Veterinary Research Communications

, Volume 37, Issue 2, pp 101–108 | Cite as

Ex vivo evaluation of imatinib mesylate for induction of cell death on canine neoplastic mast cells with mutations in c-Kit exon 11 via apoptosis

  • Giacomo Rossi
  • Chiara Bertani
  • Subeide Mari
  • Carlotta Marini
  • Giacomo Renzoni
  • Gregory Ogilvie
  • Gian Enrico MagiEmail author
Original Article


Several studies of canine spontaneous mast cell tumours have described mutations in the c-kit proto-oncogene. These mutations produce a constitutively activated product and have been suggested to play a role in the malignant transformation of mast cells. We hypothesize that the selective tyrosine kinase inhibitor imatinib mesylate inhibits signal transduction and induces apoptosis when tested in cutaneous canine mast cell tumour samples positive for mutation in c-kit exon 11. Three-dimensional ex vivo cultures of canine grade II mast cell tumour treated with STI-571 at 48, 72, and 96 h and tested for signal transduction and apoptosis using appropriate assays were used. There was a progressive and significant increase in caspase-3 and TUNEL-positive mast cells compared to the untreated cultures. Additionally, a concurrent reduced expression of Ki67 and BCL-2 was observed. Furthermore, the treated cultures showed a marked reduction of Kit expression. Our results demonstrate that STI-571 induces Caspase-dependent apoptosis in a canine neoplastic mast cells possessing mutations in c-kit exon 11.


