Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells
2007; Elsevier BV; Volume: 35; Issue: 10 Linguagem: Inglês
10.1016/j.exphem.2007.06.005
ISSN1873-2399
AutoresKaroline V. Gleixner, Laura Rebuzzi, Matthias Mayerhofer, Alexander Gruze, Emir Hadzijusufovic, Karoline Sonneck, Anja Vales, Michael Kneidinger, Puchit Samorapoompichit, Tuddow Thaiwong, Winfried F. Pickl, Vilma Yuzbasiyan‐Gurkan, Christian Sillaber, Michael Willmann, Peter Valent,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoAggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by 3H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC50 values: imatinib: 269 ± 180 nM, midostaurin: 157 ± 35 nM, nilotinib: 55 ± 24 nM, dasatinib: 12 ± 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC50 values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors. Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by 3H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC50 values: imatinib: 269 ± 180 nM, midostaurin: 157 ± 35 nM, nilotinib: 55 ± 24 nM, dasatinib: 12 ± 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC50 values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors. Mast cell (MC) tumors are frequent neoplasms in dogs [1Macy D.W. Canine mast cell tumors.Vet Clin North Am Small Anim Pract. 1985; 15: 783-803PubMed Google Scholar, 2London C.A. Seguin B. Mast cell tumors in the dog.Vet Clin North Am Small Anim Pract. 2003; 33: 473-489Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 3Misdorp W. Mast cells and canine mast cell tumours. A review.Vet Q. 2004; 26: 156-169Crossref PubMed Scopus (70) Google Scholar]. Similar to human mastocytosis [4Lennert K. Parwaresch M.R. Mast cells and mast cell neoplasia: a review.Histopathology. 1979; 3: 349-365Crossref PubMed Scopus (227) Google Scholar, 5Metcalfe D.D. Classification and diagnosis of mastocytosis: current status.J Invest Dermatol. 1991; 96: 2S-4SAbstract Full Text PDF Google Scholar, 6Valent P. Biology, classification and treatment of human mastocytosis.Wien Klin Wochenschr. 1996; 108: 385-397PubMed Google Scholar, 7Valent P. Akin C. 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Canine mast cell tumors.Vet Clin North Am Small Anim Pract. 1985; 15: 783-803PubMed Google Scholar, 2London C.A. Seguin B. Mast cell tumors in the dog.Vet Clin North Am Small Anim Pract. 2003; 33: 473-489Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 3Misdorp W. Mast cells and canine mast cell tumours. A review.Vet Q. 2004; 26: 156-169Crossref PubMed Scopus (70) Google Scholar]. Therefore, current attempts focus on identification of new drug targets in neoplastic MC, and the development of respective targeted drugs [9Valent P. Ghannadan M. Akin C. et al.On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts.Eur J Clin Invest. 2004; 34: 41-52Crossref PubMed Google Scholar]. A well-established survival factor and target of neoplastic MC is the protein-product of the KIT proto-oncogene [7Valent P. Akin C. Sperr W.R. et al.Diagnosis and treatment of systemic mastocytosis: state of the art.Br J Haematol. 2003; 122: 695-717Crossref PubMed Scopus (180) Google Scholar, 8Akin C. Metcalfe D.D. Systemic mastocytosis.Annu Rev Med. 2004; 55: 419-432Crossref PubMed Scopus (194) Google Scholar, 9Valent P. Ghannadan M. Akin C. et al.On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts.Eur J Clin Invest. 2004; 34: 41-52Crossref PubMed Google Scholar]. The encoded protein, KIT, is the tyrosine kinase (TK) receptor for MC growth factor/stem cell factor (SCF). Mice lacking a functional KIT receptor (W/Wv) are virtually MC-deficient [10Kitamura Y. Go S. Hatanaka S. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.Blood. 1978; 52: 447-452Crossref PubMed Google Scholar]. A number of previous and more recent data have shown that neoplastic human MC frequently display KIT mutations [11Furitsu T. Tsujimura T. Tono T. et al.Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of the c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (730) Google Scholar, 12Nagata H. Worobec A.S. Oh C.K. et al.Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci U S A. 1995; 92: 10560-10564Crossref PubMed Scopus (785) Google Scholar, 13Longley B.J. Tyrrell L. Lu S.Z. et al.Somatic c-kit activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.Nat Genet. 1996; 12: 312-314Crossref PubMed Scopus (551) Google Scholar, 14Longley B.J. Metcalfe D.D. Tharp M. et al.Activating and dominant inactivating c-kit catalytic domain mutations in distinct forms of human mastocytosis.Proc Natl Acad Sci U S A. 1999; 96: 1609-1614Crossref PubMed Scopus (489) Google Scholar, 15Fritsche-Polanz R. Jordan J.H. Feix A. et al.Mutation analysis of C-KIT in patients with myelodysplastic syndromes without mastocytosis and cases of systemic mastocytosis.Br J Haematol. 2001; 113: 357-364Crossref PubMed Scopus (131) Google Scholar, 16Feger F. Ribadeau Dumas A. Leriche L. Valent P. Arock M. Kit and c-kit mutations in mastocytosis: a short overview with special reference to novel molecular and diagnostic concepts.Int Arch Allergy Immunol. 2002; 127: 110-114Crossref PubMed Scopus (122) Google Scholar]. Such mutations are often associated with ligand-independent phosphorylation (activation) of the receptor and, thus, with autonomous growth of MC [11Furitsu T. Tsujimura T. Tono T. et al.Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of the c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (730) Google Scholar]. Mutations or duplications of KIT have recently also been described in canine mastocytomas [17London C.A. Galli S.J. Yuuki T. et al.Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit.Exp Hematol. 1999; 27: 689-697Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 18Jones C.L. Grahn R.A. Chien M.B. et al.Detection of c-kit mutations in canine mast cell tumors using fluorescent polyacrylamide gel electrophoresis.J Vet Diagn Invest. 2004; 16: 95-100Crossref PubMed Scopus (51) Google Scholar, 19Zemke D. Yamini B. Yuzbasiyan-Gurkan V. Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs.Vet Pathol. 2002; 39: 529-535Crossref PubMed Scopus (168) Google Scholar, 20Riva F. Brizzola S. Stefanello D. et al.A study of mutations in the c-kit gene of 32 dogs with mastocytoma.J Vet Diagn Invest. 2005; 17: 385-388Crossref PubMed Scopus (36) Google Scholar]. Based on these observations, KIT represents an attractive therapeutic target in MC neoplasms. During the past few years, a number of potent TK inhibitors have been developed, with a highlight being imatinib, a drug that effectively counteracts the TK activity of the leukemia-specific oncoprotein BCR/ABL [21Mauro M.J. Druker B.J. STI571: a gene product-targeted therapy for leukemia.Curr Oncol Rep. 2001; 3: 223-227Crossref PubMed Scopus (33) Google Scholar, 22Deininger M.W. Druker B.J. Specific targeted therapy of chronic myelogenous leukemia with imatinib.Pharmacol Rev. 2003; 55: 401-423Crossref PubMed Scopus (277) Google Scholar]. Imatinib also counteracts the TK activity of wild-type (WT) KIT as well as growth of human neoplastic MC exhibiting WT KIT or certain KIT mutants [23Pardanani A. Tefferi A. Imatinib targets other than bcr/abl and their clinical relevance in myeloid disorders.Blood. 2004; 104: 1931-1939Crossref PubMed Scopus (109) Google Scholar, 24Frost M.J. Ferrao P.T. Hughes T.P. Ashman L.K. Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant.Mol Cancer Ther. 2002; 1: 1115-1124PubMed Google Scholar, 25Akin C. Brockow K. D'Ambrosio C. et al.Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated forms of c-kit.Exp Hematol. 2003; 31: 686-692Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26Ma Y. Zeng S. Metcalfe D.D. et al.The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory type mutations.Blood. 2002; 99: 1741-1744Crossref PubMed Scopus (404) Google Scholar, 27Akin C. Fumo G. Yavuz A.S. Lipsky P.E. Neckers L. Metcalfe D.D. A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib.Blood. 