Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1

British Journal of Haematology, 2001, 114, 581±590

Cells from chronic myelogenous leukaemia patients at

presentation exhibit multidrug resistance not mediated by

either MDR1 or MRP1

Anne Carter,1 Eldad J. Dann,1 Tamar Katz,1 Yael Shechter,1 Ana Oliven,1 R. Regev,2 Esther Eytan,1

Jacob M. Rowe1 and Gera D. Eytan2 1Haematology Department and Blood Bank, Rambam Medical Centre,

and 2Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Received 27 January 2001; accepted for publication 23 April 2001

Summary. Tetramethylrosamine (TMR) is excluded from P-glycoprotein (MDR1)-enriched cell lines, but it stainsefficiently MDR1-poor parent lines. Application of theTMR resistance assay to cells obtained from chronicmyelogenous leukaemia (CML) patients revealed, in allindividuals, a significant resistance compared with healthydonors (P , 0´001). Cells from the same patients at laterphases exhibited a further increase in TMR resistance.Doxorubicin was excluded from all cell samples obtainedfrom CML patients at presentation. The resistance to TMRand doxorubicin was energy-dependent, and was notmodulated by inhibitors of MDR1 and multidrug-resistanceprotein-1 (MRP1). Transcription of mRNAs suspected asrelevant to multidrug resistance was assessed usingcomparative reverse transcription polymerase chain reac-

tion. All cells from the CML patients transcribed high levelsof MRP3, MRP4 and MRP5 compared with healthy donors.Low levels of MDR1, MRP1, MRP2, MRP6, lung resistance-related protein and anthracycline resistance-associatedprotein were equally transcribed in cells from healthydonors and CML patients. These results indicated thatneither MDR1 nor MRP1 mediate the resistance in thesecells. Our results shed light on a resistance mechanismoperative in CML patients, which, together with theresistance to apoptosis, is responsible for the lack of responseof CML patients to induction-type protocols used to treatacute myeloid leukaemia patients.

Keywords: chronic myelogenous leukaemia, multidrugresistance, MDR1, MRP1, doxorubicin.

The mechanisms of resistance and progression are poorlyunderstood in chronic myeloid leukaemia (CML) (Giles et al,1999). Disease evolution in CML has been associated withcytogenetic clonal evolution; overexpression of interleukin1; deletions and mutations of the p53 suppressor gene;overexpression of c-myc, p16 and Rb abnormalities; andboth general and site-specific increases in DNA methylation(Faderl et al, 1999a; Sawyers, 1999). The chronic phase ofCML can be controlled by combination of interferon-a withcytotoxic drugs, such as hydroxyurea, cytarabine, busul-phan and homoharringtonine, but survival is extremelyshort after the onset of blastic crisis, which usually occurswithin a few years after the onset of CML (Fader et al,1999a; Sawyers, 1999). Leukaemia cells in this phase areextremely resistant to antileukaemic agents (Fader et al,1999b; Fang et al, 2000; Sawyers, 1999). This resistance

has been ascribed to inhibition of apoptosis in cells fromCML patients and cells transformed with the BCR±ABLoncogene. In leukaemic blasts, the expression of CML-associated BCR±ABL tyrosine kinase inhibits antileukaemiadrug-induced mitochondrial membrane potential dissipa-tion and cytochrome c release, thereby blocking theactivation of the downstream caspases and apoptosis(McGahon et al, 1994; Bedi et al, 1995; Amarante-Mendeset al, 1998). In the present study, we show that CML cells, atinitial diagnosis, are already capable of actively excludingdrugs such as doxorubicin. Resistance mediated by drugexclusion could complement defective drug-induced apop-tosis as the mechanism that allows CML cells to thrive,despite cytotoxic chemotherapy.

