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作者：Laurence Pirsona,b, Frdric Barona,b, Nathalie Meurisa,b, Olivier Gieta,b, Emilie Castermansa,b, Roland Greimersc, Ivano Di Stefanoa,b, Andr Gothota,d, Yves Beguina,b作者单位：aCenter for Cellular and Molecular Therapy, University of Lige, Lige, Belgium;bDepartment of Medicine, Division of Hematology, University of Lige, Lige, Belgium;cLaboratory of Pathological Anatomy and Cytology, University of Lige, Lige, Belgium;dDepartment of Clinical Biology, Division of Laboratory
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【摘要】
$ e' }/ t4 b9 K9 y1 a; f5 s4 { There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT), and particularly allogeneic HCT with a nonmyeloablative regimen, to the tyrosine kinase inhibitor imatinib (Glivec; Novartis, Basel, Switzerland, http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However, because imatinib inhibits c-kit, the stem cell factor receptor, it could interfere with bone marrow engraftment. In this study, we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133 cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133 cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4, VLA-5, and CXCR4 of CD133 cells was not modified by imatinib, but imatinib decreased the ability of CD133 cells to migrate. Finally, imatinib did not decrease engraftment of CD133 cells into irradiated nonobese diabetic/severe combined immunodeficient/ß2mnull mice conditioned with 3 or 1 Gy total body irradiation. In summary, our results suggest that, despite inhibition of hematopoietic progenitor cell growth in vitro, imatinib does not interfere with hematopoietic stem cell engraftment. " Q" ]; d' C5 r X
【关键词】 Allogeneic stem cell transplantation Chronic myeloid leukemia Imatinib
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The Philadelphia chromosome results from the balanced t(9;22) chromosomal translocation, which leads to an aberrant BCR/ABL fusion gene .
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SCF is constitutively expressed by bone marrow endothelial cells and fibroblasts. It suppresses apoptosis and induces cell cycle entry ." _; K5 h) U, P2 L
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It is generally accepted that, after allogeneic hematopoietic cell transplantation (HCT), cure of CML and Philadelphia chromosome-positive ALL depends on the contribution of the graft-versus-leukemia (GVL) effect . However, although this strategy seems very promising for more indolent diseases, results in advanced-phase CML and in Ph-positive ALL remain disappointing. These observations provide a rationale for combining allogeneic HCT, and particularly allogeneic HCT with a nonmyeloablative regimen, to imatinib in order to maximize antileukemic activity against Philadelphia chromosome-positive leukemias. However, because SCF plays a major role in homing and proliferation of hematopoietic stem cells (HSCs), preclinical studies are needed to assess the impact of c-kit and PDGFR inhibition on hematopoiesis and on hematological reconstitution after allogeneic HCT. These are the aims of this report.' t( x& b2 u0 V p; O( d$ E
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MATERIALS AND METHODS/ u4 J3 _: f/ s; t+ q+ b
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! w$ u/ u: @# A8 P7 q% ]' b" Q$ {After informed consent was obtained, G-CSF-mobilized peripheral blood (PB) mononuclear cells were obtained from normal adult volunteers according to the guidelines established by the Ethical Committee of the University of Li¨¨ge (Li¨¨ge, Belgium). Mobilization was achieved by daily G-CSF administration at 10 µg/kg for 5 consecutive days. Cells were collected by apheresis on day 5. HSCs were subsequently isolated by CD133 selection with the Clinimacs device (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer's instructions. Mean CD133 cell positivity in the final product was more than 95%. Cells were cryopreserved in small aliquots in 90% fetal calf serum (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and 10% dimethyl sulfoxide (Sigma, Bornem, Belgium, http://www.sigmaaldrich.com).+ }* { X, s( x3 l
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Cord blood (CB) samples were collected in acid citrate dextrose solution in accordance with standard procedures for CB banking. All samples were processed within 24 hours of delivery. All material was obtained after informed consent and used according to the guidelines established by the Ethical Committee of the University of Li¨¨ge. Mononuclear low-density cells were isolated by centrifugation over Ficoll-Paque (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com).7 |1 z; m1 z9 e9 [$ d# I
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The murine stromal MS-5 cells were cultivated in -minimal essential medium (Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium, http://www.cambrex.com) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37¡ãC.
