The long range objectives are to determine etiologic factors and mechanisms involved in leukemogenesis. To this end, the study of the c-kit protooncogene was undertaken. This protooncogene is a member of a subclass of growth factor receptors. As a protooncogene, it may play a role in oncogenesis. Identification of this gene as the mouse dominant white spotting locus illustrates its functional importance in embryogenesis, melanogenesis and hematopoiesis. Identification of the c-kit ligand as Stem Cell Factor shows that this receptor is crucial in the growth and survival of hematopoietic stem cells. The purpose of this proposal is two- fold. Using the available reagents of the human c-kit gene, cDNA and a monoclonal antibody, the specific aims are: 1. Determine the cis-acting DNA elements important for c-kit expression - The cis-acting elements which are responsible for cell specific expression will be defined by testing a series of chimeric reporter plasmids in a transient expression assay utilizing c-kit expressing and nonexpressing cells. This will include defining 5' flanking region elements as well as potential intron 1 elements by the use of """"""""mini intron"""""""" constructs. Once potential elements have been identified, they will be mutated and functionally tested to confirm their importance in the regulation of c-kit expression. 2. Determine the oncogenic potential of c-kit and define its role in a leukemic cell model - Using site directed mutagenesis, specific mutations will be introduced into the receptor and tested for their ability to act as dominant positive or dominant negative mutations in an NIH-3T3 cell transformation assay. A recently described alternatively spliced form of c-kit found in leukemic cell lines will also be tested. Dominant mutant receptors will be stably introduced into human leukemic cell lines which express endogenous c-kit. Their effects on the proliferation and in vitro differentiation of these cells will be investigated. The results of these studies will define the expression and transformation potential of c-kit. They will set the stage to test if altered expression of a normal receptor or normal expression of an activated receptor will induce leukemia in an in vivo model. this will begin to define the possible role of this receptor in the induction and maintenance of the leukemic phenotype.
| Vandenbark, G R; Chen, Y; Friday, E et al. (1996) Complex regulation of human c-kit transcription by promoter repressors, activators, and specific myb elements. Cell Growth Differ 7:1383-92 |