Cancer cells that overexpress drug-transporting proteinsmay become resistant to a wide spectrum of drugs withdifferent structures or cellular targets, a phenomenon calledmultidrug resistance (MDR) (Ambudkar et al, 1999). ATP-dependent trans membrane drug transporters, such as P-glycoprotein (MDR1) (Ambudkar et al, 1999) and the

q 2001 Blackwell Science Ltd 581

Correspondence: Gera D. Eytan, Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. E-mail:

[email protected]

multidrug resistance protein (MRP1) (Cole & Deeley, 1998),can render cells multidrug resistant. Whereas, the functionof MDR1 in normal tissues appears to be limited to thedefence against drugs and other xenotoxins, MRP1 plays arole in extruding drugs across some specialized epithelia(Wijnholds et al, 1998). It is the major transporter forendogenous leukotriene C4, an important mediator of theinflammatory response. A homologue of MRP1, known asMRP2 and cMOAT, is expressed mainly in the canalicularmembrane of hepatocytes (Buchler et al, 1996; Paulusmaet al, 1996, 1997; Ito et al, 1997). Absence of MRP2 leads totransport deficient canalicular membranes of hepatocytesfound in the TR2 and the Eisai hyperbilirubinaemic rats andin patients with Dubin±Johnson syndrome, all characterizedby a mild chronic conjugated hyperbilirubinaemia (Jansenet al, 1985; Takikawa et al, 1991; Kartenbeck et al, 1996).MRP2 has been shown to confer low-level resistance to anti-cancer drugs cisplatin, etoposide, vincristine and metho-trexate (Cui et al, 1999; Evers et al, 1998). Several otherMRP family members may play a role in MDR. MRP3 hasbeen shown to confer resistance to etoposide, vincristineand methotrexate (Kool et al, 1999a; Young et al, 1999;Zeng et al, 1999). MRP4 and MRP5 confer resistance tonulcleoside analogues such as thiopurines and the anti-HIVdrug 9-(2-phosphonylmethoxyethyl)adenine (PMEA)(Schuetz et al, 1999; Wijnholds et al, 2000).

Lung resistance-related protein (LRP) (Scheper et al,1993) is frequently overexpressed in MDR cancer cells andits expression has been correlated with poor prognosis (Borget al, 1998; Izquierdo et al, 1995; List et al, 1996). LRP is thehuman major vault protein (Scheffer et al, 1995) thatconstitutes over 70% of the mass of vault ribonucleoproteinparticles. These particles are present in all eukaryotic cells,and their structure and protein composition are highlyconserved (Kedersha et al, 1990). The vault function, bothin cells and in MDR, remains an enigma. Association of asubpopulation of vault particles with oestrogen receptors inthe MCF-7 breast cancer cell lines suggests a role for thevault particles in intracellular traffic, possibly between thecytoplasm and the nucleus (Abbondanza et al, 1998).

To investigate resistance phenomena in cells from CMLpatients at presentation, samples were analysed for func-tional MDR phenomenon with MDR-type dye and drug. Allcells exhibited resistance that was energy dependent, N-ethylmaleimide sensitive and not modulated by knowninhibitors of MDR1 and MRP1. Of nine proteins known to beassociated with MDR phenomena in other cells, thetranscription levels of MRP3, MRP4 and MRP5 were highcompared with levels in cells from healthy donors. One ormore of these proteins might mediate the MDR phenomenonexhibited by cells from CML patients at presentation.

MATERIALS AND METHODS

Materials. TMR was purchased from Molecular Probes,Eugene, Oregon. Rhodamine 123 was purchased fromSigma, St Louis, MO, USA. Lymphoprep was purchasedfrom Nycomed Pharmacia Biothech AB, Uppsala, Sweden.Anti-MDR1 (clone MRK16), anti-MRP1 (clone MRPm6)

and anti-LRP (clone LRP-56) were purchased from KamiyaBiomedical, Seattle, WA, USA.

Preparation and flow cytometry of cell samples. Peripheralblood samples of healthy donors, acute myeloid leukaemia(AML) and CML patients donors were obtained from thelocal blood bank and from the Haematology Department, atthe Rambam Medical Centre. Mononuclear cells (MNC)were collected from peripheral blood using either a Ficoll±Hypaque gradient centrifugation or an apheresis machine.Apheresis product cells obtained from CML patients andfrom healthy donors had a similar content of myeloid cellsand lymphocytes. The pattern of TMR staining anddoxorubicin uptake was similar in cells prepared fromperipheral blood samples using Ficoll-Hypaque gradientcentrifugation and in cells collected by apheresis from thesame patients. The diagnosis of AML was establishedaccording to immunophenotypic, morphological and kar-yotypic analyses in each patient. The diagnosis of CML wasbased on typical morphology of the peripheral blood andbone marrow, and confirmed with cytogenetic and mole-cular analyses (Bennett et al, 1994) documenting thepresence of the Philadelphia chromosome (Ph) and theBCR±ABL fusion signal.

