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NEOPLASIA
From the Laboratoire Greffe de Moelle, Université
Victor Segalen, Bordeaux, France; Laboratoire de
Pathologie Moléculaire et Thérapie Génique Bordeaux,
France; and Department of Haematology, Imperial College of
Science, Technology and Medicine, Hammersmith Hospital, London,
United Kingdom.
Inappropriate expression of the multidrug resistance
(MDR1) gene encoding the P-glycoprotein (Pgp) has been
frequently implicated in resistance to different chemotherapeutic
drugs. We have previously generated chronic myeloid leukemia (CML) cell
lines resistant to the tyrosine kinase inhibitor imatinib mesylate
(STI571), and one line (LAMA84-r) showed overexpression not only of the
Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp.
Thus, cells from the K562/DOX line, described as resistant to
doxorubicin due to MDR1 gene overexpression, grew
continuously in the presence of 1 µM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the
imatinib-treated culture. Analysis of cell proliferation by the
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC50)
for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 µM for K562/DOX, K562/DOX plus verapamil, and K562,
respectively. Retroviral-mediated transfection of the
BCR-ABL+ AR230 cell line with the
MDR1 gene decreased its sensitivity to imatinib, an effect
that was also reversed by verapamil. The possible role of MDR
overexpression in clinical resistance to imatinib remains to be
defined. We therefore confirm that imatinib should be added to the
extensive list of drugs that can be affected by the MDR phenomenon.
(Blood. 2003;101:2368-2373) Multidrug resistance (MDR) is a well-known
phenomenon of cross-resistance of mammalian cells to a number of
anticancer agents following exposure to one such drug.1 A
diverse range of agents involved in MDR include alkaloid compounds and
bacterial and fungal antibiotics, such as anthracyclines and etoposide.
An accepted mechanism of MDR is a reduced cellular accumulation and an
altered subcellular distribution of cytotoxic drugs. In many cases,
this is mediated by increased expression at the cell surface of the MDR1 gene product, P-glycoprotein (Pgp), a 170-kDa
energy-dependent efflux pump.2,3 MDR mediated by Pgp is
thought to be primarily due to reduced intracellular concentrations and
failure of the drug to reach its target.
The causative event in the initiation of chronic myeloid leukemia (CML)
is the formation of the BCR-ABL oncogene, which codes for a
constitutively active Bcr-Abl tyrosine kinase, on the Philadelphia (Ph)
chromosome (22q STI571 or imatinib mesylate, a 2-phenylaminopyrimidine
derivative, inhibits the Bcr-Abl tyrosine kinase with high
selectivity.5 Preliminary results from 2 phase 1/2
clinical trials were recently reported.6,7 Of the
54 patients treated with at least 300 mg in chronic phase (CP),
98% showed hematologic and 53% cytogenetic responses, which have so
far been largely durable. When given in advanced phase of CML or in
Ph+ ALL, imatinib was able to induce hematologic responses,
but these were nearly invariably followed by relapse, suggesting
development of de novo resistance to the drug.7
In anticipation of drug resistance, we and other groups have isolated
BCR-ABL+ cell lines with differential
sensitivity to imatinib.8-10 Our initial studies showed
that in some cell lines amplification of the BCR-ABL gene
plays a major role in the development of imatinib resistance.
Furthermore, one resistant line showed overexpression not only of the
Bcr-Abl protein but also of the MDR Pgp, indicating that at least 2 mechanisms of resistance operate in this cell line.10
In the current study, we have investigated further the role of Pgp in
the resistance to imatinib. We show here that the K562/DOX cell line,
which displays resistance to several drugs and overexpresses Pgp, also
exhibits a reduced sensitivity to imatinib as compared to the parental
K562 cells, and that this resistance can be modulated by blockers of
the Pgp pump. We also show that retroviral-mediated introduction and
overexpression of the MDR1 gene in AR230, another CML cell
line, leads to resistance to imatinib, also reversible by Pgp inhibitors.
Cell lines
Cell proliferation assay
Cell viability assay Cells were plated at a density of 2 to 2.5 × 105cells/mL in RF-10 with or without the inhibitor and, in some experiments, with or without 5 µg/mL verapamil (Isoptine, Laboratoires Knoll, Levallois-Perret, France) or 1 µM PSC833 (Novartis Pharma, Basel, Switzerland). Aliquots were taken out at 24-hour intervals for assessment of cell viability by trypan blue dye exclusion.Apoptosis assay For determination of apoptotic death the level of caspase-3 activation was assessed by measurement of its capacity to cleave an Ac-Asp-Glu-Val-Asp-amino-4-methyl-coumarine (Ac-DEVD) substrate, as modified from a previously published method.10 Aliquots of 7.5 × 104 cells were cultured in triplicate in RF-10 into 96-well plates in the presence or absence of 1 µM imatinib. Triplicate wells with RF-10 only were used as background control. After 3 days, the plate was centrifuged at 1500 rpm for 5 minutes, the culture supernatant was removed, and the cells were resuspended in 50 µL of a buffer containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 5 mM dithiothreitol (DTT), 2 mM EDTA (ethylenediaminetetraacetic acid), 0.02% saponin (Sigma Aldrich, St Quentin Fallavier, France), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL pepstatin A, 10 µg/mL leupeptin, and 1.4 µM fluorogenic Ac-DEVD substrate (UBI Euromedex, Souffelweyerssheim, France). Development of the reaction was read on an automatic spectrofluorometer (Victor 2 Multilabel Counter, Wallac & Perkin Elmer, Akron, OH), using exc = 380 nm and em = 480 nm. After
this reading, 200 µL of a 15-µg/mL propidium iodide solution was
added to each well and a new reading was taken at
exc = 360 nm and em = 600 nm, to
evaluate DNA content in the wells. A calibration curve was performed
using increasing amounts of cells per well and the cell number in the
wells was calculated from this curve. The caspase-3 activity was
calculated for 105 cells.
