Published online before print September 2, 2003
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,1
* Department of Pediatrics, Shinshu University School of Medicine, Matsumoto,
Shinshu University Graduate School of Medicine, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Matsumoto,
Research & Development, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo,
Research Division, Tsukuba Research Institute, Novartis Pharma, Tsukuba, Japan.
1Correspondence: Department of Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan. E-mail: koikeken{at}hsp.md.shinshu-u.ac.jp
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: stem cell factor • CD34+ cells • c-kit • cord blood
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The c-kit receptor belongs to the type III receptor tyrosine kinase subfamily and consists of an extracellular domain with five Ig-like motifs, a single short membrane-spanning domain, and a cytoplasmic domain with tyrosine kinase activity [3 , 4 ]. Ligation of SCF promotes c-kit receptor homodimerization, followed by induction of multiple intracellular signaling pathways. The ATP binding site locates in the kinase domain proximal to the cell membrane. Activating mutations of c-kit have been reported in several human neoplasms, including mastocytosis, mast cell leukemia, and gastrointestinal stromal tumor [5 6 7 8 9 ].
STI571 (imatinib mesilate) is a competitive inhibitor of a few tyrosine kinases, including bcr-abl, abl, c-kit, and platelet-derived growth factor receptors. It binds to the ATP binding site of the target kinase and prevents the transfer of phosphate from ATP to the tyrosine residues of various substrates. Recent studies showed that STI571 is highly active in patients with chronic myeloid leukemia and other Philadelphia chromosome-positive leukemias [10 11 12 13 14 15 ]. Heinrich et al. [16 ] demonstrated that STI571 can inhibit c-kit-dependent signaling and proliferation of a human mast cell leukemia cell line (HMC-1). The c-kit mutation affecting the structure of the catalytic portion of the kinase is indicated to be resistant to the compound [17 ]. Several clinical trials provided a significant efficacy of STI571 in advanced gastrointestinal stromal tumors [18 , 19 ]. In addition, the compound has been reported to block the growth of some human cancers that coexpress c-kit and SCF [20 , 21 ]. These lines of evidence prompted us to evaluate the effects of STI571 on the growth and function of normal mast cells in humans. For this purpose, we used a serum-deprived culture system that permitted the selective growth of a large number of mast cells from CD34+ human cord blood under stimulation with SCF [22 ].
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STI571 was obtained from Novartis Pharma, Basel, Switzerland. The compound was solubilized in phosphate-buffered saline (PBS) at a concentration of 10-2 M, and stored at -80°C. All-trans retinoic acid (ATRA) was obtained from Sigma (St. Louis, MO) and dissolved in ethanol at a concentration of 10-2 M, and stored in light-protected vials at -80°C.
For immunocytochemical staining, purified mAb for tryptase (MAB1222) was purchased from Chemicon International Inc. (Temecula, CA). For the flow cytometric analysis, the mAbs for CD34 (8G12, fluorescein isothiocyanate, FITC), c-kit (104D2, phycoerythrin, PE), CD11a (HI 111, FITC), CD18 (6.7, FITC), CD29 (MAR4, PE), CD49c (C3 II.1, PE), CD49d (9F10, PE) and CD49e (IIA, PE) were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). The mAbs for CD13 (SJ1D1, FITC) and CD41 (SZ22, FITC) were from Immunotech S.A. (Marseilles, France). The mAbs for CD11b (2LPM19c, PE) and glycophorin A (GPA, JC159, FITC) were from Dako (Glostrup, Denmark). For Western blotting and immunoprecipitation, the mAbs for c-kit (NU-c-kit) and for phosphotyrosine (4G10) were purchased from Nichirei (Tokyo, Japan) and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively.