MNC were either assayed immediately or cryopreservedfor future analysis. Cells used immediately were suspendedin Roswell Park Memorial Institute (RPMI) 1640 mediumsupplemented with 5% fetal calf serum (FCS). For cryopre-servation, cells suspended in 10% dimethyl sulphoxide(DMSO) and 90% FCS were frozen slowly in liquid nitrogen.Prior to assay, frozen samples were rapidly thawed at 378Cand diluted 10-fold with pre-warmed RPMI 1640 mediumcontaining 40% FCS and 2 mmol/l EDTA. The cells werewashed with 10% FCS and 1 mmol/l EDTA in phosphate-buffered saline (PBS), suspended in RPMI 1640 mediumsupplemented with 5% FCS and 5 mmol/l MgSO4 to a finalcell concentration of 106 cells/ml. The cells were pre-incubated for 1 h at 378C, different concentrations (2´5±25000 nmol/l) of TMR dye were added, and the cells werethen incubated again for 1 h. Subsequently, the cells werewashed, suspended in cold PBS supplemented with 5% FCS,incubated with propidium iodide (2 mg/ml) at 48C for30 min and kept on ice until dye uptake into the cells wasmonitored. Flow cytometry was performed using either aBecton Dickinson FACS Calibur or FACStar instrument withstandard fluorescence filters (Becton Dickinson, San Jose,Ca, USA). The TMR fluorescence was compared with theautofluorescence of the cells, and transcribed as the ratio ofthe fluorescence measured in cells incubated with TMR tothe fluorescence of cells incubated in absence of dye. Thecontribution of dead cells was routinely subtracted fromfluorescence assays by gating out cells stained withpropidium iodide. Although the fluorescence of propidiumiodide stained-DNA could not be separated from the redfluorescence of either TMR or doxorubicin, the highfluorescence levels observed in cells permeable to propidiumiodide allowed for total gating out of dead cells. Samplescontaining more than 5% dead cells, defined as propidiumiodide permeable, were ignored.

Comparative reverse transcription polymerase chain reaction

582 A. Carter et al

q 2001 Blackwell Science Ltd, British Journal of Haematology 114: 581±590

(RT-PCR). Total RNA was extracted from cell samples usingthe acid guanidine thiocyanate chloroform method (Chomc-zynski & Sacchi, 1987). Reverse transcription reactions wereperformed using 1 mmol/l random hexamers (Promega,Madison, WI, USA) with Molony-murine leukaemia virus(M-MLV) reverse transcriptase according to the manu-facturer's instructions (Promega). First, to confirm theharvested RNA quality and titrate its amount, the reverse-transcribed cDNA synthesized from the same amount of totalRNA in each sample was subjected to PCR using b-actinprimers. The PCR was performed in a 25 ml volume using0´8 mmol/l primers, 2´5 mmol/l magnesium chloride,0´2 mmol/l dNTPs, various amounts of cDNA up to 10 mgand Taq DNA polymerase (Desai & Pfaffle, 1995) in anamount predetermined as optimal in a buffer containing50 mmol/l potassium chloride, 10 mmol/l Tris-HCl pH 9´0and 0´1% Triton-x-100. After pre-denaturation at 958C for5 min, the reaction mixture was subjected to 30 amplifica-tion cycles, comprising denaturation at 948C for 1 min,annealing at 588C for 1 min and extension at 728C for1 min, followed by a final incubation at 728C for 5 min. ThecDNA samples were diluted to yield a constant b-actintranscription. The transcription of the various proteins wasassayed with the cDNA amounts quoted in the legend to thefigures using the published primer pairs for b-actin, b2M,BCR-ABL and MDR1 (Noonan et al, 1990); forMRP1(Abbaszadegan et al, 1994); for MRP2 (Oguri et al, 1998);for MRP3, MRP4 and MRP5 (Kool et al, 1997); for MRP6(Kool et al, 1999b); for ARA (Belinsky & Kruh, 1999); andfor LRP (Scheffer et al, 1995), yielding fragments of 199,120, 460, 167, 260, 275, 262, 239, 381, 237, 475 and217 basepairs respectively.

