QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.125237v1 324/1/128    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jensen, B. M. Articles by Gilfillan, A. M. Search for Related Content PubMed PubMed Citation Articles by Jensen, B. M. Articles by Gilfillan, A. M.

INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Concurrent Inhibition of Kit- and FcRI-Mediated Signaling: Coordinated Suppression of Mast Cell Activation

Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (B.M.J., S.I., D.D.M., A.M.G.); and Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland (M.A.B.)

Received May 3, 2007; accepted October 4, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcRI-mediated signaling would be an attractive approach for targeting mast cell-driven allergic reactions. To explore this concept, we examined the effects of hypothemycin, a molecule that we identified as having such properties, in human and mouse mast cells. Hypothemycin blocked Kit activation and Kit-mediated mast cell adhesion in a similar manner to the well characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle for a coordinated approach for the suppression of mast cell activation and provide a rationale for the development of compounds with a similar therapeutic profile.

Stem cell factor (SCF)-mediated Kit activation is required for mast cell growth and differentiation and subsequent survival of the mature mast cells (Metcalfe et al., 1997; Kirshenbaum et al., 1999; Okayama and Kawakami, 2006). Furthermore, by the processes of chemotaxis and cell adhesion, SCF may contribute to the homing of mast cells to their eventual sites of residence (Okayama and Kawakami, 2006). It is also now recognized, however, that SCF dramatically enhances antigen-dependent mast cell degranulation, as indicated by studies conducted in both rodent and human mast cells (Bischoff and Dahinden, 1992; Hundley et al., 2004; Tkaczyk et al., 2004). Likewise, in the absence of SCF, antigen only minimally elevates cytokine message and protein levels in human mast cells, whereas in the presence of SCF, the levels are markedly enhanced (Hundley et al., 2004; Tkaczyk et al., 2004). Whether such synergy occurs in vivo is unclear, although due to the absolute requirement for SCF in mast cell homeostasis, this is likely to be the case. Indeed, under conditions of low FcRI aggregation, the relative contribution of SCF to degranulation may be greater than that of antigen (Tkaczyk et al., 2004). Therefore, significant degranulation and cytokine production may occur even at the low level of antigen-dependent FcRI aggregation that could be achieved following the anti-IgE treatment or similar approach to dampen only the FcRI-mediated response. Thus, concurrent targeting of both Kit- and FcRI-mediated signaling would be an attractive therapeutic approach for the treatment of mast cell-driven disorders. Thus, we wished to identify a molecule that would concurrently inhibit Kit kinase activity and the pathway described above for FcRI with the aim of providing a basis for further investigation of this concept and potentially the development and optimization of novel compounds, which may have a similar mode of action.

Both FcRI- and Kit-dependent responses in mast cells are initiated by activation of tyrosine kinases. In the case of FcRI, the principal tyrosine kinases responsible for these early events are the Src kinase Lyn and Syk. Lyn-dependent activation of Syk results in the phosphorylation of the transmembrane adaptor molecule LAT. This orchestrates the recruitment and thereby activation of phospholipase (PL) C₁, a critical enzyme for the generation of the calcium signal required for degranulation (Gilfillan and Tkaczyk, 2006; Rivera and Gilfillan, 2006). A complementary pathway regulated by the Lyn-related kinase Fyn leads to phosphoinositide 3-kinase (PI3K) activation, thus providing membrane docking sites for signaling molecules, such as the tyrosine kinase, Btk, and PLC₁. These two pathways act in conjunction for optimal degranulation and cytokine production. This latter response also requires activation of the Ras/Raf/MAP kinase cascade. Many of these terminal events are also regulated by Kit. However, inherent tyrosine kinase activity and multiple docking sites on Kit preclude the requirements for recruitment of cytosolic tyrosine kinases, such as Syk, and LAT-mediated recruitment of PLC₁ in these responses (Gilfillan and Tkaczyk, 2006; Rivera and Gilfillan, 2006).

Molecules that target Kit activity have been investigated for their potential therapeutic utility in the treatment of mastocytosis, and several such compounds have been described in the literature (Zermati et al., 2003; Growney et al., 2005; Petti et al., 2005; Gleixner et al., 2006; Potapova et al., 2006; Schirmer et al., 2006; Schittenhelm et al., 2006; Shah et al., 2006; Verstovsek et al., 2006; Pan et al., 2007). Of these, the most widely used compound is imatinib mesylate (imatinib), also known as Gleevec, Glivec, and STI571 (Schindler et al., 2000; Scheinfeld, 2006). Because of the selective nature of these compounds (Jensen et al., 2007), it is unlikely that they will be similarly efficacious in their ability to inhibit FcRI-mediated signaling events. However, we have identified a molecule, the resorcylic acid lactone, hypothemycin (Schirmer et al., 2006), which irreversibly inhibits a specific subset of protein kinases, including Kit, with a conserved cysteine in the ATP-binding site (Schirmer et al., 2006; Winssinger and Barluenga, 2007). Because hypothemycin had the desired pharmacological profile, we have accordingly utilized this compound to examine the manifestations of concurrently inhibiting Kit- and FcRI-mediated signaling in mast cells in culture and in vivo. As will be reported here, hypothemycin blocks SCF-induced mast cell activation, but, unlike imatinib, hypothemycin also effectively blocks FcRI-mediated responses. As a consequence, not only is the antigen-mediated component of mast cell degranulation blocked but also the ability of SCF to potentiate these responses. This profile translated into an inhibition of mast cell-induced anaphylactic responses in vivo. Thus, this study provides proof of principle for a coordinated approach for targeting mast cell-driven allergic reactions and provides a basis for the development and optimization of other compounds with a similar therapeutic profile.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Isolation and Culturing of Mast Cells. Human mast cells (HuMCs) were developed from CD34⁺ peripheral blood mononuclear cells as described (Kirshenbaum et al., 1999). In brief, CD34⁺ cells, obtained after informed consent on a protocol approved by the NIAID Institutional Review Board committee, were cultured in StemPro-34 culture media (Invitrogen, Carlsbad, CA) containing L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 µg/ml) (Biofluids, Rockville, MD), IL-6 (100 ng/ml) (PeproTech Inc., Rocky Hill, NJ), and SCF (100 ng/ml) (PeproTech). IL-3 (30 ng/ml) was included for the 1st week of culture. Experiments were conducted 6 to 10 weeks after the initiation of culture, at which point the purity of the mast cell culture was more than 99% as verified by toluidine blue staining and detection of CD117- and FcRI-positive cells by fluorescence-activated cell sorting analysis.