2004; 103: 3222-3225Crossref PubMed Scopus (288) Google Scholar]. However, the most frequently occurring mutant, KIT-D816V, is resistant against imatinib [24Frost M.J. Ferrao P.T. Hughes T.P. Ashman L.K. Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant.Mol Cancer Ther. 2002; 1: 1115-1124PubMed Google Scholar, 25Akin C. Brockow K. D'Ambrosio C. et al.Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated forms of c-kit.Exp Hematol. 2003; 31: 686-692Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26Ma Y. Zeng S. Metcalfe D.D. et al.The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory type mutations.Blood. 2002; 99: 1741-1744Crossref PubMed Scopus (404) Google Scholar]. Therefore, current research focuses on novel TK inhibitors. In human neoplastic MC, initial promising results have been reported with midostaurin, nilotinib, and dasatinib [28Growney J.D. Clark J.J. Adelsperger J. et al.Activation mutations of human c-KIT resistant to imatinib are sensitive to the tyrosine kinase inhibitor PKC412.Blood. 2005; 106: 721-724Crossref PubMed Scopus (207) Google Scholar, 29Gotlib J. Berube C. Growney J.D. et al.Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation.Blood. 2005; 106: 2865-2870Crossref PubMed Scopus (213) Google Scholar, 30von Bubnoff N. Gorantla S.H. Kancha R.K. Lordick F. Peschel C. Duyster J. The systemic mastocytosis-specific activating cKit mutation D816V can be inhibited by the tyrosine kinase inhibitor AMN107.Leukemia. 2005; 19: 1670-1671Crossref PubMed Scopus (64) Google Scholar, 31Gleixner K.V. Mayerhofer M. Aichberger K.J. et al.The tyrosine kinase-targeting drug 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. 2006; 107: 752-759Crossref PubMed Scopus (215) Google Scholar, 32Shah N.P. Lee F.Y. Luo R. Jiang Y. Donker M. Akin C. Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in the majority of patients with systemic mastocytosis.Blood. 2006; 108: 286-291Crossref PubMed Scopus (233) Google Scholar, 33Schittenhelm M.M. Shiraga S. Schroeder A. et al.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. 2006; 66: 473-481Crossref PubMed Scopus (423) Google Scholar]. However, the effects of these drugs on growth of neoplastic MC in canines have not been examined yet. Imatinib (STI571), midostaurin (PKC412) [34Fabbro D. Ruetz S. Bodis S. et al.PKC412—a protein kinase inhibitor with a broad therapeutic potential.Anticancer Drug Des. 2000; 15: 17-28PubMed Google Scholar], and nilotinib (AMN107) [35Weisberg E. Manley P.W. Breitenstein W. et al.Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl.Cancer Cell. 2005; 7: 129-141Abstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar] were kindly provided by Dr. Elisabeth Buchdunger, Dr. Doriano Fabbro, and Dr. Paul W. Manley (Novartis Pharma AG, Basel, Switzerland). Dasatinib (BMS-354825) [36Shah N.P. Tran C. Lee F.Y. Chen P. Norris D. Sawyers C.L. Overriding imatinib resistance with a novel ABL kinase inhibitor.Science. 2004; 305: 399-401Crossref PubMed Scopus (1528) Google Scholar] was kindly provided by Dr. Francis Y. Lee (Bristol-Meyers-Squibbs, New Brunswick, NJ, USA). Stock solutions of TK inhibitors were prepared by dissolving in dimethyl sulfoxide (Merck, Darmstadt, Germany). Fetal calf serum (FCS) was purchased from PAA laboratories (Pasching, Austria), Iscove's modified Dulbecco's medium from Gibco Life Technologies (Gaithersburg, MD, USA), 3H-thymidine from Amersham (Buckinghamshire, UK), and propidium iodide from Sigma (St Louis, MO, USA). The canine mastocytoma cell line C2 [37DeVinney R. Gold W.M. Establishment of two dog mastocytoma cell lines in continuous culture.Am J Respir Cell Mol Biol. 1990; 3: 413-420Crossref PubMed Scopus (49) Google Scholar] was kindly provided by Dr. Warren Gold (Cardiovascular Research Institute, University of California, San Francisco, CA, USA). C2 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 5% FCS and antibiotics at 5% CO2 and 37°C. Cells were passaged every 3 to 5 days and rethawed from an original stock every 6 to 8 weeks. In three mastocytoma patients (Table 1) MC were isolated using collagenase following a published protocol [38Valent P. Ashman L.K. Hinterberger W. et al.Mast cell typing: demonstration of a distinct hematopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes.Blood. 