Competition between innate multidrug resistance and intracellular binding of rhodamine dyes Daniella Yeheskely-Hayon, Ronit Regev, Hagar Katzir and Gera D. Eytan Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel A major obstacle impeding the success of chemother- apy is multidrug resistance (MDR). MDR in patients is exhibited as a resistance to a wide variety of struc- turally unrelated drugs. This is caused by several factors, one of which is ‘classical’ MDR characterized by diminished cellular uptake of the drugs due to active export by one or more ABC transporters. The ABC transporter most often over-expressed in vitro in cells exposed to increasing drug concentrations is plasma glycoprotein (Pgp, ABCB1) [1–3]. This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic partially positive dyes. The ABC transporter superfamily is represented in humans by 48 members [4,5], of which 24 are known to function as drug transporters relevant to various diseases. In addition to Pgp, the multidrug resistance-associated protein (MRP1, ABCC1) [6] and breast cancer resistance protein (BCRP⁄ MXR ⁄ ABCP, Keywords innate multidrug resistance; MDR; MRP1; multidrug resistance; rhodamines Correspondence G. D. Eytan, Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel Fax: +972 4 8225153 Tel: +972 4 8293406 E-mail: eytan@tx.technion.ac.il Website: http://biology.technion.ac.il (Received 7 September 2008, revised 12 November 2008, accepted 20 November 2008) doi:10.1111/j.1742-4658.2008.06812.x The present study aimed to elucidate the contribution of the intracellular binding of drugs to multidrug resistance. For this purpose, uptake of rhod- amines was studied in cells whose mitochondria had been uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of drug-untreated cells, presumed to be sensitive to multidrug resistance-type drugs, rhodamines were excluded from entering the cells. Thus, the amount of rhodamine 123 taken up into parental untreated K562 cells was less than the amount bound to the cell exterior. Rhodamine uptake was prevented by an active efflux pump. The efflux was inhibited by 4-chloro-7-nitro- 2,1,3-benzoxadiazole (NBD-Cl) and MK571 and, to a lesser extent, by ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors, apart from NBD-Cl, are known to modulate multidrug resistance-associ- ated protein (MRP) 1. Because MRP1 was expressed in all the cell lines tested and the efflux of rhodamines in MRP1 over-expressing cells was abolished by NBD-Cl, it appears that rhodamines are excluded from these cells by MRP1. On the other hand, the uptake of rhodamines into cells respiring with their coupled mitochondria demonstrated diminished sensi- tivity to NBD-Cl and MK571. Thus, active pumping into the mitochondria allowed enhanced uptake into the cells, overcoming the innate resistance. The innate resistance provided by MRP1 to cells prevents rhodamine dyes, and possibly drugs such as doxorubicin, from achieving equilibration of their concentration in the cytoplasm with their concentration in the exter- nal medium. The protection provided to multidrug resistance cells by ABC transporters has to overcome competition by passive uptake of the drugs and binding ⁄ uptake of the drugs into intracellular targets. Abbreviations CCCP, carbonyl cyanide m-chlorophenylhydrazone; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NBD-Cl, 4-chloro-7-nitro-2,1,3-benzoxadiazole; Pgp, plasma glycoprotein; TMR, tetramethylrhodamine; TMRM, tetramethylrhodamine methyl ester. FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 637 ABCG2) [7–9] have been well-established as efflux pumps preventing the penetration of anticancer-drugs into tumor cells in vitro and in patients. For MDR research, highly resistant cells have been generated by the exposure of sensitive parent cell lines to increasing concentrations of an anticancer drug. The resulting resistant cells expressed high levels of ABC transporters compared to the minute expression of these proteins in parent lines. It has been assumed that the low levels of ABC transporters present in the parent cell lines cannot cope with significant drug con- centrations and, as a result, drug uptake into sensitive cells is not affected by pump activity but is due to pas- sive transmembrane transport [10–12]. In addition, upon exposure of cells to drugs, a steady-state is achieved, with the free drug concentration in the cyto- plasm being equivalent to its concentration in the extracellular medium [10,13,14]. Pgp is a key player in the defense of the body against amphipathic xenotoxins. At the blood–brain barrier, and in placental