Western blot analysis Protein lysates were prepared according to Kabarowski et al.13 Protein concentrations were determined by the Bradford method (DC Protein Assay, Bio-Rad, Hercules, CA). Approximately 250 µg protein was resolved on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, blotted onto polyvinylidene (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA) by semidry electrophoretic transfer, probed with individual antibodies, and visualized by the enhanced chemiluminescence (ECL) system (Amersham, Little Chalfont, United Kingdom). The following antibodies were used: PY-99 antiphosphotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA), Ab-3 anti-Abl (Calbiochem-Novabiochem, Nottingham, United Kingdom), antiphosphorylated STAT5 (Santa Cruz), and A-2066 antiactin (Sigma Chemical, St Louis, MO). The secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG and swine antirabbit IgG (Dako, Glostrup, Denmark).Flow cytometric analysis A phycoerythrin (PE)-conjugated monoclonal antibody (MoAb) Fab' fragment of a murine antihuman Pgp (clone UIC2; Immunotech, Marseille, France) was used to determine the expression of the MDR1 gene product on the different cell lines. For detection of the tyrosine phosphorylated proteins, cells were fixed in 1% paraformaldehyde/phosphate-buffered saline (PBS) for 10 minutes at room temperature, permeabilized with 0.3% saponin, and stained with the PY-99 antiphosphotyrosine antibody followed by a fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (Becton Dickinson, San Jose, CA) as the secondary reagent. Appropriate isotypic controls were used in all experiments. Stained cells were analyzed with an XL Coulter instrument (Coultronics, Margency, France).Transduction of BCR-ABL+ cell line The proviral plasmid pSF-MDR (kindly donated by Dr Christopher Baum, Heinrich-Pette-Institut, Hamburg, Germany) was transfected by calcium phosphate precipitation into the ecotropic packaging cell line CRE as described.14 Filtered
supernatants from the ecotropic packaging line were then added to the
amphotropic cell line -CRIP in the presence of 4 µg/mL protamine
sulfate. -CRIP clones were selected in the presence of colchicine
(20 ng/mL; Sigma), and titered using NIH3T3 fibroblasts as targets. The
highest titer clone (105 virus particles/mL) was used in
these experiments. Viral supernatant was harvested, filtered, and
frozen in ready-to-use aliquots. The supernatant was tested for the
presence of replication competent virus and confirmed to be free of
helper virus. A 105 cell aliquot from the AR230 CML cell
line in log-phase growth was exposed to viral supernatant at a
multiplicity of infection of 5 in the presence of 4 µg/mL protamine
sulfate. Nontransduced control cells were kept for the same length of
time in RF-10 medium and protamine sulfate. Cells were selected and
propagated in the presence of 10 ng/mL colchicine.
CFU assay of human imatinib-resistant cells Peripheral blood or bone marrow cells from CML patients treated with imatinib in the Novartis protocols 0102 or 0115 (blast crisis) were obtained at the time of the relapse. None of these patients exhibited amplification of the BCR-ABL gene or point mutations in its tyrosine kinase domain that could account for the imatinib-resistant phenotype. Mononuclear cells (MNCs) were separated on Lymphoprep (Nycomed, Oslo, Norway) and plated at 5 × 104 cells/mL in Iscove methylcellulose medium (Methocult H4230 or H4330; Stem Cell Technologies, Meylan, France) supplemented with 20 ng/mL recombinant human interleukin-3 (rhIL-3), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, (Amgen, Thousand Oaks, CA), and 100 ng/mL Flt3 ligand (R & D Systems, Abingdon, Oxon, United Kingdom). All clonogenic assays were carried out in quadruplicate. Imatinib (1 µM) or PSC833 (1 µM) or both was added directly to the methylcellulose. Granulocyte-macrophage colony-forming units (CFU-GMs) or CFU-blast colonies were evaluated after 14 days of culture.
The K562/DOX cell line overexpresses Pgp and is resistant to imatinib Fluorocytometric analysis confirmed that K562/DOX exhibited a significant overexpression of Pgp; more than 80% of cells showed a strong reaction with the anti-Pgp antibody, in contrast to only 2% of the parental K562 line (Figure 1A). There was no Bcr-Abl overexpression in K562/DOX as compared to the parental clone (data not shown).
A 24-hour exposure of the 2 cell lines to imatinib resulted in
increased caspase-3 activity in K562 but not in K562/DOX (Figure 1B).