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& }' f: r# ^" X; } x9 Q' ~Short-Term Suspension Cultures6 C$ B m8 {% K7 q' x Y& Y8 T1 s
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CD133 cells were plated in Iscove's medium supplemented with 20% BIT (bovine serum albumin , insulin, and human transferrin) serum substitute (StemCell Technologies, Meylan, France, http://www.stemcell.com), 100 µg/ml penicillin, and 100 µg/ml streptomycin (Cambrex Bio Science Verviers S.p.r.l.). Cells were stimulated by a combination of 100 ng/ml SCF (Amgen, Thousand Oaks, CA, http://www.amgen.com), 50 ng/ml thrombopoietin (Amgen), and 100 ng/ml flt3-ligand (FL; R&D Systems, Abingdon, U.K., http://www.rndsystems.com) and maintained at 37¡ãC in a 100% humidified atmosphere with 5% CO2.
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& d+ N# ^4 ^$ s, N' iColony-Forming Cell and Long-Term Culture-Initiating Cell Assays5 T/ B I0 ?) \& W- O5 p
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Colony-forming unit-granulocyte/macrophages (CFU-GM) and BFU-E assays were performed in Methocult H4435 (StemCell Technologies). In a first set of experiments, imatinib mesylate was added to Methocult medium at 0, 0.5, 1, and 2 µM on day 0. In a second set of experiments, CD133 cells were first cultured in vitro with 0, 0.5, 1, and 2 µM imatinib (see Short-Term Suspension Cultures) for 48 hours and then washed and cultured in Methocult without imatinib.5 t5 T: m! }! y- C8 t: k. @3 `
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Colony-forming unit-megakaryocyte (CFU-Mk) assays were performed with the Mega Cult-C kit (StemCell Technologies) as recommended by the manufacturer.
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Absolute frequencies of long-term culture-initiating cells (LTC-ICs) were determined by limiting dilution analysis as previously described . In a first set of experiments, imatinib at 0, 1, 2, and 5 µM was added on the day of addition of CD133 cells as well as with each weekly half medium change (n = 3). In a second set of experiments, CD133 cells were first cultivated in vitro with 0, 1, 2, and 5 µM imatinib (see Short-Term Suspension Cultures) for 48 hours and then washed and assayed for LTC-IC without any imatinib (n = 3). The percentage of LTC-IC was calculated using L-calc software (StemCell Technologies).
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FACS Analysis
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For cell cycle analysis, CD133 cells recovered after 48 hours of culture with or without imatinib were incubated in phosphate-buffered saline (PBS) 0.6% IGEPAL CA-630 (Sigma) containing 100 µg/ml propidium iodide (PI). After a 30-minute incubation in the dark, cells were run on a FACSort flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), collecting FL-2 channel values. The percentage of cells in the different phases of the cell cycle was determined with Modfit software (Becton, Dickinson and Company). The percentage of cells in cycle was calculated as follows: percentage = x 100.
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For analysis of integrin (very late antigen -4 and VLA-5) and CXCR4 expression, fluorescein isothiocyanate (FITC)-conjugated anti-VLA-4 (CD49d; Coulter Immunotech, Marseille, France, http://www.immunotech.fr) or FITC-conjugated anti-VLA-5 (CD49e; BD Biosciences PharMingen, San Diego, http://www.bdbiosciences.com/pharmingen) were used in combination with phycoerythrin-conjugated anti-CXCR4 (BD Biosciences PharMingen). CD133 cells recovered after 48 hours of culture with or without imatinib were incubated with antibodies or isotype-matched control immunoglobulin G for 20 minutes on ice in the dark. Cells were washed in PBS 1% calf serum (Cambrex Bio Science Verviers S.p.r.l.) and fixed in PBS 1% paraformaldehyde (Sigma).
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( Z/ x& o+ ~( I2 k) q4 S" Y4 kData were acquired on a FACSort flow cytometer. CXCR4 and integrin density were expressed as the mean channel fluorescence ratio (MCFR) defined as MCF of CXCR4 or integrin expression divided by MCF of fluorescence-matched isotypic control.