Mouse bone marrow-derived mast cells (BMMCs) were developed from progenitor cells obtained from the femurs of 2- to 6-month-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME). The cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, L-glutamine (4 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), nonessential amino acids (1 mM), HEPES (25 mM), β-mercaptoethanol (50 µM), and mouse recombinant IL-3 (30 ng/ml) (Peprotech). The cell cultures were spun down and resuspended in fresh culture medium twice weekly. Experiments were conducted 4 to 6 weeks after the initiation of culture, at which point the purity of the mast cells was more than 99% determined as above. All animal experiments and protocols were approved by and conducted according to the Institute of Laboratory Animal Resources (1996).

Adhesion Assay. HuMCs were cultured overnight in SCF- and IL-6-depleted medium. BMMCs were cultured overnight in IL-3-depleted culture medium. Cells were washed twice in HEPES buffer + 0.04% bovine serum albumin (BSA) (Hundley et al., 2004), and 1 x 10⁵ human mast cells or 2.5 x 10⁵ BMMCs were seeded per well in a 96-well tissue culture plate (Falcon; BD Bioscience, San Jose, CA) precoated with 5 µg/ml fibronectin (Sigma-Aldrich, St. Louis, MO) and blocked with 4% BSA. The cells were preincubated with inhibitors for 15 min and then stimulated with 0.01 to 100 ng/ml SCF for 1 h. Nonadherent cells were removed by carefully washing the wells once. Wells used for total cells were not washed. Cells were lysed with 0.1% Triton X-100, and 100 µl of the lysate was then transferred to a 96-well plate for β-hexosaminidase determination (Brown et al., 2007). The percent adherent cells were calculated as [(absorbance of sample – background)/(absorbance of total cell lysates – background)] x 100%.

IgE Sensitization of Cells for Degranulation, Cytokine Release, Signaling Studies, and Calcium. HuMCs were sensitized overnight in regular culture medium or SCF- and IL-6-depleted medium (for analysis of the SCF effect) with 100 ng/ml biotinylated human myeloma IgE (Furumoto et al., 2006) (IgE from Calbiochem, biotinylation performed by NIAID Custom Antibody Facility, National Institutes of Health, Bethesda, MD). BMMCs were sensitized overnight in IL-3-depleted culture medium with 100 ng/ml anti-mouse monoclonal dinitrophenyl (DNP)-IgE (Sigma-Aldrich). After sensitization, both types of cells were washed three times with HEPES buffer + 0.04% BSA, before conducting the experiments.

Degranulation. Degranulation was monitored by β-hexosaminidase release (Brown et al., 2007) performed in a U-bottom 96-well plate (100-µl final volume). IgE-sensitized HuMCs (7 x 10³ cells per well) or BMMCs (2.5 x 10⁵ cells per well) were preincubated with inhibitors (0.001–10 µM) for 15 min before the addition of the stimuli. HuMCs were stimulated for 30 min with streptavidin (0–100 ng/ml) for cross-linking FcRI and/or human SCF (0–100 ng/ml) for triggering Kit. BMMCs were similarly triggered by the addition of DNP-human serum albumin (HSA) (Sigma-Aldrich) (0–100 ng/ml) and/or murine SCF (0–100 ng/ml). The reactions were terminated by centrifugation (1000 rpm) at 4°C, and aliquots of the supernatants were then analyzed for β-hexosaminidase content (Brown et al., 2007). The cell pellets were lysed by adding 0.1% Triton X-100 and then also analyzed for β-hexosaminidase content. Degranulation was calculated as the percentage of total β-hexosaminidase content found in the supernatants following challenge.

Cytokine Release and Measurement. HuMCs or BMMCs (2 x 10⁵ cells per sample) were sensitized as before, preincubated with inhibitors for 15 min, and antigen (10 ng/ml) and/or SCF (30 ng/ml) were added. The cells were then incubated for 15 (HuMCs) or 9 (BMMCs) h. The supernatants were harvested, and the cytokine content was measured by using the DuoSet enzyme-linked immunosorbent assay system (R&D Systems, Minneapolis, MN).

Cell Lysates and Western Blot. BMMCs (IgE-sensitized) were incubated at 1 x 10⁶ cells per sample in HEPES buffer + 0.04% BSA at 37°C. Cells were preincubated with inhibitors (0.001–10 µM) for 15 min, then stimulated with DNP-HSA (10 ng/ml) and/or SCF (30 ng/ml) for 2 or 30 min. The reaction was terminated by cell lysis as described (Tkaczyk et al., 2002). Aliquots of the lysates were loaded onto 4 to 12% NuPage BisTris gels (Invitrogen), and the proteins were separated by electrophoresis in MES buffer according to the manufacturer's protocol. After transfer onto nitrocellulose membranes, the proteins were probed with the following phospho (p)-antibodies: p-Kit 568/570, p-Kit730, p-Kit 823, and p-PLC1 [Tyr(P)-783] from Biosource (Invitrogen); p-Kit 703, p-Kit 721, and p-Kit 936 from Zymed (Invitrogen); p-Kit719, p-Src [Tyr(P)-416], p-AKT [Ser(P)-473], p-ERK [Thr(P)-202 and Tyr(P)-204], p-p38 [Thr(P)-180], p-JNK [Thr(P)-183 and Tyr(P)-185], p-NFB [Ser(P)-536], and p-c-Jun [Ser(P)-73] from Cell Signaling (Beverly, MA); p-LAT [Tyr(P)-191] from Upstate (Waltham, MA); p-Btk [Tyr(P)-551] from BD Pharmingen (BD Bioscience); and c-Jun from Santa Cruz (Santa Cruz, CA).

