A novel energy dependent mechanism reducing ... - Nature

Leukemia (1997) 11, 48–53  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

A novel energy dependent mechanism reducing daunorubicin accumulation in acute myeloid leukemia DW Hedley1, S Xiu-Yan Xie1, MD Minden1, CH Choi1,2, H Chen1,3 and V Ling1,4 1 Ontario Cancer Institute/Princess Margaret Hospital, Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada

Using cyclosporin A (CsA) to inhibit P-glycoprotein (P-gp) function we showed previously that there was a discordance between the ability of acute myeloid leukemic (AML) blast cells to accumulate daunorubicin and P-gp antigen expression (Xie et al, Leukemia 1995; 9: 1882–1887). This discordance suggests that a CsA-sensitive drug efflux mechanism distinct from P-gp is expressed in many clinical samples. In the present study using the ATP depleting agents cyanide, azide, or dinitrophenol to inhibit energy dependent transport processes, we observed even larger increases in daunorubicin accumulation than were seen with CsA. Similar patterns were seen in a wide range of Pgp negative human cancer cell lines. Also the observed cyanide effect did not correlate with the expression of mRNA for multidrug resistance-associated protein (MRP), the only other member of the ABC family of membrane transporters that is known to be capable of effluxing daunorubicin. Thse results suggest that daunorubicin accumulation in many cases of AML is modulated by one or more novel energy-dependent processes that are distinct from P-gp or MRP. We speculate that this novel drug transport mechanism(s) may influence the response of AML patients to daunorubicin and other therapeutic agents. Keywords: leukemia; drug resistance; P-glycoprotein; MRP; ABC transporter

Introduction P-glycoprotein is a member of a large class of membrane transporters coded for by the ATP-binding cassette (ABC) gene superfamily.1,2 The physiological function of most of these ABC transporter gene products is unknown, but an additional member, multidrug resistance associated protein (MRP), has recently been shown to produce resistance to a range of natural products such as anthracyclines and vinca alkaloids by an active transport mechanism similar to that of P-gp.3,4 Although expression of MRP has been observed in leukemic blasts its clinical significance is still unknown.5 When we compared the amount of P-gp antigen expressed on the cell membrane to the modulating effects of the P-gp inhibitor cyclosporin A (CsA) on daunorubicin accumulation, we found little correlation between the two assays in a series of AML patients.6 Compared to the results obtained with P-gp expressing cell lines, the AML samples showed generally greater transport function in relation to Pgp antigen expression. This suggests that the modulating effect of CsA on daunorubucin accumulation, commonly regarded as a P-gp functional assay, can detect drug efflux mechanisms that are antigenically distinct from classical Pg-p. Because CsA might be a poor inhibitor of some of these putative non-P-gp transporters, the functional assay was modified by replacing CsA with agents that inhibit Correspondence: DW Hedley Present addresses: 2Department of Pharmacology, Chosun University, Kwangju City, Korea; 3Allelix Corporation, Mississauga, Ontario, Canada; 4BC Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada Received 30 May 1996; accepted 27 September 1996

oxidative phosphorylation, such as cyanide and azide, thereby depleting cellular ATP and inhibiting all energy-dependent processes. This strategy was used originally to demonstrate the ATP dependence of P-gp,7 and more recently has been shown to inhibit drug efflux via MRP.8,9 We now report that it also appears to show the presence of high levels of non-P-gp mediated daunorubicin efflux in cell lines and blast cells obtained from patients with acute myeloid leukemia. Materials and methods

Cell lines The following human cell lines were used to study energydependent daunorubicin efflux: CCRF-CEM T lymphoblasts, the vinblastine-resistant lines VLB 0.1 and VLB 1.0,8 and the AML lines OCI/AML 1–5, established in this Institute. In addition, we investigated the human carcinoma lines: MGHU1 (bladder), MCF7 (breast), SKOV3 (ovary), and HCT8 (colon). Parental HeLaS3 cervical carcinoma cells, and the multidrug resistance-associated protein (MRP) expressing variant HeLaJ2,4 were kindly supplied by Dr Raymond Baker, Roswell Park Cancer Institute, Buffalo, NY, USA. With the exception of the CCRF-CEM VLB 0.1 and 1.0 cells, none of these lines showed increased expression of P-glycoprotein as assessed using Western blotting with monoclonal antibody C219,10 or surface immunolabelling with the MRK-16 antibody.11