trophoblasts, testis and bone marrow, it provides protection of vital body parts and, in the gut, liver and kidney, Pgp helps to eliminate tox- ins from the body [15–18]. On the other hand, MRP1 is present in virtually all human tissues and in most human tumor cell lines and tumor samples [4]. Using mice genetically deficient in both the mdr1a and mdr1b genes [mdr1a ⁄ 1b() ⁄ )) mice], the mrp1 gene [mrp1() ⁄ )) mice] and the combined genes mdr1a ⁄ 1b and mrp1 [mdr1a ⁄ 1b() ⁄ )), mrp1() ⁄ )) mice], it has been shown that the innate levels of these ABC transporters, expressed in mice, confer MDR [19–21]. BCRP is expressed specifically in human placenta, liver and breast [22]. The evidence presented below indicates that a variety of ‘sensitive’ human cell lines exhibit MDR activity capable of preventing the accumulation of rhodamine dyes in the cell cytoplasm. These cationic, relatively hydrophobic dyes, especially rhodamine 123, have been widely used as probes for MDR ex vivo. Inside the cells, these dyes are actively driven into the mitochondria by the respiratory electrochemical poten- tial [23]. In the presence of uncouplers that abolish this active accumulation, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP), rhodamine dyes entering the cells remain free in the cytoplasm and are passively bound to various intracellular sites. Likely sites are the intracellular membranes because they are hydrophobic and negatively charged. A comparison of rhodamine uptake, in the presence and the absence of uncouplers, allows the estimation of the effect of intracellular binding on the MDR phenomenon. The data detailed obtained in the present study indicate that innate cellular MDR interferes successfully with the accumulation of dyes, and probably certain drugs, in the cytoplasm. On the other hand, high-affinity binding of these agents to intracellular receptors over- whelms the low levels of ABC transporters present in ‘sensitive’ cells, and higher expression levels of ABC transporters are required to prevent binding to these receptors. Results The amount of rhodamine dyes associated with K562 cells was determined. The cells were separated from the external medium by centrifugation through an oil cushion and dissolved prior to determination of the amount of dyes associated with them. The advantages of such an assay are that: (a) it is quantitative; (b) it is not affected by intracellular quenching of the dyes; and (c) it measures the total dye amount associated with the cells, including the dye adsorbed to the sur- face of the cells. To avoid complication of the uptake studies by active pumping of the dyes into the mitochondria, dye uptake was measured routinely in the presence of the mitochondrial uncoupler, CCCP, The quantities of rhodamine 123, tetramethylrhodamine (TMR), tetram- ethylrhodamine methyl ester (TMRM) and rhodamine 6G adsorbed to the cell surface under these conditions were equivalent to the rhodamine amounts dissolved in medium volumes equal to 1.3, 9.1, 2.3 and 4.8 cell vol- umes, respectively (Fig. 1 and Table 1). TMR exhib- ited the highest affinity toward the cell plasma membrane, whereas rhodamine 123 exhibited the low- est affinity. Within the limitations imposed by rhoda- mine dye solubility in the medium, the apparent volume of dye adsorbed to the cells was independent of the dye concentrations used (Fig. 2). Thus, it appears that the rhodamine dyes are bound to sites abundant on the cell surface, rather than to specific receptors. As expected, the omission of mitochondrial poisons did not affect the amount of dye adsorbed to the cell surface. Incubation of cells at room temperature with rhoda- mine dyes resulted in dye uptake into the cells that reached apparent quasi-equilibrium within < 1 h. In agreement with an earlier study [14], the cellular uptake of rhodamine 123 was slow and the cells reached quasi-equilibrium within 1 h when incubated at 37 °C. Under conditions designed to eliminate the active uptake of the dyes into mitochondria by poison- ing the latter with CCCP, azide or cyanide, the amount of rhodamine dye accumulated within the cells was low (Fig. 1). The amount of rhodamine 123 taken Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al. 638 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS up into the cells was less than the amount adsorbed to the cell surface. The amounts of the other rhodamine dyes were somewhat higher, but still < 3.5 times the amounts adsorbed to the cell surface. Because the amount of intracellular membranes exceeds that of the plasma membrane, it was expected that, under quasi-equilibrium