The immunoreactive proteins were visualized by probing with horseradish peroxidase-conjugated anti-mouse (Sigma-Aldrich) or anti-rabbit IgG (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), then visualized by enhanced chemiluminescence (PerkinElmer Life Sciences, Shelton, CT). Protein loading of the samples was normalized by probing for β-actin (Sigma-Aldrich). To quantitate changes in protein phosphorylation, membranes were scanned using ChemiDoc XRS and Quantity One software (Bio-Rad Laboratories, Hercules, CA).

Measurement of Intracellular Calcium. BMMCs (IgE sensitized, 1 x 10⁶ cells/ml) in culture medium were loaded with Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at 37°C, rinsed, then resuspended in HEPES buffer containing 0.04% BSA and sulfinpyrazone (0.3 mM) (Sigma-Aldrich). The BMMCs were then placed in a 96-well black culture plate (10,000 cells/well) (CulturPlat-96 F; PerkinElmer Life Sciences), preincubated with the inhibitors for 15 min, then stimulated with antigen (10 ng/ml) and/or SCF (30 ng/ml). The fluorescence was determined in a Wallac Victor² 1420 Multilabel Counter (PerkinElmer Life Sciences) with excitation wavelengths (340 and 380 nm) and an emission wavelength of 510 nm. After subtraction of background fluorescence of non-Fura-2-loaded cells, data were calculated as the ratio of fluorescence at 340- and 380-nm excitation wavelengths.

Passive Cutaneous Anaphylaxis. BALB/c mice (6–8 weeks old, 30 g; Jackson Laboratories) received intradermal injections of 1 µg of mouse monoclonal anti-DNP-IgE in 25 µl of PBS in the left ear and 25 µl of PBS in the right ear as a control. After 14 to 16 h, the mice were injected i.p. with 500 µg of hypothemycin in 200 µlofthe carrier hydroxylpropyl-β-cyclodextran (CTD Inc., High Springs, FL) or with 200 µl of hydroxylpropyl-β-cyclodextran + DMSO (control for hypothemycin solvent). This concentration and dosage regimen was selected based on personal communication with Dr. Sumati Murli (Kosan Biosciences Incorporated, Hayward, CA). After 8 h, the mice were challenged with antigen by i.v. injection of 500 µg/ml DNP-HSA and 0.5% Evans blue in 100 µl of saline in the tail vein. The mice were euthanized 30 min after the i.v. injection, the ears were removed, and the Evans blue was extracted in 200 µl of formamide at 55°C for 24 h. Extravasation of Evans blue dye was quantitated by spectrophotometric analysis at 620 nm.

Other Materials. Hypothemycin (KOS-1949) was kindly provided by Kosan Biosciences Incorporated (Schirmer et al., 2006). Imatinib was purified by Dr. Elizabeth Greiner (Laboratory of Medical Chemistry, NIDDK, NIH) (Seggewiss et al., 2005).

Statistics. Data were analyzed by a two-tailed Student's t test. The levels of significance were as follows: *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; and ***, p < 0.001.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Hypothemycin and Kit Phosphorylation. Hypothemycin (Fig. 1A) has been reported to inhibit Kit activity in a cell-free assay (Schirmer et al., 2006). Therefore, we initially confirmed that hypothemycin could block Kit activation and function in mast cells. Kit activation, following ligation by SCF, results in the phosphorylation of several tyrosine residues in the cytosolic tail of Kit, which in human mast cells (or mouse) includes Tyr568 (567) and Tyr570 (569) in the juxtamembrane region; 703 (701), 721 (719), and 730 (728) in the kinase insert domain; 823 (821), 900 (898), and 936 (934) in the distal kinase domain (Linnekin, 1999; Roskoski, 2005a, b; Reber et al., 2006). Thus, we compared the effects of hypothemycin with those of imatinib in its ability to inhibit Kit phosphorylation following SCF challenge in HuMCs and BM-MCs. As seen in Fig. 1, B and C, hypothemycin inhibited the phosphorylation of Kit (tyrosine 823; 821 in mouse) when stimulated with 30 ng/ml SCF in a concentration-dependent manner, with complete inhibition observed at 10 µM. These responses were comparable with those obtained with imatinib as shown in Fig. 1D, where 10 µM also completely blocked the phosphorylation of Kit. The optimal concentration of hypothemycin (10 µM) also blocked the phosphorylation of the other known tyrosine phosphorylation sites on human (or mouse) Kit: residues 568/570 (567/569), 703 (701), 721 (719), 730 (728), and 936 (934) (Fig. 1E).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Hypothemycin and its effect on Kit phosphorylation. A, molecular structure of hypothemycin (KOS-1949). Inhibition of the SCF-induced phosphorylation of Kit in HuMCs and BMMCs by hypothemycin (B, C, and E) or imatinib (D). Cells were pretreated with inhibitors 15 min before SCF (30 ng/ml) challenge for 2 min. The blots are representative of n = 3. DMSO in a concentration equal to 10 µM hypothemycin is used as solvent control. β-Actin was used as loading control. The graphs, which represent the means ± S.E.M. of n = 3 experiments, were generated by scanning of blots and then normalized to the SCF response in the absence of inhibitors.

