of membrane potential after having checked that the MRP1-mediated efflux of drug (in this case, daunorubicin) was not potential-dependent.
To characterize the MRP1-mediated efflux of rhodamines, the parameter ka was calculated. As shown (Materials and Methods, this article; and Marbeuf-Gueye et al., 1998, 1999), ka is proportional to the ratio VM/Km and is very convenient to evaluate the efficiency of a transporter for any substrate. The determination of ka requires the measurement of the gradient of concentration, i.e., Ce vs. i, which is generated by the presence of the pump. Thanks to the use of two independent fluorometric techniques, macrofluorescence and flow-cytometry, it is possible to directly determine the free rhodamine concentration in the cytosol and in the extracellular medium. Actually, our data clearly show that the cytofluorometric signal, in cells without membrane potential, is proportional to the amount of rhodamine free in the cytosol. This observation allows the further determination of the concentration of drug free in the cytosol of resistant cells. The determination of ka also requires the measurement of the rate of passive diffusion of the dye through the plasma membrane. For this reason, we have determined the ratio of the drug bound to the drug free in the cytosol, which subsequently allows the determination of the real number of molecules that penetrate per second into one cell and therefore the true rate of passive diffusion of the dye.
As can be seen in Table 1, the ka values determined for the four molecules are of the same order of magnitude, ranging from 0.8 to 2.6 x 10-13 L cell-1 s-1. One of our aims was to compare the MRP1-mediated efflux of these rhodamine analogs to that of other substrates obtained previously using the same cell line. To help this comparison, the values of i/Ce, k, and ka for daunorubicin are reported in Table 1, together with the ka value for GSH, calcein, and As(OH)3. Compared to previously published data, the efficiency of the MRP1-mediated rhodamine pumping is 10-fold less than for anthracycline (Marbeuf-Gueye et al., 1999) but 100-fold more than for GSH (Salerno and Garnier-Suillerot, 2001), calcein (Essodaïgui et al., 1998), and As(OH)3 (Salerno et al., 2002). In addition, the present study shows that the efficiency of the MRP1-mediated rhodamine pumping is 10-fold less than the efficiency of the P-gp-mediated rhodamine pumping, whereas the efficiency of the anthracycline pumping was very similar for both transporters.
The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine analogs in intact cells.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Submitted on December 5, 2002; accepted for publication April 21, 2003.
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REFERENCES |
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Daoud, R., C. Kast, P. Gros, and E. Georges. 2000. Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1). Biochemistry. 39:15344–15352.[Medline]
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Essodaïgui, M., H. Broxterman, and A. Garnier-Suillerot. 1998. Kinetic analysis of calcein and calcein—acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein. Biochemistry. 37:2243–2250.[Medline]
Feller, N., C. M. Kuiper, J. Lankelma, J. K. Ruhdal, R. J. Scheper, H. M. Pinedo, and H. J. Broxterman. 1995. Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry. Br. J. Cancer. 72:543–549.[Medline]
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Loetchutinat, C., S. Chantarawan, C. Marbeuf-Gueye, and A. Garnier--Suillerot. 2003. New insights into the P-glycoprotein-mediated effluxes of rhodamines. Eur. J. Biochem. 27:476–485.
Lu, P. H., R. H. Liu, and F. J. Sharom. 2001. Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state. Eur. J. Biochem. 268:1687–1697.[Medline]
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Marbeuf-Gueye, C., H. Broxterman, F. Dubru, W. Priebe, and A. Garnier-Suillerot. 1998. Kinetics of anthracycline efflux from multidrug resistance protein-expressing cancer cells compared with P-glycoprotein-expressing cancer cells. Mol. Pharmacol. 53:141–147.
Marbeuf-Gueye, C., D. Ettori, W. Priebe, H. Kozlowski, and A. Garnier-Suillerot. 1999. Correlation between the kinetics of anthracycline uptake and the resistance factor in cancer cells expressing the multidrug resistance protein or the P-glycoprotein. Biochim. Biophys. Acta. 1450:374–384.[Medline]
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Salerno, M., and A. Garnier-Suillerot. 2001. Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cell. Eur. J. Pharmacol. 421:1–9.[Medline]
Salerno, M., M. Petroutsa, and A. Garnier-Suillerot. 2002. The MRP1-mediated effluxes of arsenic and antimony do not require arsenic-glutathione and antimony-glutathione complex formation. J. Bioenerg. Biomembr. 34:135–145.[Medline]
Twentyman, P. R., T. Rhodes, and S. Rayner. 1994. A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes. Eur. J. Cancer. 30A:1360–1369.[Medline]
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); GLC4 cells (
); and GSH-depleted GLC4/ADR cells (•). The data points are from a representative experiment.
). The data points are from a representative experiment.