1989; 73: 1778-1785Crossref PubMed Google Scholar]. In brief, tissue specimens were first cut into small pieces and washed thoroughly in Tyrode's buffer. Tissue was then incubated with collagenase type II (Worthington, Lakewood, NJ, USA) at 37°C for 180 minutes. Thereafter, isolated cells were recovered by filtration through Nytex cloth and placed in FCS-containing tubes. After washing, cells were examined for viability and percentage of MC (Wright-Giemsa stain). In control experiments, normal bone marrow cells (five dogs without hematopoietic neoplasm) and normal peripheral blood leukocytes (three dogs) were examined. Mononuclear cells were prepared by layering over Ficoll.Table 1Patients' characteristicsNo.BreedAge (y)SexStageaStaging according to the histologic classification of mastocytomas (Patnaik et al. [58]): I, one tumor lesion confined to the dermis; II, dermis and regional lymph nodes; III, large infiltrating tumor with or without regional lymph nodes or multiple sites in the skin; IV, tumor with distant metastasis or relapsing disease with metastasis; a, without systemic symptoms; b, with systemic symptoms (pruritus; collapse, others).Histologic grade1Golden retriever07F/SIIIbIII2Beagle10FIIIaII3Labrador09F/SIIIaIF = female; S = spayed.a Staging according to the histologic classification of mastocytomas (Patnaik et al. 58Patnaik A.K. Ehler W.J. MacEwen E.G. Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs.Vet Pathol. 1984; 21: 469-474Crossref PubMed Scopus (574) Google Scholar): I, one tumor lesion confined to the dermis; II, dermis and regional lymph nodes; III, large infiltrating tumor with or without regional lymph nodes or multiple sites in the skin; IV, tumor with distant metastasis or relapsing disease with metastasis; a, without systemic symptoms; b, with systemic symptoms (pruritus; collapse, others). Open table in a new tab F = female; S = spayed. To confirm expression of KIT in neoplastic MC, flow cytometry and immunocytochemistry (ICC) were carried out. Flow cytometry was performed as described previously [31Gleixner K.V. Mayerhofer M. Aichberger K.J. et al.The tyrosine kinase-targeting drug 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. 2006; 107: 752-759Crossref PubMed Scopus (215) Google Scholar, 39Rebuzzi L. Willmann M. Sonneck K. et al.Detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR in canine mastocytoma cells.Vet Immunol Immunopathol. 2007; 115: 320-333Crossref PubMed Scopus (45) Google Scholar], using the mouse anti-KIT monoclonal antibody 104D2 (Becton Dickinson Biosciences, San Jose, CA, USA) and a FACScan (Becton Dickinson). Prior to ICC, cells were spun on cytospin-slides and fixed in acetone. ICC was performed as described previously [39Rebuzzi L. Willmann M. Sonneck K. et al.Detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR in canine mastocytoma cells.Vet Immunol Immunopathol. 2007; 115: 320-333Crossref PubMed Scopus (45) Google Scholar, 40Agis H. Krauth M.T. Böhm A. et al.Identification of basogranulin (BB1) as a novel immunohistochemical marker of basophils in normal bone marrow and patients with myeloproliferative disorders.Am J Clin Pathol. 2006; 125: 273-281Crossref PubMed Scopus (39) Google Scholar], using a polyclonal rabbit anti-KIT antibody (dilution 1:500) (Dako, Glostrup, Denmark). Analysis for KIT mutations was performed on DNA extracted from paraffin-embedded tumor tissue sections obtained in the three patients. Tissue sampling and digestion, DNA extraction, amplification of exon 11 of KIT, and sequencing were performed as described previously [19Zemke D. Yamini B. Yuzbasiyan-Gurkan V. Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs.Vet Pathol. 