Clinical samples Peripheral blood samples containing at least 50% leukemic blasts were obtained from 49 patients with acute myeloid leukemia, either at initial presentation (27 patients) or with recurrent disease following prior chemotherapy (22 patients). Induction chemotherapy consisted of a standard protocol of high dose cytosine arabinoside and daunorubicin. Blasts were separated by density gradient centrifugation, washed, counted and resuspended in PBS at 1 × 106/ml.

Flow cytometric measurement of P-gp Cell surface expression of Pgp was detected using the MRK16 antibody (gift of Dr T Tsuruo, University of Tokyo), which recognizes an external epitope of the human MDR-1 gene product.11 Because epitope masking can impair labelling of AML blasts with this antibody,6,12 cells were incubated with 0.2 U neuraminidase (type V, from Clostridium perfringens; Sigma Chemicals, St Louis, MO, USA) per 2 × 106 cells in 1 ml at 37°C for 30 min, and washed in PBS prior to antibody labelling. Cells were resuspended at 1 × 107/ml in PBS, and

Energy-dependence of daunorubicin uptake in AML DW Hedley et al

incubated for 30 min with a 1:10 dilution of goat gammaglobulin on ice, to block non-specific binding sites. They were then incubated with 20 mg/ml MRK-16 antibody for a further 30 min on ice. An MRK-16 dilution curve using CCRF-CEM VLB 0.1 cells showed that this was a saturating concentration of antibody. After washing twice with cold PBS, the cells were incubated with a 1:25 dilution of phycoerythrin (PE)-conjugated F(ab′)2 goat anti-mouse immunoglobulin (Dakopatts, Denmark) on ice for 30 min, and washed twice.6 Fluorescence calibration beads were used to obtain an estimate of the number of antigenic sites per cell. These calibration beads (SpheroTech, Libertyville, IL, USA), consist of a mixture of particles at six different fluorescent intensities, calibrated to give the mean equivalent soluble fluorochrome (MESF). Thus the mean number of phycoerythrin molecules bound to the cell surface can be estimated.

Flow cytometric measurement of daunorubicin accumulation Cells were incubated in 3 mg/ml daunorubicin, which is naturally fluorescent, at a concentration of 1 × 106 cells/ml, ±3 mg/ml CsA at 37°C for 30 min. Increased efflux of daunorubicin by Pgp causes a decrease in daunorubicin accumulation that can be reversed by the addition of CsA. The mean fluorescence from each daunorubucin and daunorubucin + CsA sample was recorded, and the Pgp functional activity expressed as the percentage increase in daunorubicin fluorescence after CsA treatment, ie: (mean fluorescence + CsA) − (mean fluorescence − CsA) × 100. (mean fluorescence − CsA)

In addition to CsA, which is a classical P-gp inhibitor, cellular ATP was depleted by the addition of 2 mm KCN, 2 mm NaN3, or 1 mm dinitrophenol, in order to inhibit all energy-dependent drug transport mechanisms. Thus a % functional activity (KCN etc) was calculated similar to the % functional activity (CsA). Flow cytometry was done using an Epics Elite cell sorter (Coulter Electronics, Hialeah, FL, USA) equipped with an aircooled argon laser emitting at 488 nm and operating at 15 mW. Forward and side scatter signals were collected using linear amplification to define the blast population, from which the fluorescence data were taken. Phycoerythrin fluorescence was measured with a 575 nm bandpass filter using logarithmic amplification, and daunorubicin fluorescence with the same filter using linear amplification. A minimum of 20 000 events were collected for each sample.