conditions, the amount of rhodamine dye located within the cells would be much higher compared to the amount adsorbed to the cell surface. Indeed, rendering cells permeable with dig- itonin allowed the enhanced uptake and binding of all the rhodamines (data not shown). To explore the pos- sibility that the rhodamine dyes were prevented from penetrating into cells by an ATP-dependent pump mechanism, we depleted the ATP content of cells by depriving them of glucose in addition to poisoning their mitochondria. Under these conditions, cellular ATP levels were reduced ten-fold (as determined by 15 200 5 10 100 Rhodamine 123 TMR + CCCP – CCCP 0 15304560 0 0102030 0 On ice + CCCP 40 60 40 60 Rhodamine 6GTMRM 0102030 0 20 0102030 0 20 Apparent concentrations ratio (cell/medium) Time (min) Fig. 1. Uptake kinetics of rhodamine ana- logues into K562 cells. K562 cells were incubated either on ice, at 37 °C (rhodamine 123) or at 23 °C (other rhodamine dyes), for 15 min in the absence or presence of CCCP (1 l M). Subsequently, 25 lM of rhodamine 123, TMR, TMRM or rhodamine 6G was added and the cells were incubated further. At various time points, cell samples were withdrawn and the amount of dye associ- ated with the cells was determined after their separation from the external medium by centrifugation through an oil cushion as described in the Experimental procedures. The apparent concentrations ratio was calculated assuming a cell volume of 2.5 l 3 [49]. Data represent the mean ± SD of four independent experiments. Table 1. Uptake kinetics of the various rhodamine analogues into K562 cells. The raw experimental results are presented in Fig. 1. The inhibitor used was either NBD-Cl or MK571. The dye amount adsorbed to the cell surface was estimated as the dye quantity associated with cells incubated on ice at the first time point. This amount was subtracted from the various curves to obtain the amount of dye taken up into cells. The uptake data were fitted using the equation: dye uptake = c · (1 – e )kt ). The amount of dye taken up by the cells at quasi-equilib- rium conditions is equal to c. The half time of the uptake curve was calculated using the constant, k. The amount of dye associated with the cells is expressed as the apparent cell volume of dye bound to a cell, which is equivalent to the concentration ratio of dye bound to the cells versus extracellular dye. The quality of the fitting is expressed in terms of R 2 . Uncoupler Uncoupler + inhibitor Control Control + inhibitor Rhodamine 123 Half time Minutes 21.7 14.5 14.1 14.7 Dye uptake Cell volumes 1.2 10.5 9.3 8.9 R 2 0.93 0.96 0.97 0.95 TMR Half time Minutes 2.6 6.8 6.6 4.8 Dye uptake Cell volumes 31.6 193.0 134.1 255.6 R 2 0.83 1.00 0.99 0.98 TMRM Half time Minutes 2.6 6.9 10.2 7.3 Dye uptake Cell volumes 5.6 51.7 48.7 56.7 R 2 0.77 1.00 1.00 0.98 Rhodamine 6G Half time Minutes 6.3 4.2 6.7 5.8 Dye uptake Cell volumes 13.9 60.8 44.2 64.0 R 2 0.98 0.96 0.98 0.98 D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 639 the luciferin–luciferase assay and confirmed in a previ- ous study [24]) and this resulted in a doubling of the amount of TMRM taken up into the cells. To charac- terize further the mechanism responsible for the exclusion of rhodamine dyes from cells, we surveyed a variety of agents known to interfere with the trans- port of anticancer drugs and dyes in and out of cells (Table 2). 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) caused a prominent increase in rhodamine dye uptake into cells (Fig. 3 and Table 2). Further- more, it had a marked effect in cells whose mitochon- dria had been poisoned and a relatively small effect in cells with unpoisoned mitochondria (Fig. 3). Fluorescence microscopy confirmed that the amount of rhodamine dye taken up into cells whose mitochon- dria had been poisoned was very low (Fig. 4). The low fluorescence of TMRM present in K562 cells was dif- fuse throughout the cytoplasm and the nucleus. As expected, omission of the uncoupler allowed a massive accumulation in the mitochondria. The fluorescence level and location of TMRM taken up into respiring cells was not altered by NBD-Cl. On the other hand, NBD-Cl enhanced the fluorescence of TMRM taken up into nonrespiring cells, but had little effect on the distribution of the dye within the cells. NBD-Cl is known to inactivate Pgp in vitro [25]. However, this inactivation was prevented by the pres- ence of ATP [25]; thus, the increased uptake of rhoda- mine dyes observed in the presence of NBD-Cl does not appear to be due to the inhibition of Pgp. A half maximal effect of NBD-Cl was observed at concentra- tions of 