This difference was also illustrated by the fact that less than 10% of
the parental K562 cells survived a 4-day treatment with 1 µM
imatinib, whereas the viability of the K562/DOX line was above 80% at
the end of the same period of exposure to the inhibitor (Figure
2A).
Resistance of K562/DOX to imatinib is reversed by verapamil or PSC833 K562/DOX cells were incubated with verapamil or PSC833, 2 known inhibitors of the Pgp pump, and assessed for viability on exposure to imatinib. A clear enhancement of sensitivity to 1 µM imatinib was observed when either verapamil (5 µg/mL) or PSC833 (1 µM) was added to the culture (Figure 2). MTS cell proliferation assays were performed on cells exposed to 0.1, 0.5, 1, 5, and 10 µM imatinib (Figure 3). On day 3 we evaluated the dose of imatinib necessary to induce a 50% decrease in the uptake of MTS (IP50), as measured by the OD index. Thus, the IP50 for K562 was 0.1 µM, whereas that for K562/DOX was 1 µM. This indicated that the concentration of imatinib needed for a 50% reduction in the number of viable cells after 3-day exposure to the compound was on average 10 times higher in the resistant as compared to the sensitive cells (Figure 3). This was, however, still lower than the respective concentration for 2 other lines resistant to imatinib, LAMA84-r (IP50 = 2 µM) and K562-r (IP50 = 3 µM).10 The resistance of K562/DOX to imatinib was partially reversed by simultaneous incubation with verapamil (IP50 = 0.5 µM), and completely reversed by PSC833 (IP50 = 0.1 µM).
Reduced inhibition of cellular tyrosine phosphorylation of K562/DOX by imatinib To analyze the effect of imatinib on the phosphorylation pattern of K562 and K562/DOX cells, we established a new assay using flow cytometry on permeabilized cells stained with a phosphotyrosine antibody. We therefore washed K652 and K562/DOX with RF-10, incubated the individual cell lines in graded concentrations of 0 to 10 mM imatinib for 2 hours, and analyzed their phosphotyrosine content by fluorocytometry (Figure 4). Phosphorylation of various proteins decreased in a dose-dependent manner in the sensitive and resistant clones (Figure 4). However, the doses of imatinib necessary to inhibit overall tyrosine phosphorylation were higher for K562/DOX than for K562. Estimation of the IC50 of the compound for inhibition of 50% of total cellular tyrosine phosphorylation from the fluorocytometric data showed that it was 5 µM for K562 and 15 µM for K562/DOX. When the latter cells were cotreated with verapamil, the IC50 was reduced to 10 µM, whereas no change was detected in the parental line on treatment with verapamil (data not shown).
K562/DOX requires higher imatinib concentrations for inhibition of STAT5 phosphorylation Because the transcription factor STAT5 is constitutively tyrosine phosphorylated and activated after transformation of hematopoietic cells by p210Bcr/Abl, we have studied the effect of imatinib on this target protein.15 The 2 K562 cell lines were incubated with graded concentrations of imatinib during 2 hours and immunoblotted with an antibody that recognizes the phosphorylated isoform of STAT5. As shown in Figure 5A, the concentration of imatinib necessary to inhibit STAT5 phosphorylation was higher in K562/DOX as compared to K562. Thus, when exposed to 5 µM imatinib the level of STAT5 phosphorylation in K562 was 38% of that observed in the control (nonexposed) cells; in contrast, under the same conditions, the amount of phospho-STAT5 in K562/DOX cells was 67% of the control and decreased to 40% after the addition of verapamil (Figure 5B).
Ectopic overexpression of Pgp in a CML cell line leads to resistance to imatinib To demonstrate a direct causal relationship between Pgp overexpression and resistance to imatinib, we infected AR230, a BCR-ABL+ CML cell line with a retrovirus carrying the MDR1 gene. Transduced cells were selected with colchicine and tested by flow cytometry for the expression of Bcr-Abl and Pgp, in parallel with control, nontransfected cells (Figure 6A). The MDR1-transduced cells showed significant overexpression of Pgp in comparison with the negligible, baseline level of expression in the parental, nontransfected line. As expected, expression of the endogenous Bcr-Abl protein was unaffected by the transduction procedure (data not shown).
The effect of imatinib on the viability of the
MDR1-transduced AR230 cell line was compared to that of the
parental AR230 cells. Whereas the latter died within 4 days of exposure
to 0.5 µM imatinib, the MDR1+ cells were still
65% viable at the end of the same period (Figure 6B). This longer
survival was, however, abrogated when verapamil was also included in
the imatinib culture. The viability of these cell lines on exposure to
DOX was also examined, with similar results: 70% of the AR230/MDR
cells were alive after a 3-day exposure to 100 ng/mL DOX but none of
the parental AR230 cells survived this treatment. Analysis of
proliferation after 3 days in 1 µM imatinib showed that the growth of
the nontransfected AR230 cells was significantly reduced to 13% of the
control (untreated cells); in contrast, proliferation of the
MDR1-transduced line was reduced to only 70% of the control
(Figure 7A). Finally the concentration of
imatinib necessary to inhibit STAT5 phosphorylation was significantly higher in AR230/MDR as compared to the parental AR230 cells (Figure 7B).