actin cDNA, the original probe was removed by boiling the blots in 0.5% SDS for 5 min. Expression of MRP in human cell lines was measured using Northern blotting. Results were compared to the level of b actin expression, and for comparison between the cell lines they were normalized with the ratio MRP:b action for HeLaS3 cells set as 1. Results

Measurement of P-gp function and antigen expression in acute leukemia samples Using the methods described above, we have previously found that in the blast cells of the majority of AML patients, P-gp antigen expression is detectable, and daunorubicin accumulation is increased in the presence of CsA, an inhibitor of P-gp function. Unexpectedly, there appeared to be little correlation between the amount of P-gp detected and the functional assay.6 In the present study we used fluorescent calibration beads to quantitate the number of phycoerythrin molecules bound per cell, thereby obtaining an estimate to the actual number of P-gp antigenic sites. Although the stoichiometry of the immunolabelling technique is unlikely to be strictly 1:1, the error introduced is small in relation to the range of values seen between individual samples, and the use of standardized measurement units facilitates comparison between the levels of P-gp found in different cell populations. As seen in Figure 1, there is a clear correlation between transport function and antigen expression in a series of CCRF-CEM human T lymphoblast lines, which are well characterized with regard to their P-gp expression. When the data for the cell lines and AML samples from patients were plotted using the same axis scales, it was observed that compared to the cell lines, the clinical samples showed relatively low levels of

Measurement of Pgp and MRP mRNA Cells grown at exponential stage were harvested and subjected to total RNA isolation using the guanidinium isothiocyanate method according to Chomczynski and Sacchi.13 Total RNA 15 mg was then size fractionated on a denaturing formaldehyde agarose (1%) gel, transferred to Hybond N Membrane (Amersham, Arlington Heights, IL, USA), and probed with a (32P) labelled MRP cDNA obtained from Dr Susan Cole, Queen’s University, Kingston, Ontario, Canada. (Fragment 10.1, comprising 1 kb from the 3′ end of MRP, including the second nucleotide binding domain, 1–5 × 105 c.p.m./ml, 105 c.p.m./mg, random priming method.) For reprobing with b-

Figure 1 Correlation between the percentage increase in daunorubicin accumulation with CsA and P-gp antigen expression determined by quantitative MRK-16 labelling. CCRF-CEM lines (I): CEM wildtype, CEM VLB 0.1 and CEM VLB 1.0; blasts from AML patients (o). Data replotted from Ref. 6.

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MRK-16 labelling but some of the samples showed disproportionately high increases in daunorubicin accumulation with CsA, relative to the number of P-gp antigenic sites per cell (Figure 1). This suggests that in a significant proportion of AML patients, CsA-inhibitable daunorubicin efflux can occur by mechanisms that are antigenically distinct from P-gp. It is now established that energy-dependent efflux of daunorubicin can result from overexpression of MRP, another member of the ABC family of membrane transporters.8,14 Furthermore, MRP expression has recently been reported in some AML patients.5,15 MRP is less sensitive than P-gp to CsA inhibition.8 We were therefore interested in determining if the concentration of CsA used in our experiments would be effective against an MRP-mediated reduction in daunorubicin accumulation. We used the well-characterized HeLaJ2 cell line containing an amplified MRP gene4 to investigate this question. We observed no measurable increase in daunorubicin accumulation when these cells were treated with 3 mg/ml CsA (data not shown). Therefore, the CsA-stimulated accumulation of daunorubicin observed in the AML samples is not likely due to expression of MRP.