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For quantification of apoptosis and necrosis, flow cytometric analysis using annexin V fluorescein and PI (Annexin-V-Fluos Staining kit; Roche, Basel, Switzerland, http://www.roche.com) was used as described by the manufacturer./ f9 Q# q! W8 \2 [( L$ d7 J
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Flow Cytometric Cell Sorting
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6 R5 R% T4 }* |0 l2 r qCultured CD133 cells were sorted on the basis of cell cycle status as previously reported . Briefly, cells were incubated in serum-free medium supplemented with 5 mg/ml Hoechst 33,342 (Molecular Probes, Eugene, OR, http://probes.invitrogen. com) during 30 minutes at 37¡ãC. Cells in G0/G1 or in S/G2/M were sorted by a FACS Vantage (Becton, Dickinson and Company). The purity of sorted cells was confirmed by DNA staining of sorted cells and always exceeded 90%.! f, U6 e6 O) |( V1 d# H' V
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Migration Assays8 @8 o/ N* \+ I+ m" a
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Migration assays were performed in 6.5-mm diameter 5-µm- pore transwells (Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences). Two million CD133 cells were plated in 100 µl of Myelocult (StemCell Technologies) in the upper chamber of the transwell. The bottom compartment was filled with Iscove's modified Dulbecco's medium (Cambrex Bio Science Verviers S.p.r.l.) supplemented with 10% FBS and 100 ng/ml stromal derived factor-1 (SDF-1) (PeproTech EC Ltd., London, http://www.peprotechec.com). After incubation at 37¡ãC during 3 hours, nonmigrating and migrating cells were harvested by two standardized washes using warm PBS containing 1% BSA. Nonmigrating and migrating cells were counted using Trypan blue exclusion. The percentage of nonmigrating and migrating cells was calculated relative to the total number of cells recovered (recovery >95%).
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Adhesion assays were carried out on fibronectin-coated plates as previously described . Adherent and nonadherent cells were counted using Trypan blue exclusion. The percentage of adherent and nonadherent cells was calculated relative to the total number of cells recovered (recovery >95%). Adhesion on BSA-coated control plates was less than 2%.3 E& K+ ?0 Z+ x1 q/ l
; K# A* E5 D* J( w: Z2 NTransplantation into Nonobese Diabetic/Severe Combined Immunodeficient/ß2mnull Mice! ?- j1 T) g7 ~+ x* d. Z: {: D8 n
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Nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/ß2mnull mice were sublethally irradiated on day 0. Four hours later, 6 x 105 human CD133 HSCs were inoculated intravenously. Mice were started on day 0 on a placebo (sterilized water, first group) or on imatinib (50 mg/kg every morning and 100 mg/kg every evening, second group) by gavage. Blood was collected 1, 2, and 4 weeks after the transplant with a heparined capillary (Hirschmann Laborgerate, Mannheim, Germany, http://www.hirschmann-laborgeraete.de) after cutting the mouse tail. Mice were sacrificed 6 weeks after xenotransplantation. Blood was collected on anticoagulant citrate dextrose by vena cava puncture. Cells were counted with an ABX automatic cell counter (ABX Hematology, Montpellier, France, http://www.horiba-abx.com) with appropriate corrections for dilution by ACD. Bone marrow cells were harvested from the two femurs, suspended into single-cell suspension, counted, and stained with anti-human CD45 (BD Biosciences PharMingen) to determine the overall level of human chimerism. Engraftment was compared between the two groups of mice.6 z( d- v; ] A+ N7 j& v
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Statistical Analyses. t9 Y3 } }- X; e1 z) e
; ?1 }* @4 l' S* F7 d4 J5 ^One-way analysis of variance tests were used to assess the impact of imatinib on colony and LTC-IC formation and migration/adhesion abilities of CD133 cells. Unpaired and paired Student's t tests were used to compare the effects of imatinib on the percentage of CD133 cells in cycle and the engraftment of CD133 cells into NOD/SCIDß2mnull mice. Welsh's correction was used in case of unequal variance. The probability of survival was studied by Kaplan-Meier methods. Statistical analyses were carried out with GraphPad Prism (GraphPad Software, San Diego, http://www.graphpad.com).
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RESULTS
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Imatinib Decreased Generation of Colony-Forming Units from CB and PB
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4 Y! ^6 Z( \* W) EResults of 10 consecutive CB cultures were analyzed for CFU-GM and BFU-E generation. Imatinib added in CFU culture assays on day 0 at 0.5, 1, and 2 µM decreased CFU-GM formation by a mean ¡À SD of 11% ¡À 16%, 20% ¡À 21%, and 44% ¡À 19%, respectively (p 9 j& x1 h; k& ~$ P
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Figure 1. Impact of imatinib in vitro (1). (A): Effect of imatinib on CFU-GM and BFU-E growth from unmanipulated cord blood cells. Results are expressed as percentages relative to experiments without imatinib (see text). (B): Effect of imatinib on CFU-GM, BFU-E, and CFU-Mk growth from PB CD133-selected cells. Results are expressed as percentages relative to experiments without imatinib (see text). (C): Effect of 48-hour preculture with or without imatinib on subsequent CFU-GM and BFU-E growth from PB CD133-selected cells. Results are expressed as percentages relative to experiments without imatinib (see text). (D): Influence of imatinib on LTC-IC generation from PB CD133-selected cells. In a first set of experiments, imatinib (when present) was maintained throughout the assay (with columns). In a second set of experiments, CD133 cells were cultured for 48 hours with or without imatinib and then washed and assayed for LTC-IC without imatinib (black columns). P values were calculated with one-way analysis of variance tests. Abbreviations: CFU-GM, colony-forming unit-granulocyte/macrophage; CFU-Mk, colony-forming unit-megakaryocyte; LTC-IC, long-term culture-initiating cell; NS, not significant; PB, peripheral blood.