RESULTS

Drug resistance exhibited by cells from CML patients wasassayed with Tetramethylrosamine (TMR), a dye that hasproven a sensitive functional assay for MDR in cell linesoverexpressing MDR1 (Eytan et al, 1997). Application of theTMR assay to MNC from blood samples of 24 CML patientsat presentation (median age 40´4 ^ 16´7 years) and 39healthy donors (38´5 ^ 13´7 years) revealed low staininglevels of MNC from the CML patients compared with thehealthy donors. The higher sensitivity of the TMR assaycompared with the standard rhodamine 123 assay enabledthe detection of resistance in CML patients. TMR fluores-cence is extraordinarily high and insensitive to bleaching bylight, thus allowing reproducible assays at TMR concentra-tions as low as 2´5 nmol/l. Although all the cells of ahealthy donor were stained with 2´5 nmol/l TMR (Fig 1A),most of the CML-patient cells completely excluded thisconcentration of the dye. Only a small subpopulation of theCML patient was stained by 2´5 nmol/l TMR. At higherTMR concentrations, the dye penetrated into all the cells,but even at higher concentrations it was still partiallyexcluded from most of the cells when compared with thehealthy donor cells. Distinct cell subpopulations differing intheir capacity to exclude TMR were observed (Fig 1A). Incontrast, the staining pattern of the same cells with

rhodamine 123 neither revealed significant differencesbetween the cells from the healthy donor and those obtainedfrom the CML patient, nor distinguished between cellsubpopulations (Fig 1B).

To test the reproducibility of the TMR assay, six separatesamples obtained on different occasions from the same CMLpatient were compared with samples from 39 healthydonors. As shown in Fig 2, the cellular levels of TMRobtained with the various CML samples from the samepatient were reproducible, especially at the low TMRconcentrations. Multiple TMR assays of the same bloodsample appeared practically identical (SD of less than 2%,data not shown). The high reproducibility of varioussamples obtained from the same patient allowed forconfident determinations of even low levels of TMRexclusion. The TMR-based assay involves one-step incuba-tion and produces reliable data without the use of specificinhibitors.

As shown in Fig 3, cells from CML patients at presenta-tion were already resistant to staining by TMR comparedwith cells from healthy donors (P , 0´001). The averageTMR fluorescence in CML cells was 3´7 ^ 1´3-fold theautofluorescence of the cells compared with 34´6 ^ 19´4 inhealthy donors. All the cell samples from the CML patientsexhibited resistance, and no overlapping was observedbetween the range of staining of these cell samples andthe range of staining of samples from healthy donors. Theresistance (1´7 ^ 0´8-fold the autofluorescence) was evenmore pronounced at the accelerated phase of CML, despitethe fact that the chemotherapy did not include MDR-typedrugs. For comparison, cell samples from AML patents werestained. The average staining of these patients (9´7 ^ 13´6)was also low compared with samples from healthy donors(P , 0´001), although significantly higher than the stain-ing levels of CML patients at presentation (P , 0´05). Thevariability in staining levels of cells from AML patients wasvery large compared with samples from CML patients, asexpected, based on the variable resistance to chemotherapyand the variety of cytogenetic abnormalities, fusion proteinsand rearrangements exhibited by AML patients comparedwith the BCR±ABL oncogene, the uniformly observedhallmark of cells from CML patients.