Effects of metabolic inhibitors on the accumulation of daunorubicin by cell lines In addition to agents such as CsA or verapamil, that probably act by competitive inhibition of substrate binding sites, drug efflux via P-gp can be reversed using ATP-depleting agents such as cyanide or azide. This strategy was originally used by Ling and colleagues7 to show the energy dependence of P-gp. More recently, depletion of cellular ATP has also been shown to inhibit MRP,8,9 and is likely to inhibit all other energydependent processes influencing daunorubicin accumulation. In other words, substitution of ATP-depleting agents for CsA in the daunorubicin accumulation assay will give a summation of all energy-dependent drug transport processes, regardless of whether these are sensitive to classical P-gp inhibitors such as CsA or verapamil. Initially, the five human AML cell lines OCI/AML 1–5 were investigated, since these have been extensively characterized in our Institution, and express very low levels of P-gp. This was confirmed in the present series of experiments by demonstrating a lack of MRK-16 staining and low levels of CsAdependent increases in daunorubicin accumulation in all five lines. Cells were treated with 2 mm KCN, 2 mm sodium azide, or 1 mm dinitrophenol (DNP) in glucose-free medium, in an assay that was otherwise identical to the P-gp functional assay described above. Similar results were obtained for all five AML lines, with a mean increase in daunorubicin accumulation of 122% ± 25 s.d. using KCN, compared to 6% ± 4 s.d. when CsA was used as the modulating agent. Similar results were obtained for all three metabolic inhibitors, and for this reason most subsequent experiments were done using 2 mm KCN only. In order to establish that the increases in daunorubicin accumulation seen with KCN treatment were due to the inhibition of an ATP-dependent efflux mechanism, 5 mm glucose was added at the end of the 30 min incubation period. The cells were maintained in daunorubicin ± KCN throughout the experiment. In the presence of continued exposure to KCN, glucose allows ATP generation to take place by glycolysis.7 As shown in Figure 2, addition of glucose to cells pre-loaded with daunorubicin in the presence of KCN resulted in the rapid loss of drug, so that by 20 min daunorubicin fluor-

Figure 2 Reversal of KCN effect by addition of glucose. AML1 cells were preloaded with daunorubicin in the presence (k) or the absence (K) of 2 mm KCN. At time = 0, glucose 5 mm was added in order to allow ATP generation by glycolysis. Note that daunorubicin fluorescence rapidly falls to that of cells loaded with drug in the absence of KCN.

escence values were similar to those of controls that were preloaded with daunorubicin in the absence of KCN. This reversal effect of glucose is consistent with our hypothesis that KCN treatment inhibits an ATP-dependent daunorubicin efflux mechanism. In addition to active extrusion via P-gp, cellular accumulation of weak bases such as daunorubicin may be influenced by the pH gradient across the surface membrane.16,17 A relative reduction in intracellular pH may cause decreased drug accumulation, because the equilibrium is shifted to the protonated form which diffuses much more slowly through lipid bilayers. To test the possibility that KCN was causing increased daunorubicin accumulation by producing cellular acidosis, thereby altering drug partitioning across the surface membrane, cells were loaded with the pH-sensitive dye SNARF-1, and relative intracellular pH values measured using a standard ratio metric flow cytometry assay.18 Within the sensitivity of the assay (approximately 0.2 pH units) there was no detectable change in intracellular pH during the 30 min exposure to KCN, suggesting that acidification of cytoplasm is unlikely to account for the increased daunorubicin uptake.

KCN-sensitive daunorubicin accumulation and MRP expression in P-gp-negative human cell lines The increased daunorubicin accumulation seen in the OCI/AML cell lines following KCN treatment suggests that energy-dependent mechanisms other than classical P-gp are expressed in these cells, and inhibit drug uptake. To date the


only other ABC transporter known to be expressed in human cells and capable of transporting daunorubicin is MRP. We therefore compared the expression of MRP to the effect of KCN on daunorubicin accumulation. In addition to the OCI/AML lines, we examined parental CCRF-CEM T lymphoblasts, and a series of human carcinoma lines including parental HeLa cells and the MRP overexpressing line HeLaJ2. None of these cell lines overexpresses P-gp. Expression of MRP was determined by comparing levels of mRNA for MRP to those for b actin, using Northern blotting, these results then being normalized to MRP expression by parental HeLaS3 cells, which was set an an arbitrary value of 1. As shown in Figure 3, there was a wide range of values between the 12 cell lines. As expected, the highest level of MRP expression was found in HeLaJ2 cells, but significant levels were also seen in the OCI/AML lines AML1 and AML4, and in the HCT8 colon cancer line. In all cases, daunorubicin uptake was increased by co-incubation with 2 mm KCN, but there was no correlation with the level of MRP expression. The results for the OCI/AML lines were particularly striking. As illustrated in Figure 3, despite expressing very low levels of MRP mRNA, the lines AML2, AML3 and AML5 showed similar increases in daunorubicin uptake with KCN treatment to those seen in the MRP expressing lines AML1 and AML4.

