RAF-1 PROTEIN IS REQUIRED FOR GROWTH FACTOR-INDUCED PROLIFERATION OF HEMATOPOIETIC-CELLS

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a varie ty of mitogens and growth factors, including hematopoietic growth fact ors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth f actor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural cl asses: (a) the hematopoietin receptor family, including interleukin (I L)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/m acrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose rece ptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activat e the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-in duced proliferation of both myeloid and T cell lines, and IL-4 activat ed Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In co lony assays, c-raf antisense oligonucleotides completely inhibited col ony formation of unseparated normal murine bone marrow cells stimulate d with either IL-3 or CSF-1 and partially inhibited cells stimulated w ith GM-CSF. In addition, c-raf antisense oligonucleotides completely i nhibited both IL-3- and GM-CSF-induced colony formation of CD34(+) pur ified human progenitors stimulated with these same growth factors. Thu s, Raf-1 is required for growth factor-induced proliferation of leukem ic murine progenitor cell lines and normal murine and human bone marro w-derived progenitor cells regardless of the growth factor used to sti mulate cell growth.