The TMR resistance exhibited by the cells of CML patientslacked the following characteristics expected of MDR1-mediated resistance. Well-established MDR1 modulators,such as verapamil, reserpine, genistein, cyclosporine A andPSC-833 did not affect the staining pattern of cells fromCML patients with TMR. Similarly, these modulators had noeffect on the uptake of rhodamine 123 into these cells (datanot shown), suggesting that the resistance in these cells isnot mediated by P-glycoprotein. The TMR resistance of cellsfrom the CML patients was not reversed by inhibitors ofMRP1, such as probenecid, genistein (Lam et al, 1992; Leieret al, 1994; Versantvoort et al, 1994) and verapamil.Moreover, double staining of the cells with the functionalTMR assay and immunostaining with anti-MDR1 or anti-MRP1 monoclonal antibodies and flow cytometry failed toreveal a correlation between TMR resistance and eitherMDR1 or MRP1 localization (data not shown).


Multidrug Resistance in CML Patients 583

Fig 1. TMR uptake into cells of a healthy donor and a CML patient. Frozen MNC from a CML patient and from a healthy donor were thawed and

subsequently incubated for 1 h at 378C with the nM concentrations indicated of either TMR (A) or rhodamine 123 (B). The cells were washed

and analysed using flow cytometry. Dead cells, stained with propidium iodide were excluded from the analysis.

Fig 2. TMR uptake into cells from a CMLpatient compared with dye uptake into cells

from healthy donors. Six separate samples

were obtained from the same CML patient

whose cells were analysed in Fig 1 andcompared with samples obtained from 39

healthy donors. All samples were frozen as

described in Materials and Methods. EachCML sample was processed for TMR stain-

ing on a different day, together with several

samples from healthy donors. Uptake of

rhodamine 123 amounts into five cellsamples from the same CML patient (X)

and samples from six healthy donors (B)

were measured. The amount of TMR stain-

ing of the CML cells was significantly lowercompared with the cells obtained from the

healthy donors (P , 0´0005 at TMR con-

centrations 2´5±2500 nmol/l). The best

corresponding differences were obtainedwith 25±250 nmol/l rhodamine 123, but

they were not statistically significant

(P � 0´08).

584 A. Carter et al


Doxorubicin resistance exhibited by cells from CML patientsTo better characterize the resistance exhibited by cellsobtained from CML patients, cells from 10 CML patients and10 healthy donors were exposed to doxorubicin. Doxorubi-cin accumulation in cells is not dependent on active uptake,as is the case for TMR and rhodamine 123. Cells incubatedwith doxorubicin in the cold bind significant amounts ofdrug. Because the amount of drug bound in the cold did notvary with time, it is presumably bound to the exterior of thecells, and constitutes the background to doxorubicin uptakeinto the cells (see Figs 4 and 5). Similar to the resistance toTMR staining, most of the cells of all CML patients exhibitedresistance to doxorubicin. Although, doxorubicin was takenup by a small subpopulation of cells incubated withdoxorubicin at 378C, most cells were resistant to the drug,and their drug content after 1 h incubation was close tothat of the background. This resistance was exhibited bysamples from all CML patients, and could be partiallyreversed by ATP depletion induced by glucose starvationand poisoning of mitochondrial function with KCN and,thus, is at least partially ATP dependent. On the other hand,all cell samples from healthy donors, apart from one, did notexhibit ATP-dependent doxorubicin resistance. After a 1 hincubation, the doxorubicin content of the cells fromhealthy donors was close to that of permeabilized cells,and was not modulated by ATP depletion. The only otheragent capable of modulating doxorubicin uptake into cellsfrom CML patients was the unspecific inhibitor of proteinfunction, NEM. Similar to the case with either TMR orrhodamine 123, competitive inhibitors (modulators) of

either MDR1 or MRP1, such as cyclosporine, PSC-833,reserpine, genistein, verapamil or probenecid, did notmodify the doxorubicin resistance exhibited by CML cells.The doxorubicin resistance was not modulated by non-toxicconcentrations of NBD chloride, a covalent inhibitor ofseveral transport proteins including MDR1 (Ferguson et al,1975; Puopolo & Forgac, 1990; Al-Shawi et al, 1994) (TableI). Other known inhibitors of various transport ATPasesinclude DCCD (Linnett et al, 1979), DIDS (MacLennan et al,1980) and quercitin (Kuriki & Racker, 1976).