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In another series of experiments, CFU-GM and BFU-E generation by PB CD133 cells was analyzed in the presence of imatinib. Imatinib added in the culture on day 0 at 1, 2, and 5 µM decreased CFU-GM formation by a mean of 67% ¡À 13%, 62% ¡À 29%, and 80% ¡À 16%, respectively (p
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2 f1 V9 ]+ `$ xFinally, PB CD133 cells were first plated in cytokine-supplemented suspension culture for 48 hours with imatinib at the same concentrations and then washed and cultured in CFU assays without imatinib (n = 3). Imatinib at 1, 2, and 5 µM decreased CFU-GM formation by a mean of 17% ¡À 9%, 11% ¡À 5%, and 38% ¡À 6%, respectively (p = .0003), and BFU-E formation by ¨C8%¡À 42%, 10% ¡À 24%, and 51% ¡À 12% (p = .08), respectively (Fig. 1C).* t. a7 n2 X$ z
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Imatinib Did Not Decrease the Development of Primitive Progenitor Cells in Long-Term Cultures
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In a first set of experiments (n = 3), imatinib was added to long-term cultures at 1, 2, and 5 µM on day 0 and at each weekly medium change for 5 weeks. Imatinib treatment had no significant impact on LTC-IC frequency (Fig. 1D). In a second set of experiments, cells were first plated in cytokine-supplemented suspension cultures for 48 hours with imatinib at the same concentrations and then washed and replated in LTC-IC assays without imatinib. Again, the frequency of LTC-IC relative to the number of plated cells was similar with or without imatinib pre-treatment at any dosage (Fig. 1D).: r0 m- U# `9 R3 Z
! ], E! }* s% m4 C0 b0 t2 A/ B6 P" QImatinib Decreased the Number of PB CD133 Cells in Cycle but Did Not Induce Apoptosis
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To determine whether the reduction of colony generation by imatinib was due to reduced proliferation, CD133 cells were analyzed by FACS after 48 hours of culture with various concentrations of imatinib in SCF-containing medium. The percentage of cells in cycle (G2/M S) was 28% ¡À 12% for control cells (without imatinib) and 18% ¡À 8% (p = .01), 19% ¡À 15% (p = .01), and 16% ¡À 11% (p = .003) for cells treated with 0.5, 1, and 2 µM imatinib, respectively (Fig. 2A¨C2C). When the same experiment was performed in a medium containing IL-3 instead of SCF, the percentage of cells in cycle was not decreased by imatinib (data not shown). S- R. v! L2 o1 O
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Figure 2. Imatinib decreased the number of peripheral blood CD133 cells in cycle but did not induce apoptosis. Cells were cultured in the absence (A) or in the presence (B) of 2 µM imatinib and then stained with Hoechst 3342 and separated into noncycling (G0/G1) and cycling (S G2/M) fractions. A representative experiment is shown. The plots are presented in FL2-A (DNA content) and FSC. (C): Effect of various imatinib concentrations on the percentage of cycling cells. (D): Effect of various imatinib concentrations on the percentage of apoptotic cells. Abbreviations: FSC, forward scatter; NS, not significant.0 A8 _/ |$ Y2 r. N g
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To determine whether the reduction of colony generation by imatinib was due to apoptosis of CD133 cells, CD133 cells after 0, 2, 4, 6, 24, 32, and 48 hours of culture with 0¨C2 µM imatinib were labeled with annexin V and PI and then analyzed by FACS. As shown in Figure 2D, imatinib did not induce apoptosis of CD133 cells. In addition, percentages of necrotic cells after 48 hours were similar without or with imatinib at 1, 2, and 5 µM in the culture (11%, 10%, 16%, and 12%, respectively) (data not shown).: @ D4 h' B+ u& f- j
8 x9 B! z6 p* C0 xImatinib Did Not Modify VLA-4, VLA-5, and CXCR4 Expression by PB CD133 Cells
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Because VLA-4, VLA-5, and CXCR4 play a major role in the homing of HSCs, we investigated the effect of imatinib on the expression of these surface receptors by FACS after 48 hours of suspension culture in three different experiments. MCFR values of VLA-4, VLA-5, and CXCR4 were not modified by imatinib (Fig. 3A).