View larger version (22K): [in this window] [in a new window]   Fig. 2. The comparative effects of hypothemycin and imatinib on mast cell adhesion. HuMCs (A and C) and BMMCs (B and D) were preincubated with hypothemycin (10 µM) (A and B) or imatinib (10 µM) (C and D) 15 min before challenging the cells with SCF (10 ng/ml). The adhesion assays were then conducted as described under Materials and Methods. The data are means ± S.E.M. of n = 4. The effect of hypothemycin or imatinib at 10 µM reached statistical significance (*, 0.01 < p < 0.05). The effects of hypothemycin were observed over a similar concentration range (data not shown) as that required to inhibit Kit phosphorylation (see Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 3. The comparative effects of hypothemycin and imatinib on mast cell degranulation. The inhibition by hypothemycin (A and B) and imatinib (C and D) of the synergistic enhancement of the antigen response by SCF in HuMCs (A and C) and BMMCs (B and D). The DMSO concentrations used to solubilize hypothemycin had no effect on degranulation (data not shown). Sensitized cells were pretreated with inhibitors for 15 min, and then HuMCs and BMMCs were challenged with streptavidin or DNP-HSA (1 ng/ml each) [antigen (Ag)], respectively, in the absence or presence of SCF for 30 min. The data are means ± S.E.M. of n = 3. , no Ag and SCF; , Ag + no SCF; , Ag + 1 ng/ml SCF; , Ag + 10 ng/ml SCF; , Ag + 100 ng/ml SCF. Where indicated, the values obtained in the presence of hypothemycin were statistically different from those in the absence of hypothemycin (*, 0.01 < p < 0.05; ***, p < 0.001).

The above effects of hypothemycin were determined not to be due to cytotoxicity because up to 9 h of incubation with hypothemycin did not affect cell viability as assessed by trypan blue exclusion. Furthermore, the effects were also not due to the DMSO carrier because DMSO at the concentrations used to deliver hypothemycin was without effect. Finally, these effects were also not due to alterations in the expression of FcRI or Kit as determined by fluorescence-activated cell sorting (above data not shown).

The Effect of Hypothemycin on Mast Cell Cytokine Release. To explore the effect of hypothemycin on cytokine production, we examined the production of IL-8 and GM-CSF in HuMCs and IL-6 and TNF- in BMMCs based on previous cytokine expression profiles (Okayama et al., 2001; Hundley et al., 2004). As reported in these studies, FcRI aggregation in the absence of SCF minimally induces cytokine production in both HuMCs (Fig. 4, A–D) and BMMCs (Fig. 4, E–H). However, in the presence of SCF, there is a marked potentiation of the release of IL-8 (Fig. 4, A and C) and GM-CSF (Fig. 4, B and D) in the HuMCs and IL-6 (Fig. 4, E and G) and TNF- (Fig. 4, F and H) in the BMMCs. The release of these cytokines when stimulated with antigen plus SCF was blocked by 10 µM hypothemycin, as shown in Fig. 4, A, B, E, and F. Again, the DMSO carrier had minimal affect on these responses. Hypothemycin also appeared to reduce the amount of cytokines produced in response to antigen, but due to the small magnitude of release, the differences did not reach statistical significance. Imatinib did not appear to affect antigen-induced cytokine release (Fig. 4, C, D, G, and H). Thus, the above data demonstrate that, unlike imatinib, hypothemycin not only effectively blocks the Kit-mediated component but also the FcRI-mediated component of the synergistically enhanced mast cell cytokine production in response to SCF and antigen.

View larger version (19K): [in this window] [in a new window]   Fig. 4. The comparative effects of hypothemycin and imatinib on FcRI- and/or Kit-induced cytokine release in mast cells. HuMCs were pretreated with hypothemycin (Hypo; 10 µM) (A and B) or imatinib (10 µM) (C and D) for 15 min and then stimulated with streptavidin (SA) (100 ng/ml) and/or SCF (30 ng/ml) for 15 h. The supernatants were measured for IL-8 (A and C) and GM-CSF (B and D). BMMCs were pretreated with Hypo (10 µM) (E and F) or imatinib (10 µM) (G and H) for 15 min and then stimulated with DNP-HSA (DNP; 10 ng/ml) and/or SCF (30 ng/ml) for 9 h. The supernatants were measured for IL-6 (E and G) and TNF- (F and H). Each cytokine value is the means ± S.E.M. of n = 3 experiments. Where indicated, the values obtained in the presence of hypothemycin were statistically different from those in the absence of hypothemycin (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001). DMSO had a minimal effect on human mast cell IL-8 release and the BMMC IL-6 release.

Hypothemycin and Its Intracellular Targets. Because hypothemycin clearly inhibits the Kit response by acting on Kit kinase but inhibits the FcRI response by an unknown mechanism, we investigated the target for hypothemycin in the FcRI signaling pathway. To facilitate these studies, we examined these responses in BMMCs in view of the identical effects of hypothemycin in human and mouse mast cells. Where examined, imatinib inhibited all responses induced by SCF but failed to influence responses mediated by antigen (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5. The effect of hypothemycin (Hypo) on early and intermediate FcRI- and/or Kit-mediated signaling events in BMMCs. Sensitized BMMCs were pretreated with 10 µM Hypo or buffer for 15 min and then stimulated for 2 min with Ag (10 ng/ml DNP-HSA) or SCF (30 ng/ml). The extracted proteins were probed for phosphoproteins: A, p-Src and p-LAT; and B, p-AKT, p-Btk, and p-PLC. DMSO in a concentration equal to 10 µM hypothemycin did not alter the phosphorylation. β-Actin was used as loading control. The histograms, which represent the means ± S.E.M. of n = 3 experiments, were generated by scanning of blots and then normalized to the antigen response. The dashed line represents the constitutive phosphorylation in the cells. Where indicated, the values obtained in the presence of hypothemycin were statistically different from those in the absence of hypothemycin (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001). BMMCs were analyzed for calcium mobilization (C). Sensitized cells were loaded with Fura-2 acetoxymethyl ester, preincubated with 10 µM hypothemycin for 15 min, and then stimulated with DNP-HSA (10 ng/ml) and/or SCF (30 ng/ml). The result is a representative of three experiments. , DNP-HSA; , SCF; , DNP-HSA + 10 µM hypothemycin; , SCF + 10 µM hypothemycin.