The non-specific covalent inhibitor, N-ethylmaleimide didreverse the doxorubicin resistance exhibited by CML cells.As shown in Fig 4, 0´1 mmol/l N-ethylmaleimide produceda doxorubicin uptake close to that observed in permeabilizedcells. Because these N-ethylmaleimide-treated cells excludedthe dye propidium iodide, the facilitation of doxorubicinuptake was not caused by cell morbidity and damage totheir plasma membrane but, instead, was probably causedby inhibition of resistance mechanism/s.

Because MDR1 inhibitors did not modulate the doxor-ubicin resistance of CML cells, it was of interest to explorethe possibility that this resistance is mediated by accumula-tion of the drug in acidic intracellular compartments and

Fig 4. Doxorubicin uptake into MNC from a CML patient. Cells

obtained from a CML patient at presentation were washed and

incubated for 1 h either in the absence (Autofluorescence) or the

presence of 10 mmol/l doxorubicin, either at 08C (Background) or at378C (Control). Other cell samples were treated similarly to the

`Control' cells, except their incubation medium contained either

1 mmol/l NEM (NEM) or 1 mmol/l KCN and no glucose (KCN). Thecells were washed and stained with propidium iodide in the cold as

described in Material and Methods. The cells stained with propidium

iodide were gated out and only the fluorescence of viable cells is

presented in the figure. Another cell sample was permeabilized with30 mg/ml digitonin, and incubated in the presence of 10 mmol/l

doxorubicin (Permeabilized).

Fig 3. TMR uptake into cells from CML and AML patients comparedwith dye uptake into cells from healthy donors. Samples of MNC

were obtained from 24 CML patients at presentation, 10 CML

patients at later stages of the disease, 23 AML patients and 39healthy donors. The cells were frozen, thawed, incubated for 1 h in

the presence of 2´5 nmol/l TMR, washed and analysed for TMR

fluorescence. The TMR fluorescence is expressed compared with the

autofluorescence measured in cells incubated in the absence of dye.Each dot in the graph represents a cell sample obtained from one

patient.



secretion by exocytosis. As shown in Table I, disrupting theacidification of intracellular compartments by either the H1

ATPase inhibitor, bafilomycin A1, or by the ionophore,monensin, had no effect on doxorubicin resistance. Like-wise, weak basic ions, such as ammonium acetate andchloroquine, did not modulate the drug resistance. Knowninhibitors of intracellular membrane trafficking, such asdansylcadaverine [inhibitor of endocytosis via clathrin-coated pits (Schlegel et al, 1982)] and cytochalasin B[inhibitor of actin assembly (Cooper, 1987)] had no effect ondoxorubicin uptake (Table I). Moreover, lowering thetemperature to 178C and thereby interfering with intracel-lular membrane traffic had no effect on doxorubicinresistance (data not shown).

Transcription of MDR-related genes in cells from CML patientsAs shown in Fig 6, transcription of nine gene productspotentially capable of mediating MDR was analysed bycomparative RT-PCR. The transcription of b-actin served forthe calibration of the amount of cDNA included in the PCR

reaction. b2M was used as an additional standard. Thetranscription levels of both MDR1 and MRP1 were low andvariable both in samples from the CML patients and fromhealthy donors. Although the transcription levels of thesegene products in some of the CML patients were higher thanin healthy donors, in others the transcription levels werecomparable or even lower than in some healthy donors. Onthe other hand, the transcription levels of MRP3, MRP4 andMRP5 were consistently higher in CML patients comparedwith their transcription levels in healthy donors. Thetranscription levels of ARA and LRP showed no variability, andwere similar in CML patients and healthy donors. Thetranscription levels of MRP2 and MRP6 were very low,variable and not significantly different in CML patients andhealthy donors. Low levels of MRP2 and MRP6 expressionin normal bone marrow cells have recently been described(van Der Kolk et al, 2000).

The transcription levels of MDR1, MRP1 and LRP in cellsfrom CML patients compared with healthy donors, asreported above, were confirmed using flow cytometry withcommercially available antibodies.