An inhibitor of the Pgp pump reduces imatinib resistance of primary CML cells We tested blast cells from 6 patients with CML in blast crisis clinically resistant to imatinib for Pgp expression by cytometry analysis. No overexpression of MDR-1 was detected using UIC2 antibody staining. Because an association between early progenitors and functional MDR-1 has been reported,16 we tested the combination of imatinib and the PSC833 pump inhibitor in a CFU assay using total MNCs from these imatinib-resistant patients. As shown on Figure 8, 1 µM imatinib induced a weak inhibition of CFU formation (77%) as compared to the clonogenicity of control untreated cells. However, addition of 1 µM PSC833 to the imatinib-treated cultures led to a significant decrease in colony formation (52%, P = .001; Student t test), whereas PSC833 alone had no effect on the CFU growth (91%). Taken together, these functional results suggest that Pgp overexpression could contribute to acquired imatinib resistance in the primary leukemic cells, even though increased levels of the protein are not demonstrable by conventional immunostaining methods.
The results on the first clinical trials with imatinib have suggested that this drug has the potential for long-term if not indefinite control of chronic phase CML.17 The same cannot be said for more advanced stages of the disease, where patients nearly always experience an initial clinical response to the inhibitor but subsequently lose it relatively early.7,18 Studies on imatinib-resistant CML cell lines suggested that BCR-ABL amplification and overexpression might be the most common mechanism by which the leukemic cells evade the action of imatinib.9,10 This together with specific mutations in the adenosine triphosphate (ATP)-binding region of the Bcr-Abl tyrosine kinase are, indeed, being progressively identified in a variable proportion of CML patients who become refractory to imatinib treatment.19-23 It appears, however, that other single or multiple mechanisms must operate in a substantial proportion of patients. Among these, MDR is probably one of the most likely candidates, because it has already been shown to participate in the phenomenon of imatinib resistance of at least one cell line, that is, LAMA84-r.10 In this line, however, the contribution of excess Pgp to the overall resistant phenotype was difficult to evaluate because the cells also overexpress Bcr-Abl. In the present study we therefore analyzed the effect of the MDR1 gene in cell lines where its overexpression is independent of previous exposure to imatinib. In one of the lines (K562/DOX), Pgp-overproducing cells were selected in response to anthracycline exposure, whereas in the other (AR230/MDR), ectopic overexpression of the pump protein was obtained by retroviral-mediated MDR1 gene transfer. Despite the difference in the origin of their MDR1 overexpression, both lines were markedly resistant to imatinib, as shown by their sustained viability and proliferation, reduced caspase-3 activity, unaltered tyrosine phosphorylation profile, and elevated STAT5 activity when exposed to the inhibitor. Furthermore, the imatinib-resistant phenotype could be significantly alleviated by Pgp pump blockers, in particular PSC833, previously shown to be more potent than verapamil.24,25 It is interesting to observe the variable degree of resistance to imatinib in CML cell lines with different mechanisms of resistance. Thus, by using the imatinib IP50 as defined, we demonstrated that K562/DOX, whose only mechanism of resistance is MDR1 overexpression, is less resistant to imatinib than LAMA84-r, which overexpresses both MDR1 and BCR-ABL and this, in turn, is less resistant than K562-r whose mechanism of resistance is still unknown.10 The possible role of Pgp overexpression in the acquired in vivo resistance of primary CML cells to the kinase inhibitor is less well defined. In none of the 6 patients who lost their initial response to the drug could MDR1 up-regulation be detected on the relapse specimens. However, addition of a pump blocker to imatinib was able to significantly increase its capacity to inhibit CFU-GM or CFU-blast (or both) proliferation, suggesting though not proving, that a pump-mediated drug resistance effect might be operating in these progenitors. In none of these patients did we observe amplification of the BCR-ABL gene or a point mutation in the tyrosine kinase domain.19 The apparent paradox may be explained by the known difficulty in accurately measuring relatively low levels of Pgp in clinical samples, as largely documented, for instance, in acute myeloid leukemia.26 Despite this problem, various workshops devoted to this phenomenon have concluded that even a weak Pgp activity may lead to an MDR phenotype.27-29 In this context, it should be noted that whereas development of imatinib resistance is the rule rather than the exception in blast crisis of CML, in chronic phase it has been only rarely documented so far. This may be related, at least in part, to the relatively increased basal level of MDR1 gene expression in blast crisis as compared to chronic phase cells, as reported by some groups.30,31 It is also possible that the apparent pump-mediated imatinib resistance in CML blasts is due to overexpression of an MDR gene other than MDR1.32 The practical implications of our study are manifold. In the first instance, it is evident that BCR-ABL+ leukemic cells that develop MDR-1 up-regulation by previous induction with another drug (eg, anthracyclines) are likely to be cross-resistant to imatinib. In clinical terms, documentation of Pgp overexpression in a patient with CML in blast crisis might preclude prescription of imatinib as an alternative to the previous chemotherapy, because the tyrosine kinase inhibitor should also be excluded from the cell by the pump protein. Another concern is based on the fact that Pgp is physiologically expressed in normal hematopoietic stem cells for their own protection against accumulation of toxic metabolites.33 Being a stem cell disorder, CML leukemic progenitors are likely to express similar if not higher levels of Pgp and this could adversely affect their total elimination by imatinib. This has, in fact, been postulated as a possible mechanism to explain the inherent resistance to imatinib of very primitive, quiescent CML stem cells.34 Finally, a similar argument may be applicable to the possible refractoriness or early relapse while on imatinib therapy of solid tumors, many of which overexpress MDR1.35-38
We are very grateful to Dr J. P. Marie (INSERM, E9912, University Paris 6, France) for providing us the K562/DOX cell line and Dr S. Mizutani (National Children's Medical Research Center, Tokyo, Japan) for AR230; to Dr Elisabeth Buchdunger (Novartis, Basel, Switzerland) for imatinib and PSC833; and to Dr Christopher Baum (Heinrich-Pette-Institut, Hamburg, Germany) for the proviral plasmid pSF-MDR. We thank Dr Patrick Berthaud and Novartis Pharma (Rueil-Malmaison, France) for supporting this work.