Intermediate Signaling Events. We next examined downstream events, namely PI3K activation, as monitored by the surrogate marker; AKT phosphorylation (Tkaczyk et al., 2003); Btk activation, as monitored by its phosphorylation (Iwaki et al., 2005); and PLC₁ activation, as monitored by its phosphorylation and calcium mobilization (Tkaczyk et al., 2003) (Fig. 5, B and C). We have previously demonstrated that the latter two responses may play a role in the integration of the signals produced by FcRI and Kit for the synergistic enhancement of mast cell degranulation (Hundley et al., 2004; Iwaki et al., 2005). As previously demonstrated (Hundley et al., 2004; Tkaczyk et al., 2004; Iwaki et al., 2005), both antigen and SCF induced the phosphorylation of AKT, Btk, and PLC₁. As with the upstream events, hypothemycin failed to inhibit the phosphorylation of AKT in response to antigen (Fig. 5B). However, consistent with the notion that hypothemycin inhibits Kit directly and FcRI-mediated signaling downstream of PI3K, SCF-induced AKT phosphorylation was substantially reduced. Thus, these data suggested that PI3K was not the target for hypothemycin in antigen-stimulated cells. In contrast to these results, however, Btk phosphorylation induced by either antigen or SCF was markedly inhibited. Furthermore, PLC₁ phosphorylation in response to antigen was also substantially reduced under these conditions (Fig. 5B). Thus, hypothemycin appears to be mediating its effects at the level of Btk phosphorylation/activation with consequential downstream inhibition of PLC₁.

To further confirm this conclusion, we examined the ability of hypothemycin to block intracellular calcium mobilization in response to antigen or SCF (Fig. 5C). As reported (Hundley et al., 2004), both antigen and SCF induced an increase in calcium mobilization, with the response to SCF being markedly lower and slower than that observed by antigen. Degranulation and critical signaling events leading to this response occur within 5 min of challenge. When examined over this time frame, antigen- and SCF-induced calcium mobilization was substantially inhibited by an optimal concentration of hypothemycin (10 µM) (Fig. 5C). In total, the data suggest that hypothemycin blocks degranulation by its ability to inhibit at the level of Btk and, hence, PLC₁ activation and calcium mobilization.

MAP Kinases and Transcription Factors. Cytokine production downstream of Btk appears to occur via activation of the MAP kinase cascades (Gilfillan and Tkaczyk, 2006). Thus, we next examined whether or not hypothemycin inhibits antigen- and SCF-mediated cytokine production through an inhibition of these events and, as a consequence, the expression/phosphorylation of downstream transcription factors that are known to promote cytokine production in mast cells (Hundley et al., 2004; Qiao et al., 2006). As seen in Fig. 6A, the increase in the phosphorylation of the MAP kinases ERK1/2, JNK, and p38 induced by antigen or ERK1/2 and JNK, enhanced by SCF, were virtually abrogated by an optimal concentration of hypothemycin (10 µM). Likewise, the induction of the AP1 transcription complex component c-Jun and its phosphorylation were also markedly inhibited by hypothemycin (Fig. 6B). Furthermore, the antigen-induced phosphorylation of NFB was also inhibited by hypothemycin (Fig. 6B). Thus, these data support the conclusion that hypothemycin inhibits cytokine production in response to antigen and SCF as a consequence of the inhibition of the activation of MAP kinases and downstream transcription factors.

View larger version (17K): [in this window] [in a new window]   Fig. 6. The effect of hypothemycin (Hypo) on FcRI- and or Kit-mediated MAP kinase activation and phosphorylation of transcription factors in BMMCs. Sensitized BMMCs were pretreated with 10 µM Hypo or buffer for 15 min (106 cells per sample) and then stimulated for 2 (MAP kinases) or 30 (transcription factors) min with Ag (10 ng/ml DNP-HSA) or SCF (30 ng/ml). The extracted proteins were probed for phosphoproteins: A, p-ERK, p-p38, and p-JNK; and B, c-Jun, p-c-Jun, and p-NFB. DMSO in a concentration equal to 10 µM hypothemycin did not alter the phosphorylation. β-Actin was used as loading control. The histograms, which represent the means ± S.E.M. of n = 3 experiments, were generated by scanning of blots and then normalized to the antigen response. The dashed line represents the constitutive phosphorylation in the cells. Where indicated, the values obtained in the presence of hypothemycin were statistically different from those in the absence of hypothemycin (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001).

View larger version (11K): [in this window] [in a new window]   Fig. 7. The effect of hypothemycin (Hypo) on in vivo passive cutaneous anaphylaxis. BALB/c mice (30 g) sensitized with IgE in the left ear and PBS in the right ear were treated for 8 h with 500 µg of Hypo or its solvent, β-cyclodextran. Antigen challenge was done by tail vein injections with 500 µg/ml DNP-HSA in 0.5% Evans blue solution. Thirty minutes after injection, the mice were sacrificed, the ears were removed, and Evans blue dye was extracted and quantitated by absorption. The figure shows the means ± S.E.M. of n = 3 experiments (each containing three control mice and three hypothemycin-treated mice). Where indicated, the values obtained in the presence of hypothemycin were statistically different from those in the absence of hypothemycin (*, 0.01 < p < 0.05).