Comparative effects of CsA and KCN on daunorubicin uptake by AML blasts A comparison was made between the effects of CsA and KCN on daunorubicin accumulation by leukemic blasts obtained from patients. In some clinical samples the effects of KCN

Figure 3 Correlation between the MRP mRNA level and percentage increase in daunorubicin accumulation after KCN treatment in a series of low P-gp expressing human leukemic and epithelial tumor cell lines.

were comparable to those seen with CsA, suggesting that Pgp overexpression or some other non-P-gp but CsA-sensitive mechanism was a major determinant of the reduced daunorubicin uptake. In others, the increase was much greater with KCN suggesting the presence of both CsA-responsive and nonresponsive energy-dependent daunorubicin transport mechanisms (Figure 4). These results are summarized in Figure 5. In the majority of cases, accumulation of daunorubicin was increased in the presence of 2 mm KCN. The magnitude of this effect varied widely between patients, ranging from no significant effect to .200% increases in daunorubicin accumulation. The trend was for larger increases in drug uptake with KCN treatment in patients with recurrent disease following previous chemotherapy using daunorubicin-containing regimens compared to patients at initial diagnosis (mean increase 123% ± 51 s.d., compared to 95% ± 57 s.d.; P . 0.05 using Student’s t-test). Discussion Using neuraminidase pretreatment and a sensitive immunofluorescence technique, P-gp could be detected in almost all cases of AML. However, the levels of expression were 30–100 times lower than those seen in P-gp overexpressing CCRFCEM cell lines. Considerable interpatient heterogeneity was seen in the levels of both P-gp antigen expression, and in cyclosporin-inhibitable daunorubicin efflux. Although the latter is widely taken to be a measure of P-gp functional activity, there was no significant correlation with P-gp antigen expression by AML leukemic blasts.6 This finding is consistent with some other recent reports for AML,19,20 and in contrast to chronic lymphocytic leukemia, where the daunorubicin accumulation assay has been shown to correlate closely with the levels of P-gp antigen and mRNA expression.12,21 Replotting the AML patient results on the same axis scale as that for the P-gp expressing cell lines shows that in many cases the patient samples have much higher activity in the functional assay than would be predicted from the amount of P-gp antigen on the cell surface (Figure 1). One explanation of this finding might be overexpression of MRP, the only other ABC transporter that is known to be expressed in AML and to be capable of effluxing daunorubicin. However, CsA is a poor inhibitor of MRP. Using a concentration of 3 mm CsA we found no significant increases in uptake of daunorubicin by the MRP amplified line HeLaJ2, or the leukemic lines OCI/AML1 and 4, both of which showed considerable MRP

Figure 4 Overlay histograms showing uptake of daunorubicin by leukemic blasts from an AML patient, comparing the effects of CsA and KCN to control values.

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Figure 5 Comparison between the modulating effects of CsA and KCN on daunorubicin accumulation by AML blasts obtained from: (a) patients at initial diagnosis, and (b) patients with recurrent disease following previous chemotherapy.