Cell preparation
Cord blood samples were aspirated in heparinized plastic syringes from the umbilical vein at normal delivery. Fully informed consent was obtained from the mothers of all neonates before harvesting the specimens. Mononuclear cells (MNCs) were separated by density centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), washed twice, and suspended in Ca2+- and Mg2+-free PBS containing 1 mmol/L EDTA-2Na and 2.5% fetal bovine serum (FBS, Hyclone, Logan, UT). After treatment with Silica (Immuno-Biological Laboratories, Fujioka, Japan) for 30 min at 37°C, CD34-positive cells were enriched using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. After treatment with 100 µL of Fc-receptor blocking reagent, 0.5-1 x 108 MNCs were mixed with 100 µL of colloidal super-paramagnetic MicroBeads conjugated to a mouse mAb specific for CD34 (QBEND/10) and incubated for 30 min at 4°C. The magnetically labeled cells were separated with a MS+/RS+ column in the magnetic field of the MACS separator (VarioMACS). More than 90% of the isolated cells were CD34-positive according to the flow cytometric analysis (data not shown).
Suspension cultures
Serum-deprived liquid cultures were carried out in 24-well culture plates (#3047; Becton Dickinson) using a modification of the technique described previously [22
23
24
]. To examine the effects of STI571 on the SCF-dependent development of mast cells, we used 10-week cultured cells grown with 20 ng/mL of SCF from CD34+ cord blood cells as target cells, because a large number of such cultured cells were positive for tryptase, as described previously [22
, 23
]. In a dose-response study, 10-week cultured cells (1x105) were incubated for 2 weeks in 24-well culture plates containing 2 mL of 
-medium supplemented with 1% bovine serum albumin (BSA), 300 µg/mL fully iron-saturated human transferrin (
98% pure, Sigma), 16 µg/mL soybean lecithin (Sigma), 9.6 µg/mL cholesterol (Nakalai Chemicals Ltd., Tokyo, Japan), 20 ng/mL of SCF with or without various concentrations of STI571. CD34+ cells (2x104) were cultured for 4 weeks in each well containing 20 ng/mL of SCF, 10 ng/mL of GM-CSF, 2 U/mL of EPO, 10 ng/mL of TPO, and different concentrations of STI571, alone or in combination. The plates were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. To prevent overgrowth and subsequent inhibition of proliferation, half of the cells and culture medium was replaced weekly with fresh medium containing the factor(s). The number of viable cells was determined by a trypan-blue exclusion test using a hemocytometer.
Clonal cell cultures
The mast cell colony assay was carried out in 35-mm Lux suspension culture dishes (#171099; Nunc, Naperville, IL) using a modification of the technique described previously [25
]. The culture consisted of CD34+ cord blood cells (1,000 cells/mL) or 10-week cultured cells (4,000 cells/mL), -medium, 0.9% methylcellulose (Shinetsu Chemical, Tokyo, Japan), 1% BSA, 300 µg/mL of fully iron-saturated human transferrin, 16 µg/mL of soybean lecithin, 9.6 µg/mL of cholesterol and 100 ng/mL of SCF with or without 10-6 M of STI571. The dishes were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. After 4 weeks, aggregates consisting of 30 or more cells were scored as mast cell colonies, and those consisting of 10 to 29 cells as mast cell clusters. Thirty individual colonies and clusters were lifted, and stained with the anti-tryptase mAb or mouse IgG1 using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. Almost all of the constituent cells were positive for tryptase.
Cytochemical and immunologic stainings
The cultured cells were spread on glass slides using a Cytospin II. A cytochemical reaction with peroxidase (POX) was performed by the conventional method. Reaction with mAb against tryptase was detected using the APAAP method (Dako APAAP Kit System, Dako Corp., Carpinteria, CA), as described previously [26
].
Immunoprecipitation and Western blotting
Immunoprecipitation and Western blotting were performed, as described previously [27
]. For immunoprecipitation, cell lysates were incubated with a relevant antibody and protein A-sepharose 4FF (Pharmacia, Uppsala, Sweden) for 1 h at 4°C. The immunoprecipitates were then washed three times with the cold lysis buffer.