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" \- ~5 X0 @$ d) ]) ~8 B+ yFigure 3. Impact of imatinib in vitro (2). (A): Impact of imatinib on VLA-4, VLA-5, and CXCR4 expression by PB CD133 cells. (B): Impact of cell cycle on expression of VLA-5 relative to VLA-4 (VLA-5/VLA-4 ratio) in cells cultured 48 hours with or without imatinib. (C): Impact of imatinib on adhesion to fibronectin of cultured PB CD133 cells. (D): Impact of imatinib on migration across fibronectin of cultured PB CD133 cells. Abbreviations: MCFR, mean channel fluorescence ratio; NS, not significant; PB, peripheral blood; VLA, very late antigen.
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, x% k# W+ P3 }6 y: WWe have previously shown an increased expression of VLA-4 and VLA-5 in cycling CD34 cells, resulting in increased binding and defective migration across fibronectin . To analyze whether this observation was also encountered in imatinib-treated CD133 cells, we analyzed VLA-5 and VLA-4 expression in cycling and noncycling cells in three different experiments. We observed a parallel upregulation of VLA-4 and VLA-5 expression in cycling cells, which was similar in imatinib-treated and control cells (Fig. 3B)./ |; Q9 x# j" k8 O, _/ j
2 \( [3 R: E9 e0 @. t" E& GImatinib Increased Adhesion to Fibronectin and Decreased Migration of CD133 Cells- [' C7 }2 i: B
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CD133 cells cultured in the presence of 0, 1, 2, and 5 µM imatinib were analyzed in adhesion assays (n = 3). As shown in Figure 3C, imatinib increased the ability of CD133 cells to adhere to fibronectin. The mean percentages of adhesion with 0, 1, 2, and 5 µM imatinib were 18.1% ¡À 3.6%, 39.6% ¡À 14.5%, 41.3% ¡À 6.9%, and 33.2% ¡À 18.3%, respectively.
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CD133 cells cultured in the presence of 0, 1, 2, and 5 µM imatinib were also analyzed in migration assays (n = 4). The mean percentages of migration through filters toward SDF-1 were 54.9% ¡À 9.8%, 42.2% ¡À 14.1%, 31.1% ¡À 4.9%, and 22.3% ¡À 6.9%, respectively (Fig. 3D).
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Imatinib Did Not Impair Engraftment of Human CD133 Cells into NOD/SCIDß2mnull Mice
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In a first set of experiments, 15 mice (six controls and nine treated with imatinib) received 3 Gy total body irradiation (TBI) before transplantation. Six of the nine mice in the imatinib group died 7, 8, 10, 10, 11, and 39 days after the transplant, from irradiation toxicity, including persistent marrow aplasia. The other nine mice were sacrificed on day 42. Among surviving animals, bone marrow cellularity was 107,600 ¡À 7,584 cells for control mice versus 105,000 ¡À 7,506 cells for imatinib-treated mice (not significant ) (Fig. 4A). Bone marrow human chimerism was 21.0% ¡À 9.4% for control mice versus 37.9% ¡À 2.8% for imatinib-treated mice (p = .021) (Fig. 4C).
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: y. i" E: Z/ W2 J- k m5 WFigure 4. Impact of imatinib in vivo. (A): Impact of continuous imatinib administration on BM cellularity 6 weeks after xenotransplantation of peripheral blood CD133 cells in mice conditioned with 3 Gy TBI. (B): Impact of imatinib on platelet recovery in mice conditioned with 1 Gy TBI. (C): Impact of continuous imatinib administration on BM human chimerism 6 weeks after xenotransplantation in mice conditioned with 3 Gy TBI. Continuous lines show the medians. (D): Impact of imatinib administration on BM human chimerism 6 weeks after xenotransplantation in mice conditioned with 1 Gy TBI. Continuous lines show the medians. Abbreviations: BM, bone marrow; NS, not significant; TBI, total body irradiation.
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In a second set of experiments, 11 mice (five controls and six treated with imatinib) received only 1 Gy TBI before transplantation. There was a trend for lower WBC count (p = .06), platelet count (p = .2), and Hb level (p = .6) in the imatinib group (Fig. 4B). All 11 mice were sacrificed on day 42, and human cells were detected in the bone marrow of all mice. Bone marrow human chimerism was 4.6% ¡À 5.4% for control mice versus 3.9% ¡À 2.9% for imatinib-treated mice (NS) (Fig. 4D).