expression by Northern blotting. A more likely explanation is that a novel CsA-sensitive mechanism is reducing daunorubicin accumulation in many AML samples. A large number of ABC transporters have now been identified in human cells on the basis of sequence homologies in the highly conserved ATP binding region.1 The function of most of these is unknown. Because of the possibility that an additional ABC transporter might be active in some AML samples, the functional assay was modified using ATP-depleting agents in order to inhibit all energy-dependent processes. This strategy was originally used to demonstrate the ATP dependence of P-gp,7 and has recently been shown to inhibit MRP.8,22 It is likely to provide a broader spectrum of activity than agents such as CsA that inhibit substrate binding sites. Generalized ATP depletion resulted in large increases in daunorubicin accumulation by a wide range of human cell lines, including some that express little or no P-gp or MRP. Addition of glucose, which allows ATP generation by glycolysis, reversed this effect. Alterations in intracellular pH have been shown to affect cellular accumulation and subcellular distribution of weakly basic drugs such as daunorubicin.16 However, no significant changes in intracellular pH were detected during 30 min exposure to 2 mm KCN. The most likely explanation for these findings is that ATP depletion inhibits an energy-dependent process that reduces daunorubicin accumulation. Comparison of the modulating effects of CsA and KCN in the AML samples shows considerable interpatient heterogeneity (Figure 5). In some cases neither agent increased drug accumulation, indicating that no energy-dependent processes were active. In others the effects of CsA and KCN were roughly comparable, suggesting the expression of P-gp or a closely related transporter. In striking contrast, however, some patient samples showed very little modulation of daunorubicin uptake by CsA, but large increases with KCN. We propose that when KCN is used as the modulating agent, the daunorubicin accumulation assay sums the activities of all energydependent processes that reduce drug uptake, whether these

be ABC transporters, or other mechanisms. If this interpretation is correct, then some AML patient samples show daunorubicin efflux rates that are similar to those seen in CCRF-CEM VLB 1.0 cells expressing .100 000 P-gp antigenic sites per cell (see Figure 1). We do not believe that the energy-dependent drug transport phenomena observed in the leukemia patients can be explained simply on the basis of variable expression of P-gp and MRP, since large increases were seen in daunorubicin accumulation following KCN treatment were also seen in AML lines that express very low levels of P-gp and MRP. More likely, this study suggests the presence of a novel mechanism(s) in some patient blast cells. Identification of new drug resistance mechanisms usually involves the use of cell lines selected by in vitro drug exposure. Stepwise increases in drug concentrations over time can result in very high levels of resistance to agents such as anthracyclines or vinca alkaloids, frequently associated with increased P-gp or MRP expression. However, drug selection is clearly not essential for this since elevated P-gp or MRP levels are also seen in some untreated cancer patients, possibly because the cancer is derived from a normal tissue that naturally expresses the resistance mechanism, or as a genetic alteration occurring as part of the neoplatic process. It is now recognized that the ABC transporter family is extremely diverse in nature,1 and that several members are capable of conferring drug resistance in a wide range of species. The human genome may contain many ABC transporters which have not yet been identified and it would not therefore be surprising if some of these as yet uncharacterized ABC transporters were constitutively expressed in AML blasts, and capable of transporting daunorubicin. The constitutive expression of high levels of non-P-gp drug transporters might be one explanation for the comparatively weak association between increased P-gp expression and treatment outcome in AML patients.23 Furthermore, the data shown here suggest that although CsA is an effective P-gpmodulating agent, it may fail to improve treatment response to anthracyclines in some patients due to the existence of drug


transporters that are relatively insensitive to CsA. A prospective clinical study to determine the prognostic significance of KCN-sensitive daunorubicin accumulation and detailed molecular characterization of this phenomenon are currently under way in our institutions.

Acknowledgements We wish to thank Dr Susan Cole, Queen’s University, Kingston, for supplying the cDNA probe for MRP, and Cynthia Fisher and Sarah Childs for critically reading the manuscript. This work was supported by grants from the National Cancer Institute of Canada and the Leukemia Research Fund of Canada. Clinical material was supplied by the core of the Leukemia Program, which is supported by a Terry Fox program project grant.