The immunoprecipitates or total cell lysates were subjected to SDS-PAGE. The protein samples separated by SDS-PAGE gels were transferred to polyvinylidene difluoride (PVDF) membranes (ImmobilonTM, Millipore Corp., Bedford, MA), identified with a relevant 1st Ab, and developed by the enhanced chemiluminescence system (Amersham Corp., Arlington Heights, IL). To remove the Abs, the membranes were submerged in stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and incubated for 30 min at 50°C with occasional agitation.
Flow cytometric analysis
For the analysis of surface markers on the cultured cells, 1-2 x 105 cells were collected in plastic tubes and incubated with appropriately diluted FITC- or PE-mAb, as described previously [22
, 28
]. The cells were washed twice, after which their surface markers were analyzed with the FACScan flow cytometer, using the Lysis 2 software program. Viable cells were gated according to their forward light scatter characteristics and side scatter characteristics, except for the analysis of DNA content of the cultured cells. The proportion of positive cells was determined by comparison with cells stained with FITC- or PE-conjugated mouse isotype-matched Ig.
For the analysis of cellular apoptosis, we carried out a flow cytometric analysis using propidium iodide (PI, Sigma) according to the procedure described previously [29
]. Approximately 5-10% of 10-week cultured mast cells grown with SCF failed to exclude trypan-blue dye. Cohen et al. [30
] reported that preapoptotic and apoptotic cells have increased density as compared with normal cells in Percoll step-density gradients. On the other hand, dead cells can be identified by their low density [31
]. Thus, the cultured mast cells were layered on 27% Percoll (Sigma) in -medium and 54% Percoll in PBS to reduce the numbers of already dead cells and dying cells. After centrifugation, the cells were harvested from the interface of the two different concentrations of Percoll solution, washed with PBS, and incubated with 20 ng/mL of SCF and/or 10-6 M STI571. The cultured cells were collected and treated with 1 mL of Ortho PermeaFixTM for 40 min at room temperature. The cells were then incubated with DNase-free RNase (Sigma) for 15 min at 37°C, and stained with PI for 15 min. The DNA content was analyzed with a flow cytometer.
DNA gel electrophoresis
The 10-week cultured mast cells (2x106) exposed to SCF or SCF+STI571 for 4 days were lysed for 10 min on ice in 100 µL of a hypotonic lysis buffer [10 mM Tris (pH 7.5), 10 mM EDTA, pH 8.0, 0.5% Triton X-100]. After centrifugation at 14,000 g for 10 min, the supernatant was transferred to a new tube and treated with 0.2 mg/mL RNase A (Sigma) and 0.2 mg/mL Proteinase K (Sigma). DNA was precipitated with 120 µL of isopropanol and 20 µL of 5M NaCl overnight at -20°C. After centrifugation at 14,000 g for 15 min, the pellets were dried and dissolved in 20 µL of Tris-EDTA. The samples were analyzed by gel electrophoresis in 2% agarose and ethidium bromide staining.
Assay of histamine and tryptase levels
Histamine and tryptase concentrations in the cell lysates obtained by the treatment of the cultured cells with 0.5% Nonidet P-40 were measured with a Histamine Radioimmunoassay (RIA) Kit (Immunotech), and a fluoroenzymeimmunoassay (UniCAP Tryptase, Pharmacia and Upjohn Diagnostics AB, Uppsala, Sweden), respectively, as described previously [22
, 23
].