2002; 39: 529-535Crossref PubMed Scopus (168) Google Scholar]. Polymerase chain reaction products were gel-purified using QIAEX II kit and submitted for direct sequencing in both directions with the amplification primers at the sequencing core, which used dideoxy termination sequencing on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Prior to Western blotting, C2 cells and primary neoplastic MC (106/mL) were incubated with control medium or TK inhibitors at 37°C for 4 hours. C2 cells were incubated with imatinib, nilotinib, dasatinib (each 1 nM to 1 μM), or midostaurin (100 nM to 2.5 μM), and primary MC with midostaurin, nilotinib, or dasatinib (each 1 μM). Western blotting and immunoprecipitation (IP) were conducted as described [31Gleixner K.V. Mayerhofer M. Aichberger K.J. et al.The tyrosine kinase-targeting drug 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. 2006; 107: 752-759Crossref PubMed Scopus (215) Google Scholar, 41Sillaber C. Gesbert F. Frank D.A. Sattler M. Griffin J.D. STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells.Blood. 2000; 95: 2118-2125Crossref PubMed Google Scholar]. IP was performed on cell lysates (107 cells) using anti-KIT monoclonal antibody 1C1 [42Bühring H.J. Ashman L.K. Gattei V. et al.Stem-cell factor receptor (p145(c-kit)) summary report (CD117).in: Schlossmann S.F. Boumsell L. Gilks W. Leucocyte Typing V. White Cell Differentiation Antigens. Oxford University Press, Oxford, UK1995: 1882-1888Google Scholar] kindly provided by Dr. Hans-Jörg Bühring (University of Tübingen, Germany), anti-p38, or anti-Jnk antibody (both from Cell Signaling Technology, Beverly, MA, USA), and protein-G Sepharose beads (Amersham). Thereafter, immunoprecipitates were separated by 7% or 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Amersham). For detection of phosphorylated proteins, anti-phospho-tyr-monoclonal antibody 4G10 (1:1,000) (Upstate Biotechnology, Lake Placid, NY, USA) was applied on immunoprecipitates. For detection of phospho-Akt, phospho-Erk, and total levels of kinases, whole protein lysates were analyzed using polyclonal antibodies (all from Cell Signaling Technology) directed against the respective phosphorylated or total kinases. Antibody-reactivity was made visible by sheep anti-mouse IgG or donkey anti-rabbit IgG and Lumiegen PS-3 detection reagent (all from Amersham). To determine growth-inhibitory drug effects, C2 cells and primary neoplastic MC were incubated with various concentrations of imatinib (1 nM to 10 μM), midostaurin (1 nM to 1 μM), nilotinib (1 nM to 1 μM), dasatinib (1 nM to 500 nM), or control medium in 96-well culture plates (TPP, Trasadingen, Switzerland) at 37°C for 48 hours. In time course experiments, C2 cells were exposed to drugs (imatinib, 300 nM; midostaurin, 300 nM; nilotinib, 100 nM; dasatinib, 10 nM) for various time periods (0, 6, 12, 24, 48, and 72 hours). After incubation, 0.5 μCi 3H-thymidine was added (37°C, 12 hours). Cells were then harvested on filter membranes (Packard Bioscience, Meriden, CT, USA) in a Filtermate 196 harvester (Packard Bioscience). Filters were air-dried, and the bound radioactivity was counted in a β-counter (Top-Count NXT; Packard Bioscience). To determine potential additive or synergistic drug effects on growth of neoplastic MC, C2 cells were exposed to various combinations of drugs (imatinib, midostaurin, nilotinib, dasatinib) at a fixed ratio of drug concentrations. All experiments were performed in triplicate. In a first step, the effects of TK inhibitors on apoptosis were analyzed by morphologic examination. In typical experiments, C2 cells were incubated with imatinib (100 nM to 1 μM), midostaurin (100 nM to 1 μM), nilotinib (50 nM to 500 nM), dasatinib (10 nM to 100 nM), or control medium in six-well culture plates in Iscove's modified Dulbecco's medium containing 5% FCS for 24 hours. The percentage of apoptotic cells was quantified on Wright-Giemsa–stained cytospin preparations, apoptosis being defined by conventional cytomorphological criteria [43Van Cruchten S. Van Den Broeck W. Morphological and biochemical aspects of apoptosis, oncosis and necrosis.Anat Histol Embryol. 