References 1 Childs S, Ling V. The MDR superfamily of genes and its biological implications. In: VT DeVita, S Hellman, SA Rosenberg (eds). Important Advances in Oncology. Lippincott: Philadelphia, 1994, pp 21–36. 2 Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Ann Rev Biochem 1993; 62: 385–427. 3 Almquist KC, Loe DW, Hipfner DR, Mackie JE, Cole SPC, Deeley RG. Characterization of the Mr 19 000 multidrug resistance protein (MRP) in drug-selected and transfected human tumor cells. Cancer Res 1995; 55: 102–110. 4 Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, Deeley RG. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992; 258: 1650–1654. 5 Schuurhuis GJ, Broxterman HJ, Ossenkoppele GJ, Baak JPA, Eekman CA, Kuiper CM, Feller N, van Heijningen THM, Klumper E, Pieters R, Lankelma J, Pinedo HM. Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-b-d-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia. Clin Cancer Res 1995; 1: 81–93. 6 Xie SX-Y, Robb D, Chow S, Hedley D. Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia. Leukemia 1995; 9: 1882–1887. 7 See YP, Carlsen SA, Till JE, Ling V. Increased drug permeability in Chinese hamster ovary cells in the presence of cyanide. Biochim Biophys Acta 1974; 373: 242–252. 8 Cole SP, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, Deeley RG. Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res 1994; 54: 5902–5910.

9 Versantvoort CHM, Broxterman HJ, Lankelma J, Feller N, Pinedo HM. Competitive inhibition by genistein and ATP dependence of daunorubicin transport in intact MRP overexpressing human small cell lung cancer cells. Biochem Pharmacol 1994; 48: 1129–1136. 10 Kartner N, Evernden-Porelle D, Bradley G, Ling V. Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 1985; 316: 820–823. 11 Ishida Y, Ohtsu T, Hamada H, Sugimoto Y, Tobinai K, Minato K, Tsuruo T, Shimoyama M. Multidrug resistance in cultured human leukemia and lymphoma cell lines detected by a monoclonal antibody, MRK16. Jap J Cancer Res 1989; 80: 1006–1013. 12 Cumber PM, Jacobs A, Hoy T, Whittaker JA, Tsuruo T, Padua RA. Increased drug accumulation ex vivo with cyclosporin in chronic lymphatic leukemia and its relationship to epitope masking of Pglycoprotein. Leukemia 1991; 5: 1050–1053. 13 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt Biochem 1987; 162: 156–159. 14 Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SPC, Deeley RG. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res 1994; 54: 357–361. 15 Burger H, Nooter K, Zaman GJ, Sonneveld P, van Wingerden KE, Oostrum RG, Stotor G. Expression of the multidrug resistanceassociated protein (MRP) in acute and chronic leukemias. Leukemia 1994; 8: 990–997. 16 Simon S, Roy D, Schindler M. Intracellular pH and the control of multidrug resistance. Proc Natl Acad Sci USA 1994; 91: 1128– 1132. 17 Roepe PD. Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cell expressing different levels of P-glycoprotein. Biochemistry 1992; 31: 12555– 12564. 18 Boyer MJ, Hedley DW. Measurement of intracellular pH. In: Darzynkiewicz Z, Robinson JP, Crissman HA (eds). Methods in Cell Biology, Vol 41. Academic Press: San Diego, 1994, pp 135–148. 19 Leith CP, Chen I-M, Kopecky KJ, Appelbaum FR, Head DR, Godwin JE, Wieck JK, Willman CL. Correlation of multidrug resistance (MDR1) protein expression with functional dye/drug efflux in acute myeloid leukemia by multiparameter flow cytometry: identification of discordant MDR−/effux+ and MDR1+/efflux− cases. Blood 1995; 86: 2329–2342. 20 Maslak P, Hegewisch-Becker S, Godfrey L, Andreeff M. Flow cytometric determination of the multidrug-resistant phenotype in acute leukemia. Cytometry 1994; 17: 84–93. 21 Herweijer H, Sonneveld P, Baas F, Nooter K. Expression of mdr1 and mdr3 multidrug-resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. J Natl Cancer Inst 1990; 82: 1133–1140. 22 Versantvoort CH, Broxterman HJ, Lankelma J, Feller N, Pinedo HM. Competitive inhibition by genistein and ATP dependence of daunorubucin transport in intact MRP overexpressing human small cell lung cancer cells. Biochem Pharmacol 1994; 48: 1129–1136. 23 Holmes JA, West RR. The effect of MDR-1 gene expression on outcome in acute myeloid leukemia. Br J Cancer 1994; 69: 382–384.

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