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In a third experiment, mice were transplanted with CD133 cells pretreated during 48 hours with (15 mice) or without (15 mice) imatinib 2 µM after 2 Gy TBI. One mouse in the imatinib group died 37 days after the transplant. On day 42, the level of human chimerism was assessed. Bone marrow cellularity was similar in the two groups. Bone marrow human chimerism was 1.5% ¡À 0.2% for control mice versus 2.0% ¡À 0.4% for imatinib-treated mice (NS) (Fig. 5).
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0 W! [' M5 o- {% P* K, g4 Z- [Figure 5. NOD/SCID repopulating activity of PB CD133 cells pre-incubated in 2 µM imatinib-supplemented medium. Mice were conditioned with 2 Gy TBI but did not receive imatinib. Human chimerism was assessed 6 weeks after transplantation. Abbreviations: NOD/SCID, nonobese diabetic/severe combined immunodeficient; PB, peripheral blood; TBI, total body irradiation.2 u) U$ D8 I$ m* \6 `
* {+ r+ z' |0 ]% bDISCUSSION, V; V: f5 E$ b" n7 d @
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Myelosuppression is common in CML patients treated with imatinib . Because the BCR/ABL clone is responsible for the major part of hematopoiesis in CML patients, myelosuppression is an expected therapeutic effect. On the other hand, because imatinib also inhibits c-kit (the receptor for SCF), myelosuppression in CML patients may also be due, in part, to the inhibition of this tyrosine kinase. In this article, we investigated the impact of imatinib on normal hematopoiesis. Several observations have been made.
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First, as reported by Bartolovic et al. .
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# X1 `( q+ V, q% Y3 R. a' oSecond, when cell proliferation is not artificially stimulated by exogenous cytokines, imatinib treatment did not significantly inhibit LTC-IC formation, providing evidence (as previously reported) that the immature stem cell compartment is less/not sensitive to the inhibitory effect of imatinib in these conditions .
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Third, we did not observe any impact of imatinib on VLA-4, VLA-5, or CXCR-4 expression. However, imatinib increased adhesion and decreased migration of CD133 cells across fibronectin. Our group .
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! T% a* z& ?8 r. m- FFinally, we showed that continuous in vivo imatinib administration did not alter engraftment of human CD133 cells into NOD/SCIDß2mnull mice. This was true even when the pre-transplant irradiation dose was reduced to 1 Gy. Also, prior incubation in imatinib-supplemented culture medium did not impair PB CD133 cell-repopulating capacity. These results suggest that imatinib has no negative impact on engraftment abilities of human CD133 cells and might support the results of Hoepfl et al., who demonstrated that imatinib, given at 25 mg/kg twice daily, has no significant influence on hematopoietic engraftment in a mice syngeneic bone marrow transplantation model , who observed that SCF neutralization increased sensitivity to radiation therapy.
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, M5 `# d% n7 P* F, gSUMMARY A- Z( d$ ? K1 ^1 K
1 m$ g" U7 y. r5 P( QOur results suggest that, despite inhibition of hematopoietic progenitor cell growth in vitro, imatinib does not interfere with hematopoietic stem cell engraftment. Further studies are needed to analyze the impact of imatinib on graft-versus-tumor effects and on post-transplant immune reconstitution .
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DISCLOSURES' ^- z3 u( G; U" p d6 J& o
: W" h$ c, ~+ {( lThe authors indicate no potential conflicts of interest.
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2 b9 M' t& ?4 s! e( iACKNOWLEDGMENTS0 @( T. u6 w ^* b( L; m. l8 X
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L.P. and F.B. contributed equally to this work. A.G. and Y.B. contributed equally to this work. F.B. and A.G. are Research Associates, and Y.B. is Research Director at the National Fund for Scientific Research (FNRS; Belgium). This work was supported in part by grants from the FNRS (T¨¦l¨¦vie), the Belgian Federation Against Cancer, the L¨¦on Fredericq Fondation and the Anticancer Center at the University of Li¨¨ge.. w4 R" Q7 N9 p
【参考文献】, A0 T* O3 g$ J* L/ F, x0 ]5 Y
5 }+ a5 X; P K& H& [6 a3 G$ U1 J; O# j
Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000;96:3343¨C3356.3 n5 b: r1 l/ C3 w0 g% C
$ u3 u4 E' c. ]2 U+ `
Kurzrock R, Kantarjian HM, Druker BJ et al. Philadelphia chromosome-positive leukemias: From basic mechanisms to molecular therapeutics. Ann Intern Med 2003;138:819¨C830.0 k9 ^; b( E9 D' i/ R
+ _! F7 s( S- u( @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¨C566.2 n# G3 j( e$ k9 s
: r- `8 q- _7 k5 T; @6 Z
Heinrich MC, Griffith DJ, Druker BJ et al. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 2000;96:925¨C932.. r% L5 y+ v' ]6 R# T" y* g
* D2 Z# E7 f# o0 B5 ^: D
Mauro MJ, O¡¯Dwyer M, Heinrich MC et al. STI571: A paradigm of new agents for cancer therapeutics. J Clin Oncol 2002;20:325¨C334.