	Discussion

Top Abstract Materials and Methods Results Discussion References

The ability of hypothemycin to inhibit Kit kinase activation was confirmed by the inhibition of the SCF-induced autophosphorylation of critical tyrosine residues within the cytosolic domain of Kit (Fig. 1). Because these serve as docking sites for SH2 domain-containing signaling molecules that mediate the downstream signaling of Kit, SCF-mediated signaling events including Btk phosphorylation, AKT phosphorylation, and PLC₁ phosphorylation leading to calcium mobilization were also inhibited (Fig. 5). The inhibition of these signaling events account for the ability of hypothemycin to effectively inhibit SCF-mediated mast cell adhesion (Fig. 2), chemotaxis (data not shown), and, as with imatinib, the ability of SCF to potentiate antigen-mediated degranulation (Fig. 3) and cytokine production (Fig. 4). Unlike imatinib, however, hypothemycin also blocked the ability of antigen to induce both degranulation and cytokine production, thus demonstrating the ability of hypothemycin to inhibit the FcRI- and Kit-mediated signaling pathway. Because hypothemycin was relatively ineffective at inhibiting Src kinases and Syk when screened against a panel of kinases (Schirmer et al., 2006), these data and our observations that hypothemycin does not block antigen-mediated phosphorylation of Src kinases and LAT (Fig. 5) indicated that the target of hypothemycin in the FcRI-dependent signaling cascade was downstream from these initial signaling events and, by inference, FcRI aggregation and phosphorylation. Moreover, because hypothemycin failed to inhibit AKT phosphorylation (Fig. 5), it appears that the target is also downstream of PI3K.

Because antigen-mediated Btk phosphorylation, as well as downstream signaling events, were markedly suppressed (Fig. 5), hypothemycin most probably inhibits FcRI-mediated degranulation at the level of Btk activation. However, resting Btk kinase activity was not blocked in an in vitro kinase assay (Schirmer et al., 2006), indicating that Btk is not directly inhibited by hypothemycin. Instead, hypothemycin may inhibit a subsidiary cryptic event associated with the phosphorylation and activation of Btk downstream of the activation of Lyn, Syk, and PI3K. Regardless, the data clearly indicate that Btk phosphorylation is the first recognizable step in the FcRI-mediated signaling cascade inhibited by hypothemycin (Fig. 8).

View larger version (40K): [in this window] [in a new window]   Fig. 8. Proposed sites of action of hypothemycin on SCF- and antigen-mediated mast cell activation. For clarity, many of the intermediate signaling events have been omitted. The readers are referred to Gilfillan and Tkaczyk (2006) for further details. Hypo inhibits Kit-mediated signaling and the potentiation of antigen-mediated degranulation and cytokine production by directly inhibiting Kit kinase activity. In contrast, hypothemycin does not inhibit early FcRI-mediated responses including activation of Src kinases, phosphorylation of the transmembrane adaptor molecules LAT and NTAL (non-T cell activation linker), and the activation of PI3K. The ability of hypothemycin to inhibit FcRI-mediated mast cell activation appears to be due to the inhibition of the phosphorylation and thus activation of Btk. This, in turn, results in the consequential inhibition of PLC1-dependent calcium mobilization, a critical signal for both degranulation and activation of specific transcription factors. It may also be possible, however, that inhibition of the MAP kinase cascade may also contribute to the down-regulation of cytokine production. Green lines, positive pathways; red lines, negative pathways.

The ability of hypothemycin to inhibit the synergistic release of mast cell activation in culture translated into a significant inhibition of the antigen-induced passive cutaneous anaphylaxis response in mice (Fig. 7), demonstrating its efficacy against a mast cell-driven allergic reaction in vivo. The residual response observed in these studies may, however, reflect the need to optimize the bioavailability and dosage regimen of potential inhibitory molecules such as hypothemycin. Future studies to expand the therapeutic repertoire for a coordinated approach targeting Kit- and FcRI-mediated signaling would require testing of hypothemycin in a more complex model, for example, in a mouse asthma mouse of allergic airway inflammation. This would provide information on how hypothemycin would affect a more complex allergic reaction involving multiple cell types. However, the further properties of hypothemycin to inhibit SCF-dependent mast cell adhesion and chemotaxis would suggest that, in addition to inhibiting mast cell degranulation, hypothemycin may also prevent mast cell infiltration into sites of inflammation.

In summary, the results presented in this study provide proof of principle for the concept of concurrent inhibition of Kit- and FcRI-mediated signaling as an approach for inhibition of mast cell-driven allergic reactions. To test this concept, we have utilized a molecule, hypothemycin, which inhibits both responses, with the aim of providing a basis for further investigation of this concept and potentially the development and optimization of novel compounds that may have a similar mode of action. Although hypothemycin irreversibly inhibits a specific subset of protein kinases, these properties may have inherent therapeutic advantages, especially when used topically in disorders such as atopic asthma and dermatitis. As noted here, the ability to suppress both Kit- and FcRI-mediated signals at multiple levels would allow maximum suppression of antigen-induced release of inflammatory mediators and possibly other physiological functions such as migration and maturation of mast cells. An alternative approach, however, could be the concurrent administration of separate compounds targeting Kit- and FcRI-specific signaling cascades. This latter approach may help to minimize potential issues with specificity of targeting.

	Acknowledgements

 
We thank Sumati Murli and others at Kosan Biosciences Incorporated for the supply of hypothemycin and advice regarding the study and Elizabeth Greiner (NIH) for supplying purified imatinib mesylate.

	Footnotes

 
This work was supported by the NIAID and National Heart, Lung, and Blood Institute Intramural Programs of the National Institutes of Health.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125237.

ABBREVIATIONS: IgE, immunoglobulin E; FcRI, high-affinity receptor for IgE; SCF, stem cell factor; LAT, linker for activation of T cells; PL, phospholipase; PI3K, phosphoinositide 3-kinase; HuMC, human mast cell; NIAID, National Institute of Allergy and Infectious Diseases; IL, interleukin; BMMC, mouse bone marrow-derived mast cell; NIH, National Institutes of Health; BSA, bovine serum albumin; DNP, dinitrophenyl; HSA, human serum albumin; p, phospho; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; NF, nuclear factor; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; GM-CSF, granulocyte macrophage colony-stimulating factor; TNF, tumor necrosis factor; MAP, mitogen-activated protein; Ag, antigen; Hypo, hypothemycin.