Adherent assays
Adherent assays were performed by a modification of the procedure described by Shimizu et al. [32
]. We coated 24-well plates (Nalge Nunc, Naperville, IL) with 400 µl PBS containing 40 µg of fibronectin (FN, Sigma) for 12 h at 4°C. As a negative control, BSA was used at 30 mg/ml. The wells were washed and blocked with 3% BSA in PBS for 40 min at 37°C. After rinsing with PBS, a 200-µl aliquot of the cell suspension (6x104 cells) was placed in each well and incubated with 20 ng/ml of SCF and/or 10-6M STI571 for 2 h at 37°C. The wells were washed with PBS twice to remove nonadherent cells. Then, the adherent cells were harvested by the treatment with 200 µl of trypsin-EDTA solution (Invitrogen Corp. Carlsbad, CA) for 5 min at 37°C. The numbers of adherent and nonadherent cells were determined by trypan-blue exclusion test using hemocytometers. The percentage of adherent cells was calculated as follows: Net % Adhesion = (FN adherent-BSA adherent) ÷ (FN adherent+FN nonadherent-BSA adherent) x 100.
Statistical analysis
All experiments were carried out at least 3 times and were shown to be reproducible. Values are expressed as means ± SD. To determine the significance of difference between two independent groups, we used the unpaired t-test, or the Mann-Whitney-U test when the data were not normally distributed. One-way ANOVA (ANOVA), followed by post hoc contrasts with Bonferroni limitation, was used for more than three independent groups.
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5 times the input quantity after 2 weeks under stimulation with SCF. Approximately 5-10% of cells failed to exclude trypan-blue dye. There were no differences in numbers of viable and dead cells between SCF and SCF+STI571 at 10-10 M to 10-8 M. The addition of STI571 at 10-6 M or higher to the culture with SCF almost completely inhibited the generation of progeny. Thus, STI571 was used at 10-6 M in the subsequent experiments, unless otherwise specified.
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Figure 1. Dose response to STI571 of mast cell growth supported by SCF Ten-week cultured mast cells (1x105) were incubated in wells containing SCF at 20 ng/mL with STI571 at 10-10 M to 10-5 M. After 2 weeks, viable cells were enumerated. The results are the mean ± SD of three independent experiments. Significantly different from SCF alone (*P<0.0001, #P<0.005).
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Figure 2. Comparison of the potential to inhibit SCF-dependent mast cell growth among STI571, IL-4, IL-6, and ATRA The cultured mast cells (5x104) were plated in wells containing 20 ng/mL of SCF alone or in combination with 10-6 M STI571, 20 ng/mL of IL-4, 50 ng/mL of IL-6, or 10-7 M ATRA. The viable cells were serially enumerated up to 2 weeks. The results are the mean ± SD of three independent experiments. Significantly different from SCF alone (*P<0.0001, #P<0.005).
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Figure 3. STI571 induces apoptotic death of cultured mast cells grown with SCF. We enriched viable 10-week cultured cells using Percoll gradient centrifugation, and then replated them into 20 ng/mL of SCF alone or in combination with 10-6 M STI571, 20 ng/mL of IL-4, 50 ng/mL of IL-6, or 10-7 M ATRA. (A) After 2 days, the DNA content of the cells was analyzed with a flow cytometer. (B) Cell cycle analysis was serially performed in the culture containing 20 ng/mL of SCF plus 10-6 M STI571. The incidences of viable cells (cells in G0/G1 phase and cells in S + G2/M phase) relative to total cells were calculated using LYSIS 2 software. Arrows indicate a significant appearance of cells with less than 2N. Events falling within 50% of the fluorescence of the G0/G1 peak were 0.2% on day 0, 7.0% on day 2, 10.6% on day 4, and 5.2% on day 7. (C) DNA was extracted from the cultured mast cells treated with 20 ng/mL of SCF alone or in combination with 10-6 M STI571 for 4 days. Lane 1, DNA molecular-weight marker, 100 bp DNA ladder; lane 2, SCF alone; lane 3, SCF + STI571.