: a2 {5 i& n7 A( M- r, K$ `; e
0 [+ E& ~( n6 h, ?9 [/ XYee NS, Paek I, Besmer P. Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: Basis for radiosensitivity of white spotting and steel mutant mice. J Exp Med 1994;179:1777¨C1787.# A9 ^% o; k. }0 M+ B
) d' P5 R! S* iOgawa M, Matsuzaki Y, Nishikawa S et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63¨C71.1 r# p4 o. o( G+ f/ X7 C
6 s9 W( {. x x/ q. ~Haylock DN, To LB, Dowse TL et al. Ex vivo expansion and maturation of peripheral blood CD34 cells into the myeloid lineage. Blood 1992;80:1405¨C1412.
/ W6 {, Q6 j7 L, x
' E* L3 Z g8 y8 Z! k, V" i1 Y/ g) ~Miller CL, Rebel VI, Helgason CD et al. Impaired steel factor responsiveness differentially affects the detection and long-term maintenance of fetal liver hematopoietic stem cells in vivo. Blood 1997;89:1214¨C1223.1 v/ o: Z# a- B8 z% B/ N1 |2 J
# x; @7 S5 T; Q% ]
Levesque JP, Leavesley DI, Niutta S et al. Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med 1995;181:1805¨C1815.) z; R% ]; o3 V' a M0 J8 x
5 A3 K; _7 i" p$ X3 T" y5 ]
Kim CH, Broxmeyer HE. In vitro behavior of hematopoietic progenitor cells under the influence of chemoattractants: Stromal cell-derived factor-1, steel factor, and the bone marrow environment. Blood 1998;91:100¨C110.
4 ^) Y: u# Q/ e- P, ~
2 O/ K4 U) j5 O! {5 _! _" }1 vPatchen ML, Fischer R, Schmauder-Chock EA et al. Mast cell growth factor enhances multilineage hematopoietic recovery in vivo following radiation-induced aplasia. Exp Hematol 1994;22:31¨C39.
: f! R3 F1 s' N8 m* P& B: ?& J( y: `( ~
Neta R, Williams D, Selzer F et al. Inhibition of c-kit ligand/steel factor by antibodies reduces survival of lethally irradiated mice. Blood 1993;81:324¨C327.
M4 V2 J. O3 ?1 \" K
( i* n8 E* Q4 d, J% jWeiden PL, Flournoy N, Thomas ED et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic marrow grafts. N Engl J Med 1979;300:1068¨C1073.6 G$ U& G h% F3 \, Q$ R# X
[) M* u3 D- Q7 [1 ^+ T
Goldman JM, Gale RP, Horowitz MM et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: Increased risk of relapse associated with T-cell depletion. Ann Intern Med 1988;108:806¨C814.1 c8 J. A* k k
3 M, G9 \& k0 w! [( {Appelbaum FR. Graft versus leukemia (GVL) in the therapy of acute lymphoblastic leukemia (ALL). Leukemia 1997;11 (suppl 4):S15¨CS17 Review.% M' b& j" N. W O0 S
/ v4 D/ T' y# i- W REsperou H, Boiron JM, Cayuela JM et al. A potential graft-versus-leukemia effect after allogeneic hematopoietic stem cell transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: Results from the French Bone Marrow Transplantation Society. Bone Marrow Transplant 2003;31:909¨C918.& y4 {5 v8 { O( K
6 n; Y. [2 e" }) mKottaridis PD, Milligan DW, Chopra R et al. In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 2000;96:2419¨C2425.; U8 t" W5 h4 s& u$ O+ h/ [ {5 W
4 P, Q% i- b% M( NOr R, Shapira MY, Resnick I et al. Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in first chronic phase. Blood 2003;101:441¨C445.& B( a3 z' |* \, E0 u/ c3 u8 d/ K& S. N8 \
; V) m& j% S: F) Z; ]+ Z$ Z8 OWong R, Giralt SA, Martin T et al. Reduced-intensity conditioning for unrelated donor hematopoietic stem cell transplantation as treatment of myeloid malignancies in patients older than 55 years of age. Blood 2003;102:3052¨C3059.
9 i! ?2 k$ U) b& b/ _, ?/ I
& v+ S1 k, @1 f3 I8 X3 uChilds R, Clave E, Contentin N et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: Full donor T-cell chimerism precedes alloimmune response. Blood 1999;94:3234¨C3241.