DISCUSSION

All samples from CML patients at presentation exhibitedMDR as measured ex-vivo with the MDR-type dye TMR andthe MDR type-drug doxorubicin. The standard functionalassay of MDR, using rhodamine 123, was not adequateto detect this resistance. Presumably, because rhodamine

Fig 5. Doxorubicin uptake into MNC from a healthy donor. Cells

obtained from a healthy donor were washed and incubated either inthe absence (Autofluorescence) or presence of 10 mmol/l doxor-

ubicin, either at 378C (Control) or at 08C (Background) for 1 h.

Other cell samples were treated similarly to the `Control' cells,

except their incubation medium contained either 1 mmol/l NEM(NEM) or 1 mmol/l KCN and no glucose (KCN). The cells were

washed and stained with propidium iodide in the cold as described

in Material and Methods. The cells stained with propidium iodide

were gated out and only the fluorescence of viable cells is presentedin the figure. Another cell sample was permeabilized with 30 mg/ml

digitonin and incubated in presence of 10 mmol/l doxorubicin

(Permeabilized).

Table I. Effect of various inhibitors on doxorubicin ATP-dependent

exclusion from CML cells.

Inhibitor Concentration Effect

Cyclosporine 5 mmo/l No effect

PSC-833 10 mmol/l No effect

Verapamil 30 mmol/l No effect

Probenecid 10 mmol/l No effectReserpine 50 mmo/l No effect

Genistein 10 mmo/l No effect

Bafilomycin A1 5 nmol/l No effectMonensin 10 mmol/l No effect

Ammonium acetate 10 mmol/l No effect

Chloroquine 10 mmol/l No effect

KCN 1 mmol/l ModulationNEM 0´1 mmol/l Modulation

Cytochalasin B 10 mmol/l No effect

Dansylcadaverine 2 mmol/l No effect

Quercitin 0´1 mmol/l No effectDIDS 1 mmol/l No effect

DCCD 0´1 mmol/l No effect

NBD chloride 0´1 mmol/l No effect

Cells obtained from CML patients at presentation were incubated

with 10 mmo/l doxorubicin and the concentrations of agentsquoted in the table for 1 h at 378C. Modulation is defined as a

significant enhancement of doxorubicin uptake compared with cells

incubated in the absence of inhibitors.

586 A. Carter et al


123-based assays of MDR1 rely on modulation of theresistance with MDR1 inhibitors, the differences observed inrhodamine 123 uptake were not statistically significant.TMR and doxorubicin uptake into the cells was notmodulated by inhibitors of either MDR1 or MRP1, thusindicating that, at least, ex-vivo neither MDR1 nor MRP1mediate the energy-dependent exclusion of drugs from thesecells. This confirms the report by Stavrovskaya et al (1998)on MDR1 functional activity in the peripheral blood of 12patients in chronic phase and five patients in acceleratedphase. None of these patients showed MDR1 activity,measured as rhodamine-123 efflux sensitive to knownMDR1 inhibitors (e.g. verapamil). MDR1 and MRP1 areexpressed in some of the CML patients and, thus, either theexpressed proteins are inactive or their activity is over-shadowed by other resistance mechanisms. A recent survey(Giles et al, 1999) of 127 CML patients in early chronicphase detected MDR1 overexpression in 73 patients.However, disease progression, response to interferon a andsurvival were independent of MDR1 expression. Lack ofcorrelation between MDR1 and MRP1 expression and exvivo functional activity has been reported for otherleukaemia types (Twentyman et al, 1994; Leith et al,1995; Pallis et al, 1999). Doxorubicin resistance, notsensitive to MDR1 inhibitors, has been observed in many,but not all, cases of AML (Hedley et al, 1997). Recently,

alternative roles to drug extrusion have been suggested forMDR1, such as modulation of an sphingomyelin-ceramideapoptotic pathway (Come et al, 1999; Pallis & Russell,2000). In contrast to rhodamine 123, the recently describedMDR-type dye, TMR proved suitable to measure resistancein cells obtained from CML patients at presentation. Thehigh sensitivity of the TMR assay enables the reproducibledetermination of resistance without reliance on the effects ofmodulators. The TMR assay revealed uniform levels ofresistance in CML patients at presentation and even higherlevels of resistance in CML patients at the accelerated phase.In contrast, TMR revealed variable levels of resistance inAML patients and variable, but lower, levels of resistance inhealthy individuals.

