Constitutively active mutant D816VKit induces megakayocyte and mast cell differentiation of early haemopoietic cells from murine foetal liver

Abstract 

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF.

Abbreviations:  AML, acute myeloid leukaemia, APAAP, alkaline phosphatase anti-alkaline phosphatase, CFU, colony forming units, DMEM, Dulbecco’s modified Eagle’s medium, FCS, foetal calf serum, FLC, foetal liver cells, GIST, gastro-intestinal stromal tumours, GM-CSF, granulocyte/macrophage colony stimulating factor, Hu-Kit, human c-Kit, IMDM, Iscove’s modified Dulbecco’s medium, mAb, monoclonal antibody, MIHC, Myb immortalised haemopoietic cells, MMCP-5, mouse mast cell protease-5, PBS, phosphate buffered saline (pH 7.5), PI3K, phosphatidylinositol 3 kinase, SCF, stem cell factor, TBS, Tris-buffered saline (pH 7.6), WTKit, wild-type Kit

Keywords:  c-Kit, D816V mutation, Transformation, Differentiation, Megakaryocyte, Mast cell