2002; 31: 214-223Crossref PubMed Scopus (418) Google Scholar]. To confirm that apoptosis occurred in C2 cells following a 24-hour exposure to imatinib (1 μM), midostaurin (1 μM), nilotinib (1 μM), or dasatinib (1 μM), a TUNEL assay was employed as reported [31Gleixner K.V. Mayerhofer M. Aichberger K.J. et al.The tyrosine kinase-targeting drug 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. 2006; 107: 752-759Crossref PubMed Scopus (215) Google Scholar]. In brief, cells were fixed in 1% formaldehyde at pH 7.4 at 0°C for 15 minutes. Cells were then treated with 70% ethanol (ice-cold) for 1 hour, washed, and incubated in terminal-transferase reaction solution containing CoCl2, DNA deoxy-nucleotidyl-exotransferase, and biotin-16-2′-deoxy-uridin-5′-triphosphate (Boehringer Mannheim, Germany) at 37°C for 10 minutes. Then, cells were washed and incubated with Streptavidin-Fluorescein (Boehringer Mannheim) (10 μg/mL) at 37°C for 20 minutes. Cells were then washed and analyzed with a Nikon Eclipse E 800 fluorescence microscope (Tokyo, Japan). For flow-cytometric determination of apoptosis and viability, combined Annexin V/propidium iodide staining was performed. C2 cells were exposed to imatinib, midostaurin, nilotinib, dasatinib (each 1 μM), or control medium at 37°C for 24 hours. Thereafter, cells were washed and incubated with Annexin V-FITC (Bender Medical Systems, Burlingame, CA, USA) in binding-buffer containing HEPES (10 mM, pH 7.4), NaCl (140 mM), and CaCl2 (2.5 mM). Then, cells were washed and propidium iodide (1 μg/mL) was added. Cells were analyzed by flow cytometry on a FACScan. For evaluation of effects of TK inhibitors on cell-cycle progression, C2 cells were incubated in control medium, imatinib (50–1000 nM), midostaurin (50–1000 nM), nilotinib (10–1000 nM), or dasatinib (1–1000 nM) at 37°C for 24 hours. Thereafter, cells were resuspended in 500 μL propidium iodide staining buffer containing 0.1% Na-acetate and 0.1% Triton X-100. Then, 20 μL propidium iodide (1 μg/mL) was added. Cell-cycle distribution was analyzed by flow cytometry on a FACScan. To determine significances in differences between proliferation and apoptosis after exposure of neoplastic MC to inhibitors, the Student's t-test for dependent samples was applied. Differences were considered statistically significant when p < 0.05. Recent data suggest that neoplastic canine MC express autophosphorylated KIT [17London C.A. Galli S.J. Yuuki T. et al.Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit.Exp Hematol. 1999; 27: 689-697Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 44Reguera M.J. Rabanal R.M. Puigdemont A. et al.Canine mast cell tumors express stem cell factor receptor.Am J Dermatopathol. 2000; 22: 49-54Crossref PubMed Scopus (61) Google Scholar]. In the present study, we were able to demonstrate expression of KIT in C2 cells and primary neoplastic MC. Expression of KIT in C2 cells was demonstrated by flow cytometry, ICC, and Western blotting. Figure 1 shows an ICC stain of primary neoplastic MC and C2 cells with an anti-KIT antibody. As assessed by IP and Western blotting, KIT was found to be autophosphorylated in primary neoplastic MC (see later) as well as C2 cells (Fig. 2). To examine whether primary neoplastic MC display any mutation involving exon 11, KIT sequence analysis was performed. However, no KIT mutation was found in the three patients examined.Figure 2Effects of tyrosine kinase (TK) inhibitors on KIT phosphorylation in C2 cells. (A–D) Tyrosine phosphorylation of KIT in C2 cells after incubation (4 hours) in control medium or various concentrations of imatinib (A), midostaurin (B), nilotinib (C), or dasatinib (D) as indicated. Immunoprecipitation was conducted using the anti
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