Address correspondence to: Dr. Alasdair M. Gilfillan, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11C206, 10 Center Drive, MSC 1881, Bethesda, MD 20892-1881. E-mail: agilfillan{at}niaid.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 

Ali K, Bilancio A, Thomas M, Pearce W, Gilfillan AM, Tkaczyk C, Kuehn N, Gray A, Giddings J, Peskett E, et al. (2004) Essential role for the p110 phosphoinositide 3-kinase in the allergic response. Nature 431: 1007–1011.[CrossRef][Medline]

Beck LA, Marcotte GV, MacGlashan D, Togias A, and Saini S (2004) Omalizumab-induced reductions in mast cell FcRI expression and function. J Allergy Clin Immunol 114: 527–530.[CrossRef][Medline]

Berlin AA, Hogaboam CM, and Lukacs NW (2006) Inhibition of SCF attenuates peribronchial remodeling in chronic cockroach allergen-induced asthma. Lab Invest 86: 557–565.[Medline]

Berlin AA and Lukacs NW (2005) Treatment of cockroach allergen asthma model with imatinib attenuates airway responses. Am J Respir Crit Care Med 171: 35–39.[Abstract/Free Full Text]

Bischoff SC and Dahinden CA (1992) c-kit ligand: a unique potentiator of mediator release by human lung mast cells. J Exp Med 175: 237–244.[Abstract/Free Full Text]

Blank U and Rivera J (2004) The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol 25: 266–273.[CrossRef][Medline]

Brown JM, Swindle EJ, Kushnir-Sukhov NM, Holian A, and Metcalfe DD (2007) Silica-directed mast cell activation is enhanced by scavenger receptors. Am J Respir Cell Mol Biol 36: 43–52.[Abstract/Free Full Text]

Brown SG (2006) Anaphylaxis: clinical concepts and research priorities. Emerg Med Australas 18: 155–169.[CrossRef][Medline]

Furumoto Y, Brooks S, Olivera A, Takagi Y, Miyagishi M, Taira K, Casellas R, Beaven MA, Gilfillan AM, and Rivera J (2006) Cutting edge: lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival. J Immunol 176: 5167–5171.[Abstract/Free Full Text]

Galli SJ, Nakae S, and Tsai M (2005) Mast cells in the development of adaptive immune responses. Nat Immunol 6: 135–142.[CrossRef][Medline]

Gilfillan AM and Tkaczyk C (2006) Integrated signalling pathways for mast-cell activation. Nat Rev Immunol 6: 218–230.[CrossRef][Medline]

Gleixner KV, Mayerhofer M, Aichberger KJ, Derdak S, Sonneck K, Bohm A, Gruze A, Samorapoompichit P, Manley PW, Fabbro D, et al. (2006) PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated variant of KIT: comparison with AMN107, imatinib, and cladribine (2CdA) and evaluation of cooperative drug effects. Blood 107: 752–759.[Abstract/Free Full Text]

Growney JD, Clark JJ, Adelsperger J, Stone R, Fabbro D, Griffin JD, and Gilliland DG (2005) Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase inhibitor PKC412. Blood 106: 721–724.[Abstract/Free Full Text]

Gu H, Saito K, Klaman LD, Shen J, Fleming T, Wang Y, Pratt JC, Lin G, Lim B, Kinet JP, et al. (2001) Essential role for Gab2 in the allergic response. Nature 412: 186–190.[CrossRef][Medline]

Hata D, Kawakami Y, Inagaki N, Lantz CS, Kitamura T, Khan WN, Maeda-Yamamoto M, Miura T, Han W, Hartman SE, et al. (1998) Involvement of Bruton's tyrosine kinase in FcRI-dependent mast cell degranulation and cytokine production. J Exp Med 187: 1235–1247.[Abstract/Free Full Text]

Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, and Zigler AJ (2000) Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 96: 925–932.[Abstract/Free Full Text]

Hundley TR, Gilfillan AM, Tkaczyk C, Andrade MV, Metcalfe DD, and Beaven MA (2004) Kit and FcRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood 104: 2410–2417.[Abstract/Free Full Text]

Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.

Iwaki S, Tkaczyk C, Satterthwaite AB, Halcomb K, Beaven MA, Metcalfe DD, and Gilfillan AM (2005) Btk plays a crucial role in the amplification of FcRI-mediated mast cell activation by Kit. J Biol Chem 280: 40261–40270.[Abstract/Free Full Text]

Jensen BM, Metcalfe DD, and Gilfillan AM (2007) Targeting Kit activation: A potential therapeutic approach in the treatment of allergic inflammation. Inflamm Allergy Drug Targets 6: 57–62.[Medline]

Kirshenbaum AS, Goff JP, Semere T, Foster B, Scott LM, and Metcalfe DD (1999) Demonstration that human mast cells arise from a progenitor cell population that is CD34⁺, c-kit⁺, and expresses aminopeptidase N (CD13). Blood 94: 2333–2342.[Abstract/Free Full Text]

Linnekin D (1999) Early signaling pathways activated by c-Kit in hematopoietic cells. Int J Biochem Cell Biol 31: 1053–1074.[CrossRef][Medline]

MacGlashan DW Jr, Bochner BS, Adelman DC, Jardieu PM, Togias A, McKenzie-White J, Sterbinsky SA, Hamilton RG, and Lichtenstein LM (1997) Down-regulation of FcRI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody. J Immunol 158: 1438–1445.[Abstract]

Mekori YA (2004) The mastocyte: the "other" inflammatory cell in immunopathogenesis. J Allergy Clin Immunol 114: 52–57.[CrossRef][Medline]

