The c-fgr proto-oncogene, a member of the src family of intracellular tyrosine kinases, is selectively expressed in normal and leukemic myeloid cells. C-fgr may play an important, but as yet undefined role in the development of the monocytic lineage from hematopoietic progenitor cells, in the induction of proliferation and functional activation in fully differentiated monocytes and macrophages, and in the development of myeloid leukemia and myelodysplasia. To dissect the functions of c-fgr in myeloid cells, a research plan has been developed with the following specific aims: 1. TO IDENTIFY AND CHARACTERIZE THE C-FGR-ENCODED PROTEIN(S) IN MONOCYTES The c-fgr kinase has not yet been identified or characterized in normal myeloid cells. The murine c-fgr cDNA may encode two distinct proteins with differing amino termini. Utilizing recently developed antisera the c-fgr- encoded protein(s) will be identified and their subcellular locations determined. It will also be determined if the c-fgr-protein(s) are differentially expressed at particular stages of monocytic differentiation or during the induction of proliferation and functional activation in fully mature monocytes/macrophages. 2.TO DETERMINE IF THE C-FGR KINASE FUNCTIONS TO INDUCE THE COMMITMENT OF HEMATOPOIETIC PROGENITOR CELLS TO THE MONOCYTIC LINEAGE. The c-fgr cDNA has been introduced into an IL-3-dependent murine myeloid progenitor cell line with a retroviral vector, resulting in the induction of expression of the c-fms tyrosine kinase (encoding the receptor for the monocytic lineage colony-stimulating factor CSF-I). Subsequent culture in CSF-1 induced monocytic differentiation in vitro, the establishing a differentiation model. It will be determined: a) whether c-fgr induces c-fms expression be transcriptional or post-transcriptional mechanisms; b) which functional domains of the c-fgr protein and essential for the induction of c-fms by creating altered forms of the c-fgr protein(s) by mutagenesis; c) whether direct introduction of c-fms bypasses the requirement for c-fgr expression; and d) whether the induction of c-fms is unique of c-fgr or may be duplicated by other related members of he src family. Studies of the in vitro effect of infecting normal hematopoietic progenitor cells with these retroviral constructs will be initiated. 3. TO DETERMINE IF THE C-FGR KINASE IS ESSENTIAL FOR THE INDUCTION OF PROLIFERATION AND/OR FUNCTIONAL ACTIVATION MONOCYTES. C-fgr is transiently expressed when differentiated monocytes/macrophages are stimulated to proliferate or when these cells are activated in diverse functional states. To determine if c-fgr is essential for these functions, expression and function of c-fgr will be disrupted in normal monocytic cells nad in a CSF- 1-dependent macrophage cell line: a) by utilizing antisense oligomers to c-fgr or an inducible expression vector which produces antisense mRNA to c- fgr; and b) by introducing vectors which drive high levels of expression of a non-functional or """"""""dead"""""""" c-fgr kinard which could compete for the cellular factors and targets associated with the normal c-fgr kinase, thereby blocking the function of the normal protein.