Submitted August 29, 2001; accepted November 19, 2002.
Supported by grants from the Association pour la recherche sur le cancer, Fondation de la recherche medicale, and Novartis Pharma France (F.-X.M., J.R. and V.L.) and the Leukaemia Research Fund, United Kingdom (J.M.G. and J.V.M.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: François-Xavier Mahon, Laboratoire Greffe de Moelle UMR CNRS 5540, Université Victor Ségalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France; e-mail: francois-xavier.mahon{at}umr5540.u-bordeaux2.fr.
1.
Arceci RJ.
Clinical significance of P-plycoprotein in multidrug resistance malignancies.
Blood.
1993;81:2215-2222 2. Dano K. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim Biophys Acta. 1973;323:466-483[Medline] [Order article via Infotrieve]. 3. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976;455:152-162[Medline] [Order article via Infotrieve]. 4. Melo JV. The molecular biology of chronic myeloid leukaemia. Leukemia. 1996;10:751-756[Medline] [Order article via Infotrieve]. 5. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566[CrossRef][Medline] [Order article via Infotrieve].
6.
Druker B, Talpaz M, Resta D, et al.
Clinical efficacy and safety of an ABL specific tyrosine kinase inhibitor as targeted therapy for chronic myeloid leukemia.
N Engl J Med.
2001;344:1031-1037
7.
Druker B, Sawyers C, Kantarjian H, et al.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in patients in blast crisis of chronic myeloid leukemia and acute leukemia with the Philadelphia chromosome.
N Engl J Med.
2001;344:1038-1042
8.
Weisberg E, Griffin JD.
Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL transformed hematopoietic cell lines.
Blood.
2000;95:3498-3505
9.
le Coutre P, Tassi E, Varella-Garcia M, et al.
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.
Blood.
2000;95:1758-1766
10.
Mahon FX, Deininger M, Schultheis B, et al.
Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the signal transduction inhibitor STI571: diverse mechanisms of resistance.
Blood.
2000;96:1070-1079 11. Drexler HG. The Leukemia-Lymphoma Cell Line Facts Book. London United Kingdom: Academic Press; 2000:733. 12. Marie JP, Faussat AM, Zhou D, Zittoun R. Daunorubicin uptake by leukemic cells: correlation with treatment outcome and mdr-1 expression. Leukemia. 1993;7:825-831[Medline] [Order article via Infotrieve]. 13. Kabarowski JH, Allen PB, Wiedemann LM. A temperature sensitive p210 BCR-ABL mutant defines the primary consequences of BCR-ABL tyrosine kinase expression in growth factor dependent cells. EMBO J. 1994;13:5887-5895[Medline] [Order article via Infotrieve].
14.
Danos O, Mulligan RC.
Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.
Proc Natl Acad Sci U S A.
1988;85:6460-6464
15.
Sillaber C, Gesbert F, Frank DA, Sattler M, Griffin JD.
STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells.
Blood.
2000;95:2118-2125 16. Chaudhary PM, Roninson IB. Expression and activity of P-plycoprotein a multidrug efflux pump in human hematopoietic stem cells. Cell. 1991;66:85-94[CrossRef][Medline] [Order article via Infotrieve].
17.
Kantarjian H, Sawyers C, Hochhaus A, et al.
Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.
N Engl J Med.
2002;346:645-652
18.
Sawyers CL, Hochhaus A, Feldman E, et al.
Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study.
Blood.
2002;99:3530-3539
19.
Gorre ME, Mohammed M, Ellwood K, et al.
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.
Science.
2001;293:876-880 20. Barthe C, Cony-Makhoul P, Melo JV, Reiffers J, Mahon FX. Roots of clinical resistance to STI-571 cancer therapy. Science. 2001;293:2163[CrossRef][Medline] [Order article via Infotrieve]. 21. Hochhaus A, Kreil S, Corbin A, et al. Roots of clinical resistance to STI-571 cancer therapy. Science. 2001;293:2163[CrossRef][Medline] [Order article via Infotrieve]. 22. von Bubnoff N, Schneller F, Peschel C, Duyster J. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-positive leukaemia to STI571. Lancet. 2002;359:487-491[CrossRef][Medline] [Order article via Infotrieve].
23.
Branford S, Rudzki Z, Walsh S, et al.