3–5 lm, irrespective of the rhodamine concen- trations that the cells were exposed to, indicating that the direct effect of the NBD-Cl was on a cellular com- ponent and not on the rhodamine dye (Fig. 5A). Moreover, incubation of rhodamine dyes with NBD- Cl, either in presence of cells or in their absence, did not affect the fluorescence spectrum of the dyes (data not shown). To exclude the possibility that the effect of NBD-Cl is due to inactivation of the uncoupler activity of CCCP, the data were reproduced with the 60 250 12 – CCCP 20 40 50 100 150 200 3 6 9 TMRMRhodamine 123 TMR + CCCP 100 10 1 0 0 100 10 1 0 0 100 10 1 0 0 Apparent concentrations ratio (cell/medium) Dye concentration (µM) On ice + CCCP Fig. 2. Effect of rhodamine dye concentrations on their uptake into K562 cells. K562 cells were incubated either on ice for 2 min or for 1 h, either at 23 °C (TMR and TMRM) or at 37 °C (rhodamine 123), in the presence of various concentrations of TMRM, TMR or rhodamine 123. The incubation medium contained CCCP in the absence or presence of NBD-Cl (20 l M). Subsequently, cell samples were withdrawn and the amount of dye associated with the cells was determined after their separation from the external medium by centrifugation through an oil cushion as described in the Experimental procedures. Data represent the mean ± SD of four independent experiments. Table 2. The effect of various agents on TMRM accumulation in K562 cells. ‘Sensitive’ cells were incubated at 23 °C for 15 min with the various agents and 1 l M CCCP. Subsequently, TMRM (10 l M) was added and the cells were incubated further for 30 min at room temperature. The amount of TMRM associated with the cells was measured as described Fig. 1. The amount of dye adsorbed to the cell surface was estimated as described in Fig. 1. Increase in TMRM uptake (%) ATP depletion Preincubation for 60 min with 10 m M deoxyglucose instead of glucose 104 ± 17 NBD-Cl 20 l M 470 ± 83 MK571 50 l M 420 ± 75 Indomethacin 0.5 m M 130 ± 42 Probenecid 3 m M 35 ± 10 Cyclosporine A 10 l M 0 Fumitremorgin C 5 l M 0 Vanadate (ortho) 10 m M 120 ± 18 n-Ethylmaleimide 0.1 m M 0 N,N¢-Dicyclohexylcarbodiimide 1 m M 0 p-Chloromercuriphenylsulfonic acid 1m M 0 Concanamycin A 20 n M 0 Glutathione depletion Growth for 48 h in presence of 50 l MDL-buthionine- (S,R)-sulfoximine [48] 0 Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al. 640 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS mitochondrial poisons, KCN (1 mm) and sodium azide (10 mm), with identical results being achieved. Similar effects of NBD-Cl on TMRM uptake into cells were observed with GLC4 (human small cell lung cancer cell), HEK293 (human embryonic kidney cell), 2008 (human ovarian carcinoma cell), and CEM- CCRF, CIR and Jurkat (human transformed T cell) cell lines (Fig. 5B), indicating that this phenomenon is not specific to K562 cells, but is evident in a variety of cells. A reversal of the innate resistance to rhodamine uptake similar to that obtained with NBD-Cl was observed with MK571 (Table 2 and Fig. 5). MK571, an established inhibitor of several MRP transporters [26–28], allowed the increased uptake of rhodamines into the multidrug-sensitive K562 cells with a half maximal effect being observed at 22 lm. The effect of MK571 on all the cell lines mentioned above was simi- lar to that of NBD-Cl (data not shown). Probenecid, another inhibitor of MRP transporters [29], increased rhodamine uptake (Table 2). Vanadate, a nonspecific inhibitor of ATPase activity, enhanced TMRM uptake into the cells. On the other hand, the Pgp chemosensi- tizer, cyclosporin A, had no effect on rhodamine uptake. Similarly, n-ethylmaleimide, N,N¢-dicyclohexyl- carbodiimide and p-chloromercuriphenylsulfonic acid, and the specific inhibitor of BCRP, fumitremorgin C [30], as well as glutathione depletion, had no effect on 15 300 5 10 100 200 Rhodamine 123 TMR +inhibitor + CCCP +inhibitor – CCCP 0 0 + CCCP 40 60 40 60 Rhodamine 6G TMRM 0102030 0153045 60 0102030 0102030 0 20 0 20 Apparent concentrations ratio (cell/medium) Time (min) Fig. 3. Effect of NBD-Cl or MK571 on uptake kinetics of rhodamine analogues into K562 cells. K562 cells were incubated either at 37 °C (rhodamine 123) or at 23 °C (other rhodamine dyes) for 15 min in the absence or presence of CCCP (1 l M), with either no addition or with the addition of either 50 l M MK571 (rhodamine 123 panel) or 20 lM NBD-Cl (other panels). Subsequently, 25 lM of rhodamine 123, TMR, TMRM or rhoda- mine 6G was added and the cells were incubated further. At various time points, cell samples were withdrawn and the amount of dye associated