Metcalfe DD, Baram D, and Mekori YA (1997) Mast cells. Physiol Rev 77: 1033–1079.[Abstract/Free Full Text]

Okayama Y, Hagaman DD, and Metcalfe DD (2001) A comparison of mediators released or generated by IFN--treated human mast cells following aggregation of FcRI or FcRI. J Immunol 166: 4705–4712.[Abstract/Free Full Text]

Okayama Y and Kawakami T (2006) Development, migration, and survival of mast cells. Immunol Res 34: 97–115.[CrossRef][Medline]

Pan J, Quintas-Cardama A, Kantarjian HM, Akin C, Manshouri T, Lamb P, Cortes JE, Tefferi A, Giles FJ, and Verstovsek S (2007) EXEL-0862, a novel tyrosine kinase inhibitor, induces apoptosis in vitro and ex vivo in human mast cells expressing the KIT D816V mutation. Blood 109: 315–322.[Abstract/Free Full Text]

Petti F, Thelemann A, Kahler J, McCormack S, Castaldo L, Hunt T, Nuwaysir L, Zeiske L, Haack H, Sullivan L, et al. (2005) Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930. Mol Cancer Ther 4: 1186–1197.[Abstract/Free Full Text]

Potapova O, Laird AD, Nannini MA, Barone A, Li G, Moss KG, Cherrington JM, and Mendel DB (2006) Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248. Mol Cancer Ther 5: 1280–1289.[Abstract/Free Full Text]

Qiao H, Andrade MV, Lisboa FA, Morgan K, and Beaven MA (2006) FcR1 and Toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells. Blood 107: 610–618.[Abstract/Free Full Text]

Reber L, Da Silva CA, and Frossard N (2006) Stem cell factor and its receptor c-Kit as targets for inflammatory diseases. Eur J Pharmacol 533: 327–340.[CrossRef][Medline]

Rivera J and Gilfillan AM (2006) Molecular regulation of mast cell activation. J Allergy Clin Immunol 117: 1214–1225,[CrossRef][Medline]

quiz 1226. Roskoski R Jr (2005a) Signaling by Kit protein-tyrosine kinase: the stem cell factor receptor. Biochem Biophys Res Commun 337: 1–13.[CrossRef][Medline]

Roskoski R Jr (2005b) Structure and regulation of Kit protein-tyrosine kinase: the stem cell factor receptor. Biochem Biophys Res Commun 338: 1307–1315.[CrossRef][Medline]

Saini SS, MacGlashan DW Jr, Sterbinsky SA, Togias A, Adelman DC, Lichtenstein LM, and Bochner BS (1999) Down-regulation of human basophil IgE and FCRI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo. J Immunol 162: 5624–5630.[Abstract/Free Full Text]

Scheinfeld N (2006) A comprehensive review of imatinib mesylate (Gleevec) for dermatological diseases. J Drugs Dermatol 5: 117–122.[Medline]

Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, and Kuriyan J (2000) Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science 289: 1938–1942.[Abstract/Free Full Text]

Schirmer A, Kennedy J, Murli S, Reid R, and Santi DV (2006) Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides. Proc Natl Acad Sci U S A 103: 4234–4239.[Abstract/Free Full Text]

Schittenhelm MM, Shiraga S, Schroeder A, Corbin AS, Griffith D, Lee FY, Bokemeyer C, Deininger MW, Druker BJ, and Heinrich MC (2006) Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies. Cancer Res 66: 473–481.[Abstract/Free Full Text]

Seggewiss R, Lore K, Greiner E, Magnusson MK, Price DA, Douek DC, Dunbar CE, and Wiestner A (2005) Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner. Blood 105: 2473–2479.[Abstract/Free Full Text]

Shah NP, Lee FY, Luo R, Jiang Y, Donker M, and Akin C (2006) Dasatinib (BMS-354825) inhibits KIT^D816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis. Blood 108: 286–291.[Abstract/Free Full Text]

Strait RT, Morris SC, Yang M, Qu XW, and Finkelman FD (2002) Pathways of anaphylaxis in the mouse. J Allergy Clin Immunol 109: 658–668.[CrossRef][Medline]

Tkaczyk C, Beaven MA, Brachman SM, Metcalfe DD, and Gilfillan AM (2003) The phospholipase C₁-dependent pathway of FcRI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase. J Biol Chem 278: 48474–48484.[Abstract/Free Full Text]

Tkaczyk C, Horejsi V, Iwaki S, Draber P, Samelson LE, Satterthwaite AB, Nahm DH, Metcalfe DD, and Gilfillan AM (2004) NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and FcRI aggregation. Blood 104: 207–214.[Abstract/Free Full Text]

Tkaczyk C, Metcalfe DD, and Gilfillan AM (2002) Determination of protein phosphorylation in FcRI-activated human mast cells by immunoblot analysis requires protein extraction under denaturing conditions. J Immunol Methods 268: 239–243.[CrossRef][Medline]

Verstovsek S, Akin C, Manshouri T, Quintas-Cardama A, Huynh L, Manley P, Tefferi A, Cortes J, Giles FJ, and Kantarjian H (2006) Effects of AMN107, a novel aminopyrimidine tyrosine kinase inhibitor, on human mast cells bearing wild-type or mutated codon 816 c-kit. Leuk Res 30: 1365–1370.[CrossRef][Medline]

Winssinger N and Barluenga S (2007) Chemistry and biology of resorcylic acid lactones. Chem Commun (Camb) 22–36.

Zermati Y, De Sepulveda P, Feger F, Letard S, Kersual J, Casteran N, Gorochov G, Dy M, Ribadeau Dumas A, Dorgham K, et al. (2003) Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms. Oncogene 22: 660–664.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.125237v1 324/1/128    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jensen, B. M. Articles by Gilfillan, A. M. Search for Related Content PubMed PubMed Citation Articles by Jensen, B. M. Articles by Gilfillan, A. M.