High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance.
Blood.
2002;99:3472-3475 24. Watanabe T, Tsuge H, Oh-hara T, Naito M, Tsuruo T. Comparative study on reversal efficacy of SDZ PSC 833 cyclosporin A and verapamil on multidrug resistance in vitro and in vivo. Acta Oncol. 1995;34:235-241[Medline] [Order article via Infotrieve]. 25. Lehne G, Angelis P, Boer M, Rugstad H. Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents. Leukemia. 1998;13:768-778. 26. Vossebeld PJM, Sonneveld P. Reversal of multidrug resistance in hematological malignancies. Blood Rev. 1999;13:67-78[CrossRef][Medline] [Order article via Infotrieve]. 27. Marie JP, Legrand O, Perrot JY, Chevillard S, Huet S, Robert J. Measuring multidrug resistance expression in human malignancies: elaboration of consensus recommendations. Semin Hematol. 1997;34:63-71[Medline] [Order article via Infotrieve].
28.
Beck WT, Grogan TM, Willman CL, et al.
Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.
Cancer Res.
1996;56:3010-3020
29.
Legrand O, Perrot JY, Simonin G, Baudard M, Marie JP.
JC-1: a very sensitive fluorescent probe to test Pgp activity in adult acute myeloid leukemia.
Blood.
2001;97:502-508 30. Giles F, Kantarjian H, Cortes J, et al. Multidrug resistance protein expression in chronic myeloid leukemia: associations and significance. Cancer. 1999;86:805-813[CrossRef][Medline] [Order article via Infotrieve]. 31. Stavrovskaya A, Turkina A, Sedyakhina NP, et al. Prognostic value of P-glycoprotein and leukocyte differentiation antigens in chronic myeloid leukemia. Leuk Lymphoma. 1998;28:469-482[Medline] [Order article via Infotrieve]. 32. Hrycyna CA. Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance. Semin Cell Dev Biol. 2001;12:247-256[CrossRef][Medline] [Order article via Infotrieve].
33.
Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham M.
Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.
Proc Natl Acad Sci U S A.
1987;84:7735-7738
34.
Graham SM, Jorgensen HG, Allan E, et al.
Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro.
Blood.
2002;99:319-325
35.
Plaat BE, Hollema H, Molenaar WM, et al.
Soft tissue leiomyosarcomas and malignant gastrointestinal stromal tumors: differences in clinical outcome and expression of multidrug resistance proteins.
J Clin Oncol.
2000;18:3211-3220 36. Efferth T, Volm M. Modulation of P-glycoprotein-mediated multidrug resistance by monoclonal antibodies, immunotoxins or antisense oligodeoxynucleotides in kidney carcinoma and normal kidney cells. Oncology. 1993;50:303-308[CrossRef][Medline] [Order article via Infotrieve]. 37. Savaraj N, Wu CJ, Xu R, et al. Multidrug-resistant gene expression in small-cell lung cancer. Am J Clin Oncol. 1997;20:398-403[CrossRef][Medline] [Order article via Infotrieve]. 38. Abe T, Mori T, Wakabayashi Y, et al. Expression of multidrug resistance protein gene in patients with glioma after chemotherapy. J Neurooncol. 1998;40:11-18[CrossRef][Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
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  D. H. Kim, L. Sriharsha, W. Xu, S. Kamel-Reid, X. Liu, K. Siminovitch, H. A. Messner, and J. H. Lipton Clinical Relevance of a Pharmacogenetic Approach Using Multiple Candidate Genes to Predict Response and Resistance to Imatinib Therapy in Chronic Myeloid Leukemia Clin. Cancer Res., July 15, 2009; 15(14): 4750 - 4758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Grosso, A. Puissant, M. Dufies, P. Colosetti, A. Jacquel, K. Lebrigand, P. Barbry, M. Deckert, J. P. Cassuto, B. Mari, et al. Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors Mol. Cancer Ther., July 1, 2009; 8(7): 1924 - 1933. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 J P. Couto, H Prazeres, P Castro, J Lima, V Maximo, P Soares, and M Sobrinho-Simoes How molecular pathology is changing and will change the therapeutics of patients with follicular cell-derived thyroid cancer J. Clin. Pathol., May 1, 2009; 62(5): 414 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Lagas, R. A.B. van Waterschoot, V. A.C.J. van Tilburg, M. J. Hillebrand, N. Lankheet, H. Rosing, J. H. Beijnen, and A. H. Schinkel Brain Accumulation of Dasatinib Is Restricted by P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) and Can Be Enhanced by Elacridar Treatment Clin. Cancer Res., April 1, 2009; 15(7): 2344 - 2351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ding, Y. Su, L. Pang, Q. Lu, Z. Wang, S. Zhang, S. Zheng, J. Mao, and Y. Zhu Inhibition of apoptosis by downregulation of hBex1, a novel mechanism, contributes to the chemoresistance of Bcr/Abl+ leukemic cells Carcinogenesis, January 1, 2009; 30(1): 35 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F.-X. Mahon, S. Hayette, V. Lagarde, F. Belloc, B. Turcq, F. Nicolini, C. Belanger, P. W. Manley, C. Leroy, G. Etienne, et al. Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression Cancer Res., December 1, 2008; 68(23): 9809 - 9816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dulucq, S. Bouchet, B. Turcq, E. Lippert, G. Etienne, J. Reiffers, M. Molimard, M. Krajinovic, and F.