The mononuclear cell populations from healthy indivi-duals and from CML patients were obtained by apheresisand had a similar content of CD341 cells, constitutingless than 3% of the cell population. As the resistancemechanism, characterized as TMR and doxorubicin excre-tion, was present in all the cells, it can be assumed that asimilar resistance mechanism is active in the stem cells aswell.

As no correlation was observed between the resistanceexhibited by all CML patients at presentation and theexpression of either MDR1 or MRP1, the transcription ofother known gene products capable of mediating MDR was

Fig 6. Semi-quantitative RT-PCR of MDR-related proteins. RNA was extracted from

cell samples obtained from two healthy

donors and three CML patients at presen-tation. The presence of mRNA of the

various proteins indicated in the figure was

assessed by semiquantitative RT-PCR as

described in Materials and Methods. Theamounts of cDNA used for the 30 cycle RT-

PCR amplification were equivalent to 0´01,

0´03 and 0´1 mg for actin and b2M; 0´3, 1

and 3 mg for MDR1, MRP1, MRP3, MRP4,MRP5, ARA, and LRP; 0, 1 and 3 mg for

BCR-ABL: and 1; 3 and 10 mg for MRP2

and MRP6.



studied. Because all CML patients exhibited resistance, itwas expected that the protein-mediating MDR in these cellswould be over-transcribed in all samples of CML patients atpresentation compared with their levels in healthy indivi-duals. Of the nine proteins studied, only MRP3, MRP4 andMRP5 met these criteria. MRP2 and MRP6 transcriptionlevels were very low and similar to those observed in healthydonors. On the other hand, the transcription levels of ARAand LRP were significant but very similar to those observedin healthy individuals. Thus, as the resistance observed inhealthy donors was limited to a small number of individuals,both ARA and LRP do not seem to mediate the resistanceobserved in CML patients. MRP3 transports organic anionsand also hydrophobic drugs, such as etoposide andmethotrexate, and thus could contribute to the resistanceto TMR and doxorubicin observed in cells from the CMLpatients. In accordance with this suggestion, Young et al(1999) have surveyed the expression levels of severalmembers of the MRP family in lung cancer samples andobserved a strong correlation of MRP3 mRNA levels withresistance of cell lines to doxorubicin. MRP4 (Schuetz et al,1999) and MRP5 (Wijnholds et al, 2000) are organic aniontransporters linked to the efflux of nucleotide analoguesfrom mammalian cells and, thus, may play a role inresistance to thiopurines in acute lymphoblastic leukaemiaand/or anti-retroviral nucleoside analogues in HIV-infectedpatients. MRP5 transports conjugates of GSH and, thus,could contribute to resistance towards hydrophobic drugs,although neither MRP4 nor MRP5 have been shown totransport such drugs. A possibility that cannot be ruled outis that although MRP3, MRP4 and MRP5 are overtranscribed in cells from the CML patients that exhibitresistance to doxorubicin and TMR, they do not necessarilyconstitute the major resistance mechanism. The resistancecould be mediated by other known mechanisms that havenot been investigated in the present study or by, as yet,unknown mechanisms.

Our findings of resistance towards TMR and doxorubicinin cells from CML patients at presentation using a functionaltest may explain the lack of response in these patients toinduction type chemotherapy used to treat AML Patients.The lack of response ex vivo to known modulators suggeststhat further investigations are needed to understand betterthe biological basis of the observed resistance. Identificationof the proteins mediating the resistance in these patientsand development of inhibitors capable of modulating thisresistance could lead to chemotherapy regimens betteradapted to CML patients at presentation.

ACKNOWLEDGMENTS

This work was supported by grants from the Lewin-MandelFoundation for Leukaemia Research and from the IsraelCancer Research Fund.

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Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1