with the cells was deter- mined after their separation from the exter- nal medium by centrifugation through an oil cushion as described in the Experimental procedures. The apparent concentrations ratio was calculated assuming a cell volume of 2.5 l 3 [49]. Data represent the mean ± SD of four independent experiments. Fig. 4. The effect of MK571 and NBD-Cl on TMRM distribution in K562 and 2008 cell lines. K562 cells and 2008 cells were incubated in the absence or presence of either 50 l M MK571 or 20 lM NBD-Cl for 15 min at room temperature. TMRM (0.1 lM) was added and the cells were incubated further in the presence or the absence of 10 l M CCCP for 30 min. The cells were photographed as described in the Experi- mental procedures. D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 641 the uptake of this rhodamine analogue. As expected for the active efflux mechanism of rhodamine dyes, agents allowing the enhanced uptake of rhodamine dyes inhibited the efflux of these dyes from the cells. The efflux of rhodamine 123 and TMRM was inhib- ited by NBD-Cl and MK571 and, to a lesser extent, by ATP depletion (Fig. 6). An alternative experimental approach to demon- strate the uptake of rhodamine dyes into cells is to follow their quenching upon entering the cells. In respiring cells, the active concentration of these dyes in the mitochondria results in quenching of their fluores- cence. On the other hand, it has been reported that, when the active uptake of rhodamine dyes into the mitochondria is disrupted, incubation of cells with such dyes does not result in quenching of their fluores- cence [14] (Fig. 7). The data obtained in the present study suggest that the reason for the observed lack of fluorescence quenching could be due to prevention of the uptake of dyes by their active efflux from the cells. Indeed, inhibition of this efflux mechanism by MK571 or NBD-Cl resulted in fluorescence quenching of 0.15 0.10 0.05 0.00 4 6 2 100 75 50 25 0 0 50 25 0 NBD-C1 (µM) Cell associated dye A B Fig. 5. NBD concentrations required to inhibit TMRM efflux from various cell lines. (A) 10 6 per mL K562 cells were incubated for 15 min at 23 °C in the presence of various NBD-Cl concentrations. Subsequently, either 2 l M (circles) or 25 lM (diamonds) TMRM was added and the cells were incubated further for 1 h. The amount of TMRM associated with the cells was determined as described in Fig. 1. The results are expressed as the fraction of the dye that became cell-associated. (B) Six cell lines were treated similarly, except that the TMRM concentration was 10 l M. Circles, GLC4; diamonds, HEK293; squares, 2008; triangles, CEM-CCRF; inverted triangles, CIR; cross haired circles, Jurkat. The results are expressed as the dye amount associated with the cells relative to the dye amount associated with cells in absence of NBD-Cl. 1.00 0.75 0.50 0.25 0.00 TMRM Rhodamine 123 Relative TMRM content 30 20 10 0 60 45 30 15 0 Time (min) AB Fig. 6. NBD-Cl and MK571 inhibit efflux of rhodamine dyes from ‘sensitive’ K562 cells. (A) K562 cells were loaded with TMRM with the addition of CCCP (1 l M) by incubation at 23 °C for 30 min in the presence of either 25 lM dye (circles), 5 lM dye and 20 lM NBD-Cl (trian- gles) or 5 l M dye and 50 lM MK571 (squares). Another cell sample was depleted of ATP by incubation in a medium containing CCCP and 10 m M deoxyglucose and in the absence of glucose for 1 h (diamonds). TMRM (10 lM) was added to this sample and the cells were incu- bated further for 30 min. The various dye concentrations presented to the cells during the loading phase were chosen in order to yield simi- lar final dye contents despite various loading conditions. Subsequently, all cell samples were pelleted and resuspended in dye-free media with the same additions as present during the loading of the cells. At various time points, cell samples were withdrawn and the amount of dye associated with the cells was determined after their separation from the external medium by centrifugation through an oil cushion as described in the Experimental procedures. (B) K562 cells were incubated for 1 h at 37 °C in the presence of 1 l M CCCP and 100 lM rhoda- mine 123. The cells were pelleted and resuspended in dye-free media in the absence (circles) or presence of either 20 l M NBD-Cl (triangles) or 50 l M MK571 (squares). At various time points, cell samples were withdrawn and processed as described above. Data represent the mean ± SD of four independent experiments. Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al. 642 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS TMRM (Fig. 7). Similarly, but to a lesser extent, indo- methacin, probenecid and vanadate allowed the enhanced uptake of TMRM, resulting in quenching of its fluorescence. Other agents, such as concana- mycin A, which had no effect on rhodamine efflux as assayed by the quantitative assay reported in Table 2, did not allow quenching of TMRM fluorescence. This phenomenon has been observed in various cell lines, including K562, CCRF-CEM, HL-60, T2 and Chinese hamster ovary cells (Fig. 7). To explore the possibility that the active efflux of the rhodamines from cells presumed to be multidrug sensitive is mediated by one or more of the commonly studied ABC transporters, we studied the effect of var- ious efflux inhibitors on TMRM efflux from cell lines over-expressing Pgp, MRP1, MRP2, MRP3, MRP4 and MRP5 (Fig. 8). Under experimental conditions optimized to observe the NBD-sensitive efflux of rhod- amines, the effect of the inhibitors on the cell lines over-expressing the various transporters was similar to that observed in their corresponding parent cell lines, except for the cells over-expressing Pgp. Over-expres- sion of Pgp reduced the amount of TMRM taken up into cells in the absence of inhibitors compared to its parent line. However, this reduction was reversed by cyclosporin A and, thus, the NBD-Cl-sensitive export observed in multidrug ‘sensitive’ cells does not appear to be due to Pgp activity. Because the over-expression of MRP 1–5 did not significantly reduce the amount of TMRM taken up into the cells, the obstacle to uptake of rhodamines into the cells can be due to one of two possibilities: (a) NBD-Cl-sensitive export is not mediated by either of these MRPs or (b) the low amount of MRPs present in ‘sensitive’ cells is sufficient to prevent the uptake of rhodamine dyes into the cyto- plasm. In the latter case, the over-expression of the transporter has no effect, but inhibition of the trans- porter present in the ‘sensitive’ cells is expected to reverse the obstacle to rhodamine uptake. Because MRP-1 is inhibited by MK571 and is an MRP that is relatively abundant in untreated ‘sensitive’ cells [4], it is a likely candidate for mediating innate resistance in these cells. Indeed, as shown in Fig. 9, NBD-Cl inhi- bited MRP1-mediated efflux, but not efflux mediated by Pgp. Discussion Upon exposure of cells whose mitochondria had been poisoned to rhodamines, quasi-equilibrium is achieved within 1 h. Contrary to our expectations, under these conditions, the free concentration of the dyes in the cytoplasm is low compared to the extracellular concen- trations of the dyes. Equilibration of the dyes across the plasma membrane appears to be prevented by an active export mechanism, which is capable of handling relatively high extracellular concentrations of dyes. This is most evident with rhodamine 123, whose intra- cellular amount at quasi-equilibrium is less than the dye amount bound to the cell surface. It is possible that the small amount of rhodamine 123 taken up into the cells is located at the inner leaflet of the plasma Time (min) Fig. 7. TMRM uptake measured as quenching of the dye fluorescence. Left: CCRF-CEM cells were incubated in fluorimeter cuvettes with stirring for 15 min at 37 °C in the presence of 10 m M sodium azide and in the absence or presence of 20 lM NBD-Cl, 50 lM MK571, 2 mM indomethacin, 2 mM probenecid, 10 mM vanadate or 20 nM concanamycin A. At the time points indicated by arrows, 5 lM TMRM was added. The TMRM fluorescence was monitored continuously with an excitation wavelength of 555 nm and an emission wavelength of 575 nm. Inhibition of the NBD-Cl-sensitive efflux allowed the uptake of TMRM and its quenching within the cells. Right: the various cell lines indicated in the figure were incubated for 15 min with the addition of 10 m M sodium azide and in the absence (upper trace) or presence (lower trace) of 20 l M NBD-Cl. Subsequently, TMRM was added and its fluorescence was monitored continuously. CHO, Chinese hamster ovary. D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 643 K562 K562/ADR GLC4 GLC4/ADR 0.3 0.2 0.1 0.0 0.3 0.2 0.1 0.0 0.3 0.2 0.1 0.0 0.3 0.2 0.1 0.0 2008 MRP1 MRP4 MRP5 MRP2 MRP3 HEK293 Cell associated TMRM (fraction of total) Control NBD-Cl MK571 Indomethacin Probenecid Cyclosporin A Control NBD-Cl MK571 Indomethacin Probenecid Cyclosporin A Fig. 8. NBD-Cl-sensitive TMRM uptake into various MDR cells and their wild-type cell lines. Cells were incubated for 15 min at 23 °Cina medium containing CCCP (1 l M) in the absence (control) or presence of 20 lM NBD-Cl, 50 lM MK571, 2 mM indomethacin, 2 mM probene- cid or 5 l M cyclosporin A. Subsequently, 10 lM TMRM was added and the cells were incubated further for 30 min. The amount of TMRM associated with the cells was determined as described in Fig. 1. The cells included the K562 cell line and its Pgp over-expressing subline (K562 ⁄ ADR), the GLC4 cell line and its MRP1 over-expressing subline (GLC4 ⁄ ADR), the 2008 cell line and its sublines over-expressing MRP1, MRP2 or MRP3, and the cell line HEK293 and its sublines over-expressing either MRP4 or MRP5. 