-X. Mahon Multidrug resistance gene (MDR1) polymorphisms are associated with major molecular responses to standard-dose imatinib in chronic myeloid leukemia Blood, September 1, 2008; 112(5): 2024 - 2027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Hiwase, V. Saunders, D. Hewett, A. Frede, S. Zrim, P. Dang, L. Eadie, L. B. To, J. Melo, S. Kumar, et al. Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications Clin. Cancer Res., June 15, 2008; 14(12): 3881 - 3888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Michor Mathematical Models of Cancer Stem Cells J. Clin. Oncol., June 10, 2008; 26(17): 2854 - 2861. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Puissant, S. Grosso, A. Jacquel, N. Belhacene, P. Colosetti, J.-P. Cassuto, and P. Auberger Imatinib mesylate-resistant human chronic myelogenous leukemia cell lines exhibit high sensitivity to the phytoalexin resveratrol FASEB J, June 1, 2008; 22(6): 1894 - 1904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fojo Commentary: Novel Therapies for Cancer: Why Dirty Might Be Better Oncologist, March 1, 2008; 13(3): 277 - 283. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Baccarani, F. Pane, and G. Saglio Monitoring treatment of chronic myeloid leukemia Haematologica, February 1, 2008; 93(2): 161 - 169. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wetzler Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia: On Target to Improve Outcome ASCO Educational Book, January 1, 2008; 2008(1): 275 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Goldman How I treat chronic myeloid leukemia in the imatinib era Blood, October 15, 2007; 110(8): 2828 - 2837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sleijfer, E. Wiemer, C. Seynaeve, and J. Verweij Improved Insight into Resistance Mechanisms to Imatinib in Gastrointestinal Stromal Tumors: A Basis for Novel Approaches and Individualization of Treatment Oncologist, June 1, 2007; 12(6): 719 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Michor Chronic Myeloid Leukemia Blast Crisis Arises from Progenitors Stem Cells, May 1, 2007; 25(5): 1114 - 1118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.=C> Miselli, P. Casieri, T. Negri, M. Orsenigo, M. S. Lagonigro, A. Gronchi, M. Fiore, P. G. Casali, R. Bertulli, A. Carbone, et al. c-Kit/PDGFRA Gene Status Alterations Possibly Related to Primary Imatinib Resistance in Gastrointestinal Stromal Tumors Clin. Cancer Res., April 15, 2007; 13(8): 2369 - 2377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Picard, K. Titier, G. Etienne, E. Teilhet, D. Ducint, M.-A. Bernard, R. Lassalle, G. Marit, J. Reiffers, B. Begaud, et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia Blood, April 15, 2007; 109(8): 3496 - 3499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Raanani, O Shpilberg, and I Ben-Bassat Extramedullary disease and targeted therapies for hematological malignancies--is the association real? Ann. Onc., January 1, 2007; 18(1): 7 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baccarani, G. Saglio, J. Goldman, A. Hochhaus, B. Simonsson, F. Appelbaum, J. Apperley, F. Cervantes, J. Cortes, M. Deininger, et al. Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet Blood, September 15, 2006; 108(6): 1809 - 1820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Wassmann, H. Pfeifer, N. Goekbuget, D. W. Beelen, J. Beck, M. Stelljes, M. Bornhauser, A. Reichle, J. Perz, R. Haas, et al. Alternating versus concurrent schedules of imatinib and chemotherapy as front-line therapy for Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) Blood, September 1, 2006; 108(5): 1469 - 1477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Rossi, I. Ehlers, V. Agosti, N. D. Socci, A. Viale, G. Sommer, Y. Yozgat, K. Manova, C. R. Antonescu, and P. Besmer Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor PNAS, August 22, 2006; 103(34): 12843 - 12848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Melo Imatinib and ABCG2: who controls whom? Blood, August 15, 2006; 108(4): 1116 - 1117. [Full Text] [PDF]
|
|
|
|
|
|
D. L. White, V. A. Saunders, P. Dang, J. Engler, A. C. W. Zannettino, A. C. Cambareri, S. R. Quinn, P. W. Manley, and T. P. Hughes OCT-1-mediated influx is a key determinant of the intracellular uptake of imatinib but not nilotinib (AMN107): reduced OCT-1 activity is the cause of low in vitro sensitivity to imatinib Blood, July 15, 2006; 108(2): 697 - 704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Copland, A. Hamilton, L. J. Elrick, J. W. Baird, E. K. Allan, N. Jordanides, M. Barow, J. C. Mountford, and T. L. Holyoake Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction Blood, June 1, 2006; 107(11): 4532 - 4539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mishra, B. Zhang, J. M. Cunnick, N. Heisterkamp, and J. Groffen Resistance to imatinib of bcr/abl p190 lymphoblastic leukemia cells. Cancer Res., May 15, 2006; 66(10): 5387 - 5393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. George, T. Chen, and C. C. Taylor Src Tyrosine Kinase and Multidrug Resistance Protein-1 Inhibitions Act Independently