An exhaustive study of tumorigenesis and development of a novel molecular targeting therapy for canine mastocytoma

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1) c-kit gene mutations in clinical samples of dog mastocytoma were widely analyzed. Gene mutations (insertion, internal tandem duplication, and deletion) in the c-kit juxtamembrane domain were found in less than 12% of all cases ; however, c-kit gene in 88% cases was a wild type.2) Although mutated c-kit gene was cloned and transfected into mock cells, neoplastic proliferation was not induced, indicating that the c-kit gene mutations may not the major inducer of mast cell tumor. We investigated the other candidates that may induce neoplastic proliferation of mast cells, and found that the over-expression of D-type cyclins and one of Bcl-2 family proteins Mcl-1. We also found that the low expression of one of BH3 family proteins Bim-1 and tumor suppressors p21, p27, and p53.3) Transcription factors NF- κ B and AP-1 were found to be activated in neoplastic mast cells and by the addition of those transcription factor inhibitors into the culture, proliferation of neoplastic mast cells was abolished. Signaling molecules down below the PI3 kinase pathway, S6 kinase, was spontaneously activated and over-expression of S6 ribosomal protein was obvious.4) We established a novel high-affinity IgE receptor-positive canine mast cell line with wild type c-kit receptors. By using cell lines and clinical samples, production of growth factors and cytokines in dog mastocytoma was analyzed and found auto-production of interleukin-3, and -6, GM-CSF, and SCF, and the expression of those receptors was found. Neutralization of those auto-produced cytokines by specific antibodies partially inhibited cell proliferation, indicating that, at least in part, auto-production of growth factors by mastocytoma cells themselves may induce cell proliferation and/or survival.