1.00 K562 K562 + NBD-Cl K562/ADR + NBD-Cl K562/ADR 0.75 GLC4 GLC4/ADR GLC4 + NBD-Cl GLC4/ADR + NBD-Cl 0.25 0.50 3020100 Relative TMRM content 604530150 0.00 Time (min) Fig. 9. NBD-Cl inhibits MRP1-mediated efflux of TMRM. Cells were incubated for 15 min at 23 °C in CCCP (1 lM) medium in the absence or presence of 20 l M NBD-Cl. Subsequently, 5 lM (to control cells) or 1.25 lM (to NBD-treated cells) TMRM was added and the cells were incubated further for 30 min. The dye concentrations presented to the cells during the loading phase were chosen in order to yield similar final dye contents despite different loading conditions. The cells were pelleted and resuspended to a concentration of 10 6 cellsÆmL )1 in dye- free medium containing CCCP. At the time points denoted, samples were withdrawn and the amount of TMRM associated with the cells was determined. The cells included the K562 cell line and its Pgp over-expressing subline (K562 ⁄ ADR), and the GLC4 cell line and its MRP1 over-expressing subline (GLC4 ⁄ ADR). Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al. 644 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS membrane and does not accumulate within the cyto- plasm at all. A logical assumption is that the affinity of rhodamines to intracellular membranes is not very different from their affinity towards the extracellular surface of the plasma membrane. Because the surface area of intracellular membranes far exceeds that of the plasma membrane, it is expected that, upon true equili- bration of dyes or drugs throughout the cells, their intracellular amount will be large compared to the amount bound to the cell surface. Active uptake of dyes into the mitochondria allows a significant increase in uptake of rhodamine 123 into MDR ‘sensitive’ cells. Presumably, the active uptake of the rhodamines into the mitochondria competes with and overcomes the efflux pump. Thus, the relative success of the active pump in preventing rhodamine 123 uptake could be due to its low affinity towards membranes and its slow transmembrane passive transport [14]. On the other hand, the relatively minor success of the efflux mecha- nism in preventing TMR uptake into the cells could be due to the high affinity of this dye to membranes and its fast transmembrane passive transport [14]. A con- clusion from our experiments is that, whether in the presence or the absence of active transport into the mitochondria, the free concentration of rhodamines in the cytoplasm of the cells remains significantly lower compared to their extracellular concentration. More- over, the role played by mitochondria in allowing an increased uptake of rhodamines into MDR ‘sensitive’ cells suggests that the efficiency of MDR in preventing the cellular uptake of hydrophobic agents depends on two competitions: (a) competition between the active pumping of the agents out of the cells and their pas- sive uptake [10,31] and (b) competition between the active pumping of the agents out of the cells and their intracellular binding to receptors and ⁄ or pumping into intracellular compartments. Because the rhodamines were excluded from all cell lines tested, it is reasonable to assume that the NBD- Cl-sensitive pump mediating this efflux is ubiquitous in cell lines, and probably also in malignant tumors and healthy tissues. The inhibition pattern of the NBD-Cl- sensitive efflux suggests that it might be mediated by a member of the ABC transporter family. The efflux is inhibited by ATP depletion; by vanadate, which has been shown to inhibit Pgp, MRP1 and MRP2 [32–34]; by MK571, which has been shown to inhibit MRP1, MRP2, MRP4 and MRP7 [26,27,35,36]; and by indo- methacin, which has been shown to inhibit MRP1, MRP3, MRP4 and MRP6 [37–40]. Of these transport- ers, MRP1 appears to be the best candidate to mediate the innate efflux from ‘sensitive’ cells for several reasons: (a) it is widely distributed in cell lines and tissues [4]; (b) it is expressed in all the cell lines tested here; (c) MRP1 is inhibited by NBD-Cl; and (d) the efflux kinetics of rhodamines from NBD-Cl-inhibited MRP1 over-expressing cells are similar to those exhib- ited