/ m2 O X# V8 t3 y0 o# b7 H$ D# N# t- e! y9 p8 V9 ^- U
McSweeney PA, Niederwieser D, Shizuru J et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: Replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 2001;97:3390¨C3400.
( A" Q1 x8 p" i% r1 y7 |) o8 r- a5 h/ J
Maris MB, Niederwieser D, Sandmaier BM et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 2003;102:2021¨C2030.
. C4 B W7 G L- h& q0 x+ x- y' t5 {% @
Khouri IF, Lee MS, Saliba RM et al. Nonablative allogeneic stem-cell transplantation for advanced/recurrent mantle-cell lymphoma. J Clin Oncol 2003;21:4407¨C4412.) b: Q1 ^' R. C/ N2 I' S
7 s( _3 y }2 `# a
Baron F, Baker JE, Storb R et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004;104:2254¨C2262.
k5 T( B% J) f( ~4 W7 d! M# T" y5 v8 X# I3 M3 i; N
Kerbauy FR, Storb R, Hegenbart U et al. Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML. Leukemia 2005;19:990¨C997.
8 @2 q8 X f: p( O1 _9 e) r5 H+ L7 w/ P9 I1 O! C
Baron F, Maris MB, Storer BE et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with chronic myeloid leukemia. Biol Blood Marrow Transplant 2005;11:272¨C279.
2 K" r8 F u5 U' i
1 r) Y+ K8 N9 q; G( {8 d$ Q2 EBaron F, Maris MB, Sandmaier BM et al. Graft-versus-tumor effects after allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning. J Clin Oncol 2005;23:1993¨C2003.
% L3 u5 }$ q1 S1 ~2 e d* N0 P! a7 x, L
Giet O, Van Bockstaele DR, Di S et al. Increased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells. Blood 2002;99:2023¨C2031., ^, ~) ~0 O4 C* i5 v0 G
4 ^6 `6 Z g; Z; ^2 b
Huygen S, Giet O, Artisien V et al. Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture. Blood 2002;100:2744¨C2752.+ r2 f: f+ d8 a& s3 H3 \
9 h" u8 Y" d3 _7 e( N! A9 hDruker BJ. STI571 (Gleevec) as a paradigm for cancer therapy. Trends Mol Med 2002;8:S14¨CS18.) X! s! D9 \6 W0 W
8 f* \; X- f) C2 y1 F/ T1 K* ~
Bartolovic K, Balabanov S, Hartmann U et al. Inhibitory effect of imatinib on normal progenitor cells in vitro. Blood 2004;103:523¨C529.2 | S9 [* _- m! C; u* E6 Y
. b( |7 R3 K5 oDeininger MW, Goldman JM, Lydon N et al. The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 1997;90:3691¨C3698.8 g% g( Y$ Z. K9 Z$ `
8 r8 I0 N8 l; C' S7 M
Holtz MS, Slovak ML, Zhang F et al. Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation. Blood 2002;99:3792¨C3800.
) L& _4 V% F0 @) Y9 y
! k; c) R: m1 R: ZGiet O, Huygen S, Beguin Y et al. Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin. Exp Hematol 2001;29:515¨C524.
0 D7 ^9 p* m4 \# |1 ?
6 I2 X C# _. QFoguenne J, Huygen S, Greimers R et al. Modulation of homing properties of primitive progenitor cells generated by ex vivo expansion. Haematologica 2005;90:445¨C451.
) S5 p. Y; H+ E0 J. M* l8 d
( S* ?3 ^7 b. D0 [Yong KL, Fahey A, Pizzey A et al. Influence of cell cycling and cell division on transendothelial migration of CD34 cells. Br J Haematol 2002;119:500¨C509.5 b+ J- f2 Z( T$ U+ G
2 M `# K! [, C7 ^, i5 [Levesque JP, Simmons PJ. Cytoskeleton and integrin-mediated adhesion signaling in human CD34 hemopoietic progenitor cells. Exp Hematol 1999;27:579¨C586.( u6 i7 H* G# z7 T' C
' b0 v3 `% e! {, X! I, [5 p& _7 l& x
Hoepfl J, Miething C, Grundler R et al. Effects of imatinib on bone marrow engraftment in syngeneic mice. Leukemia 2002;16:1584¨C1588.: W3 i2 X! \4 \, d
9 G% K4 ?. c) K- QMohty M, Blaise D, Olive D et al. Imatinib: The narrow line between immune tolerance and activation. Trends Mol Med 2005;11:397¨C402. |
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