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Figure 4. Comparison of cell survival between treatment with STI571 and deprivation of SCF. The cultured mast cells (1x105) were incubated with 20 ng/ml of SCF + 10-6 M STI571 or with no SCF. (A) Total viable cell numbers were serially calculated. (B) After 2 or 4 days, the cells exposed to SCF + STI571 were washed and replated into the medium containing 20 ng/ml of SCF; the cells grown with no SCF were recultured into SCF. SCF + STI571, circles; no SCF, squares. Day 0 indicates the beginning of the second culture when SCF was added back. The results are the mean ± SD of three independent experiments.
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Figure 5. STI571 inhibits tyrosine phosphorylation of c-kit in cultured mast cells under stimulation with SCF. (A) The cultured mast cells were incubated in the absence of SCF for 8 h, then plated in wells containing 20 ng/mL of SCF alone (S) or in combination with 10-6 M STI571 (S+STI) for the indicated time (0-60 min). Cell lysates from 1 x 106 cells were subjected to 10% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-phosphotyrosine mAb. Bars indicate molecular weight evaluated by marker proteins. An arrow indicates 145 kDa. (B) To confirm the effects of STI571 on c-kit tyrosine phosphorylation, cell lysates from 2 x 106 cultured mast cells were immunoprecipitated with anti-c-kit mAb. The immunoprecipitates were subjected to 10% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-c-kit mAb or anti-phosphotyrosine mAb. Lane 1, the cells 10 h after SCF deprivation; lane 2, the cells re-exposed to SCF for 10 min; lane 3, the cells exposed to SCF plus STI571 for 10 min.
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Effects of STI571 on maturation property and function of the cultured mast cells
To examine the effects of STI571 on the intracellular mediator content of the cultured mast cells, the cells were treated with or without the compound for 2 days. The histamine concentration in 1x105 viable cultured mast cells became significantly lower after incubation with SCF+STI571, compared with the control value (171.8±10.7 ng/mL in SCF vs. 121.0±3.4 ng/mL in SCF+STI571, P=0.0001). Similar results were obtained in the tryptase content: its concentration in the lysate of the cultured mast cells exposed to SCF + STI571 was 80% of the level obtained with SCF alone.
On the basis of the flow cytometric analysis, the cultured mast cells were positive for CD29, CD49c, CD49d, and CD49e (Fig. 6
), but virtually negative for CD11a and CD18 (data not shown). The exposure to SCF+STI571 for 2 h did not alter the expression of CD29, CD49c, CD49d, and CD49e (Fig. 6)
. We then examined whether STI571 influenced the adhesion of mast cells to FN in the presence of SCF. As presented in Fig. 7
, the frequency of the cells adherent to FN-conjugated wells was 40% at 2 h. On the other hand, >99% of the cells did not adhere to the BSA-conjugated wells even after 24 h. The treatment with STI571 significantly inhibited mast cell adhesion to FN in the presence of SCF. There was no difference in the sum total of adherent cells and nonadherent cells between SCF and SCF+STI571.
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Figure 6. Expressions of integrin receptors on cultured mast cells treated with or without STI571 Expressions of CD29, CD49c, CD49d, and CD49e on the cultured mast cells grown with 20 ng/ml of SCF and on those exposed to 20 ng/ml of SCF + 10-6 M STI571 for 2 h were analyzed by flow cytometry. (—), labeled with PE-conjugated mAb against CD29, CD49c, CD49d or CD49e. (...), labeled with PE-conjugated mouse IgG.
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Figure 7. Effects of STI571 on adhesion of cultured mast cells to fibronectin in the presence of SCF The cultured mast cells were incubated for 2 h in 20 ng/mL of SCF with or without 10-6 M STI571, using FN-conjugated wells or BSA-conjugated wells. Net percent adhesion was calculated, as described in Materials and Methods. The results are the mean ± SD of three independent experiments.