but Cooperatively to Restore Paclitaxel Sensitivity to Paclitaxel-Resistant Ovarian Cancer Cells Cancer Res., November 15, 2005; 65(22): 10381 - 10388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Theou, S. Gil, A. Devocelle, C. Julie, A. Lavergne-Slove, A. Beauchet, P. Callard, R. Farinotti, A. Le Cesne, A. Lemoine, et al. Multidrug Resistance Proteins in Gastrointestinal Stromal Tumors: Site-Dependent Expression and Initial Response to Imatinib Clin. Cancer Res., November 1, 2005; 11(21): 7593 - 7598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. White, V. Saunders, A. B. Lyons, S. Branford, A. Grigg, L. B. To, and T. Hughes In vitro sensitivity to imatinib-induced inhibition of ABL kinase activity is predictive of molecular response in patients with de novo CML Blood, October 1, 2005; 106(7): 2520 - 2526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Crossman, B. J. Druker, M. W. N. Deininger, M. Pirmohamed, L. Wang, and R. E. Clark hOCT 1 and resistance to imatinib Blood, August 1, 2005; 106(3): 1133 - 1134. [Full Text] [PDF]
|
|
|
|
|
|
M. Deininger, E. Buchdunger, and B. J. Druker The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood, April 1, 2005; 105(7): 2640 - 2653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Ilaria Jr. Pathobiology of Lymphoid and Myeloid Blast Crisis and Management Issues Hematology, January 1, 2005; 2005(1): 188 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Thomas, L. Wang, R. E. Clark, and M. Pirmohamed Active transport of imatinib into and out of cells: implications for drug resistance Blood, December 1, 2004; 104(12): 3739 - 3745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Burger, H. van Tol, A. W. M. Boersma, M. Brok, E. A. C. Wiemer, G. Stoter, and K. Nooter Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump Blood, November 1, 2004; 104(9): 2940 - 2942. [Abstract] [Full Text] [PDF]
|
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|
|
|
D. Mahadevan and A. F. List Targeting the multidrug resistance-1 transporter in AML: molecular regulation and therapeutic strategies Blood, October 1, 2004; 104(7): 1940 - 1951. [Abstract] [Full Text] [PDF]
|
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|
|
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L. Legros, C. Bourcier, A. Jacquel, F.-X. Mahon, J.-P. Cassuto, P. Auberger, and G. Pages Imatinib mesylate (STI571) decreases the vascular endothelial growth factor plasma concentration in patients with chronic myeloid leukemia Blood, July 15, 2004; 104(2): 495 - 501. [Abstract] [Full Text] [PDF]
|
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|
 S. Parmar, E. Katsoulidis, A. Verma, Y. Li, A. Sassano, L. Lal, B. Majchrzak, F. Ravandi, M. S. Tallman, E. N. Fish, et al. Role of the p38 Mitogen-activated Protein Kinase Pathway in the Generation of the Effects of Imatinib Mesylate (STI571) in BCR-ABL-expressing Cells J. Biol. Chem., June 11, 2004; 279(24): 25345 - 25352. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 C. Ozvegy-Laczka, T. Heged""s, G. Varady, O. Ujhelly, J. D. Schuetz, A. Varadi, G. Keri, L. Orfi, K. Nemet, and B. Sarkadi High-Affinity Interaction of Tyrosine Kinase Inhibitors with the ABCG2 Multidrug Transporter Mol. Pharmacol., June 1, 2004; 65(6): 1485 - 1495. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 R. J. Jones, W. H. Matsui, and B. D. Smith Cancer Stem Cells: Are We Missing the Target? J Natl Cancer Inst, April 21, 2004; 96(8): 583 - 585. [Full Text] [PDF]
|
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|
|
|
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P. La Rosee, K. Johnson, A. S. Corbin, E. P. Stoffregen, E. M. Moseson, S. Willis, M. M. Mauro, J. V. Melo, M. W. Deininger, and B. J. Druker In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines Blood, January 1, 2004; 103(1): 208 - 215. [Abstract] [Full Text] [PDF]
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P. T. Ferrao, M. J. Frost, S.-P. Siah, and L. K. Ashman Overexpression of P-glycoprotein in K562 cells does not confer resistance to the growth inhibitory effects of imatinib (STI571) in vitro Blood, December 15, 2003; 102(13): 4499 - 4503. [Abstract] [Full Text] [PDF]
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N. Widmer, S. Colombo, T. Buclin, and L. A. Decosterd Functional consequence of MDR1 expression on imatinib intracellular concentrations Blood, August 1, 2003; 102(3): 1142 - 1142. [Full Text] [PDF]
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| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
), which is itself the result of the t(9;22)(q34;q11) reciprocal translocation. This Ph chromosome and BCR-ABL oncoprotein are also found in about 25% of adults and 5% of children with acute
lymphoblastic leukemia (ALL).
, control) or with (1 µM) imatinib (
). Results
represent the mean ± SD of triplicate cultures. The difference
between the 2 treated cultures is statistically significant
(P = .004; Student t test).
); AR230/MDR (×); AR230/MDR + imatinib (*); AR230 + imatinib (
); AR230/MDR + imatinib + VP (
); AR230 + imatinib + VP (
).