by ‘sensitive’ cells. Thus, the MRP1 levels present in MDR ‘sensitive’ cells are sufficient to prevent equili- bration throughout the cells of rhodamines, and probably also of hydrophobic anticancer drugs, such as doxorubicin. As mentioned above, the data obtained in the pres- ent study suggest that MRP1 interferes with the accu- mulation of rhodamines and drugs participating in the MDR phenomenon inside a wide variety of healthy and malignant cells. Presumably, drugs exhibiting a high affinity toward their intracellular targets over- come this effect of MRP1 by tight binding to their receptors. Thus, the resistance provided by the over- expression of ABC transporters, such as Pgp, to MDR cells could be due to their ability to compete with the tight binding of the drugs to their respective targets, rather than their capacity to compete with the passive uptake of the drugs across the plasma membrane. It is possible that the capacity of MRP1 to reduce the intracellular concentrations of certain drugs in ‘sensi- tive’ cells is more relevant to the clinical side-effects of the drugs and less relevant to their chemotherapeutic effect upon binding to their specific targets. Experimental procedures K562, a human leukemia cell line established from a patient with chronic myelogeneous leukemia in blast trans- formation [41], was purchased from ATCC (Rockville, MD, USA) and maintained in RPMI medium (Biological Industries, Beit-Haemmek, Israel). The K562 Pgp over- expressing subline was obtained by sequential exposure of cells to increasing concentrations of doxorubicin and was maintained in the presence of 0.5 lm doxorubicin. A total of 2008 parental cells and their MRP1, MRP2 and MRP3 over-expressing cell lines, as well as HEK293 parental cells and HEK293 over-expressing MRP4 [42] or MRP5 [43], were kindly provided by P. Borst (Netherlands Cancer Institute, Amsterdam, the Netherlands) and grown in RPMI-1640 (Sigma-Aldrich, Rehovot, Israel). The CIR [44], CCRF-CEM [45] and the Jurkat [46] cell lines were purchased from ATCC (Rockville, MD, USA) and main- tained in RPMI-1640 (Sigma-Aldrich). GLC4 cells and MRP1 over-expressing GLC4 ⁄ ADR cells [47] were cultured in RPMI 1640. All media were supplemented with 10% fetal bovine serum, 100 IUÆmL )1 penicillin and 100 lgÆmL )1 streptomycin (Invitrogen, Rehovot, Israel) and the cells were grown at 37 °C under 5% CO 2 ⁄ humi- dified air. D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 645 TMRM, rhodamine 123, rhodamine 6G, silicone oil AR200, NBD-Cl and mineral oil were purchased from Sigma. TMR was purchased from Molecular Probes (Invi- trogen). For determination of the amount of rhodamine associ- ated with cells, cells were incubated with the dyes in a medium containing NaCl (132 mm), KCl (3.5 mm), CaCl 2 (1 mm), MgCl 2 (0.5 mm), glucose (10 mm), CCCP (1 lm) and Hepes–Tris buffer (20 mm, pH 7.4). Samples contain- ing 4 · 10 5 cells in 0.4 mL of medium were withdrawn and placed in an Eppendorf-style microfuge above a 0.2 mL cushion composed of 95 parts silicone oil AR 200 (d 20 = 1.049) and five parts mineral oil (d 20 = 0.89). After centrifugation for 4 min at 13 226 g at room temperature, the oil cushion was washed three times with water by suction. Subsequently, all of the upper phase, including part of the oil cushion, was removed, leaving a fraction of the oil above the cell pellets. The cell pellets were dissolved by addition of 0.1 mL of guanidine HCl (5 m) buffered with Hepes–Tris (50 mm, pH 7.4), centrifugation for 5 min, and incubation for at least 1 h at room tempera- ture. The dissolved samples were mixed thoroughly with 0.5 mL of water and centrifuged for 5 min. Samples (0.4 mL) were withdrawn from the pellets dissolved in the aqueous phase. The fluorescence of TMR, TMRM, rhoda- mine 123 and rhodamine 6G was determined using an excitation wavelength of 552, 563, 496 or 527 nm and an emission wavelength of 576, 583, 526 or 552 nm, respec- tively. To ensure fidelity of the assay, dye-free cell samples were mixed with known amounts of rhodamines and pro- cessed as above. The rhodamine yield, thus obtained, matched the amount expected. To determine the volume of incubation medium carried through the oil cushion together with the cells, a cell sample was incubated on ice with 10 lm acidic dye (calcein) and processed as above. The amount of calcein associated with the cells was equiv- alent to < 0.05% of the sample volume. The time period required to separate cells from the external medium was equivalent to 1.5 min. All curves were adjusted accord- ingly. 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