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Figure 8. Effects of STI571 on SCF-, GM-CSF-, EPO-, or TPO-dependent cell growth from CD34+ cord blood cells CD34+ cord blood cells (2x104) were cultured with 20 ng/mL of SCF, 10 ng/mL of GM-CSF, 2 U/mL of EPO, 10 ng/mL of TPO, and 10-6 M STI571 alone or in combination. The viable cells were enumerated at 2 weeks. The results are the mean ± SD of three independent experiments. Black bar, c-kit+ cells; black hatched bar, CD11b+/CD13+ cells; white hatched bar, GPA+ cells; white dotted bar, CD41b+ cells. Significantly different from no STI571 (*P<0.0001, #P<0.01).
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Although IL-4, IL-6, and ATRA suppressed the SCF-dependent progeny production, the total viable cell numbers on day 14 were higher than the input value. On the basis of the flow cytometric analysis, the IL-4-, IL-6- and ATRA-mediated inhibition of mast cell growth may result from a decrease in proliferation rate rather than an increase in apoptosis. On the other hand, STI571 almost completely abrogated the SCF-dependent progeny generation of the cultured mast cells up to 14 days. In cell cycle analysis, 2-day exposure to SCF + STI571 resulted in an emergence of cells with less than 2N as well as a decrease in percentage of cells in the S + G2/M phase. The effect of STI571 was due to apoptosis induction, as indicated by the gel electrophoresis. After 7 days, a majority of the cells fell into apoptotic death, suggesting that exposure time is crucial for the compound to complete apoptosis of mast cells. On day 4, half of cells exposed to the two-factor combination were at the G0/G1 phase. This may be related to a recovery of cell proliferation after removal of ST1571 from the culture media. Coupled with similar regrowth after the delayed addition of SCF to culture wells with no factor, it is likely that some mast cells can "hibernate" for a period of time in the absence of their survival factor.
STI571 markedly decreased the tyrosine phosphorylation of c-kit with no alteration of the total cellular protein level. The time-course study showed that the surface expression of c-kit of the cultured mast cells was down-modulated after the re-exposure to SCF, whereas the c-kit expression remained unchanged up to 6 h after the treatment with SCF + STI571. It is generally held that binding of SCF induces rapid internalization of the SCF/c-kit receptor complex [33 , 34 ]. In parallel, the c-kit receptor is ubiquitinated and targeted for degradation by the proteasome proteolytic pathway. Using mutant c-kit receptors, Yee et al. [34 ] demonstrated that SCF-induced internalization and ubiquitination/degradation require the active c-kit receptor kinase. Therefore, no change in the surface c-kit expression of cultured mast cells after the exposure to SCF + STI571 may result from a failure of internalization and ubiquitination/degradation of the c-kit receptor.
STI571 exerted its inhibitory effects at the early phase of mast cell development, as well as on the growth of the late-appearing mast cells. In contrast, GM-CSF-, EPO-, and TPO-dependent proliferation of CD34+ cord blood cells was hardly affected by STI571. These results are in line with those reported by Druker et al. [11 ], that the numbers of erythroid and GM colonies from normal bone marrow cells are equal in the presence or absence of STI571 at 10-6 M. The addition of the compound significantly hindered the interaction of SCF with the other growth factors. STI571 reduced GM-CSF + SCF-dependent and TPO + SCF-dependent progeny production to the level obtained by GM-CSF and TPO alone, respectively. On the other hand, EPO + SCF-dependent erythroid cell production was substantially inhibited by the addition of STI571, but only in part. These distinct results may be due to the difference in the potential to generate progeny from CD34+ cord blood cells among the two-factor combinations; EPO plus SCF exerted the most potent synergism. Thus, the antiproliferative action of STI571 appears to be restricted to a SCF-dependent cell production system in human hematopoiesis. The results described above may also account for the occurrence of thrombocytopenia and neutropenia caused by the treatment with STI571 in patients who had chronic myeloid leukemia and Ph-positive acute lymphoblastic leukemia [12 ]. Therefore, further studies are required for the clinical application of this compound in allergic disorders.
Received June 8, 2002; revised July 11, 2003; accepted July 16, 2003.
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