Apoptosis STI-571 Signal transduction Immunohistochemistry c-Kit 


  1. Barzanti F, Zoli W, Susino MD, Ricotti L, Tesei A, Papa S, Reno F, Amadori D (2001) Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells. J Biol Regul Homeost Agents 15:359–365PubMedGoogle Scholar
  2. Bennett MH, Wilson GD, Dische S, Saunders MI, Martindale CA, Robinson BM, O’Halloran AE, Leslie MD, Laing JH (1992) Tumour proliferation assessed by combined histological and flow cytometric analysis: Implications for therapy in squamous cell carcinoma in the head and neck. Br J Cancer 65:870–878PubMedCrossRefGoogle Scholar
  3. Demetri GD, Von Mehren M, Blanke CD, Van Den Abbeele AD, Eisenberg B, Roberts PJ, Heinrich MC, Tuveson DA, Singer S, Janicek M, Fletcher JA, Silverman SG, Silberman SL, Capdeville R, Kiese B, Peng B, Dimitrijevic S, Druker BJ, Corless C, Fletcher CD, Joensuu H (2002) Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 347:472–480PubMedCrossRefGoogle Scholar
  4. Dixon SC, Soriano BJ, Lush RM, Borner MM, Figg WD (1997) Apoptosis: its role in the development of malignancies and its potential as a novel therapeutic target. Ann Pharmacother 31:76–82PubMedGoogle Scholar
  5. Downing S, Chien MB, Kaas PH, Moore PE, London CA (2002) Prevalence and importance of internal tandem duplications in exons 11 and 12 of c-kit in mast cell tumors of dogs. Am J Vet Res 63:1718–1723PubMedCrossRefGoogle Scholar
  6. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville R, Talpaz M (2001) Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J M 344:1038–1042CrossRefGoogle Scholar
  7. Fain O, Stirnemann J, Eclache V, Barete S, Casassus P, Hermine O, Lorholary O (2005) Systemic mastocytosis. Presse Med 34:681–687PubMedCrossRefGoogle Scholar
  8. Gleixner KV, Rebuzzi L, Mayerhofer M, Gruze A, Hadzijusufovic E, Sonneck K, Vales A, Kneidinger M, Samorapoompichit P, Thaiwong T, Pickl WF, Yuzbasiyan-Gurkan V, Sillaber C, Willmann M, Valent P (2007) Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells. Exp Hematol 35:1510–1521PubMedCrossRefGoogle Scholar
  9. Goldschmidt MH, Hendrick MJ (2002) Tumors of the skin and soft tissue. In: Meuten DJ (ed) Tumors in domestic animals, 4th edn. Blackwell, Ames, pp 105–107Google Scholar
  10. Gore SD, Weng LJ, Burke PJ (1993) Validation of flow-cytometric determination of Ki67 expression as a measure of growth factor response in acute myelogenous leukemia. Exp Hematol 21:1702–1708PubMedGoogle Scholar
  11. Gown A, Willingham MC (2002) Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem 50:449–454PubMedCrossRefGoogle Scholar
  12. Hahn KA, Ogilvie G, Rusk T, Devauchelle P, Leblanc A, Legendre A, Powers B, Leventhal PS, Kinet JP, Palmerini F, Dubreuil P, Moussy A, Hermine O (2008) Mastinib is safe and effective for the treatment of canine mast cell tumors. J Vet Intern Med 22:1301–1309PubMedCrossRefGoogle Scholar
  13. Isotani M, Ishida N, Tominaga M, Tamura K, Yagihara H, Ochi S, Kato R, Kobayashi T, Fujita M, Fujino Y, Setoguchi A, Ono K, Washizu T, Bonkobara M (2008) Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs. J Vet Intern Med 22:985–988PubMedCrossRefGoogle Scholar
  14. Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108:153–164PubMedCrossRefGoogle Scholar
  15. Kiupel M, Webster JD, Kaneene JB, Miller R, Yuzbasiyan-Gurkan V (2004) The Use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors. Vet Path 41:371–377CrossRefGoogle Scholar
  16. Li N, Margolis LB, Hoffman RM (1991) Skin toxicity determined in vitro by three-dimensional, native-state histoculture. Proc Natl Acad Sci USA 88:1908–1912PubMedCrossRefGoogle Scholar
  17. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC, Geissler EN (1999) Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Exp Hematol 27:689–697PubMedCrossRefGoogle Scholar
  18. Michels GM, Knapp DW, DeNicola DB, Glickman N, Bonney P (2002) Prognosis following surgical excision of canine cutaneous mast cell tumors with histopathologically tumor-free versus nontumor-free margins: a retrospective study of 31 cases. J Am Anim Hosp Assoc 38:458–466PubMedGoogle Scholar
  19. Morin PJ (2003) Drug resistance and the microenvironment: nature and nurture. Review. Drug Resist Updat 6:169–172PubMedCrossRefGoogle Scholar
  20. Nakamura T, Kato Y, Fuji H, Horiuchi T, Chiba Y, Tanaka K (2003) E-cadherin-dependent intercellular adhesion enhances chemoresistance. Int J Mol Med 12:693–700PubMedGoogle Scholar
  21. Nocka K, Buck J, Levi E, Besmer P (1990) Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors. EMBO J 9:3287–3294PubMedGoogle Scholar
  22. Nocka K, Majumder S, Chabot B, Ray P, Cervone M, Bernstein A, Besmer P (1989) Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice—evidence for an impaired c-kit kinase in mutant mice. Genes Dev 3:816–826PubMedCrossRefGoogle Scholar
  23. Patnaik AK, Ehler WJ, MacEwen EG (1984) Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Vet Path 21:469–474Google Scholar
  24. Qiu FH, Ray P, Brown K, Barker PE, Jhanwer S, Ruddle FH, Besmer P (1988) Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family—oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J 7:1003–1011PubMedGoogle Scholar
  25. Rahmani M, Reese E, Dai Y, Bauer C, Kramer LB, Huang M, Jove R, Dent P, Grant S (2005) Cotreatment with suberanoylanilide hydroxamic acid and 17-allylamino 17-demethoxygeldanamycin synergistically induces apoptosis in Bcr-Abl+ Cells sensitive and resistant to STI571 (imatinib mesylate) in association with down-regulation of Bcr-Abl, abrogation of signal transducer and activator of transcription 5 activity, and Bax conformational change. Mol Pharmacol 67:1166–1176PubMedCrossRefGoogle Scholar
  26. Reguera MJ, Rabanal RM, Puigdemont A, Ferrer L (2000) Canine mast cell tumors express stem cell factor receptor. Am J Dermatopathol 22:49–54PubMedCrossRefGoogle Scholar
  27. Resendes AR, Majo N, Segales J, Espadamala J, Mateu E, Chianini F, Nofrarias M, Domingo M (2004) Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues. Vet Immunol Immunopathol 99:203–213PubMedCrossRefGoogle Scholar
  28. Simoes JPC, Schoning P (1994) Canine mast cell tumors: a comparison of staining techniques. J Vet Diagn Invest 6:458–465PubMedCrossRefGoogle Scholar
  29. Strasser A, Harris AW, Huang DC, Krammer PH, Cory S (1995) Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 14:6136–6147PubMedGoogle Scholar
  30. Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R, Cortes J, O’Brien S, Nicaise C, Bleickardt E, Blackwood-Chirchir MA, Iyer V, Chen TT, Huang F, Decillis AP, Sawyers CL (2006) Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. New Engl J Med 354:2531–2541PubMedCrossRefGoogle Scholar
  31. Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281:1312–1316PubMedCrossRefGoogle Scholar
  32. Truong T, Sun G, Doorly M, Wang JY, Schwartz MA (2003) Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl. Proc Natl Acad Sci USA 100:10281–10286PubMedCrossRefGoogle Scholar
  33. Tsai M, Takeishi T, Thompson H, Langley KE, Zsebo KM, Metcalfe DD, Geissler EN, Galli SJ (1991) Induction of mast cell proliferation, maturation and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc Natl Acad Sci USA 88:6382–6386PubMedCrossRefGoogle Scholar
  34. Vail DM (1994) Mast cell tumors. In: Withrow SJ, MacEwen EG (eds) Small animal clinical oncology, 2nd edn. WB Saunders, Philadelphia, p 192Google Scholar
  35. Van Etten RA (2004) Mechanisms of transformation by the BCR-ABL oncogene: new perspectives in the post-imatinib era. Leuk Res 1:21–28CrossRefGoogle Scholar
  36. Yee NS, Paek I, Besmer P (1994) Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: basis for radiosensitivity of white spotting and steel mutant mice. J Exp Med 179:1777–1787PubMedCrossRefGoogle Scholar
  37. Yu C, Bruzek LM, Meng XW, Gores GJ, Carter CA, Kaufmann SH, Adjei AA (2005) The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43–9006. Oncogene 24:6861–6869PubMedCrossRefGoogle Scholar
  38. Zemke D, Yamini B, Yuzbasiyan-Gurkan V (2002) Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs. Vet Path 39:529–535CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Giacomo Rossi
    • 1
  • Chiara Bertani
    • 2
  • Subeide Mari
    • 1
  • Carlotta Marini
    • 1
  • Giacomo Renzoni
    • 1
  • Gregory Ogilvie
    • 3
  • Gian Enrico Magi
    • 1
    Email author
  1. 1.School of Veterinary Medical ScienceUniversity of CamerinoMatelicaItaly
  2. 2.Clinica Veterinaria FlaminiaAnconaItaly
  3. 3.CVS Angel Cancer CenterSan MarcosUSA

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