RAF-1 PROTEIN IS REQUIRED FOR GROWTH FACTOR-INDUCED PROLIFERATION OF PRIMITIVE HEMATOPOIETIC PROGENITORS STIMULATED WITH SYNERGISTIC COMBINATIONS OF CYTOKINES

Raf-l is a serine/threonine kinase that has been identified as a compo nent of growth factor-activated signal transduction pathways, and is r equired for growth factor-induced proliferation of leukemic cell lines and colony formation of hematopoietic progenitors stimulated,vith sin gle colony-stimulating factors, which promote the growth of committed hematopoietic progenitor cells, However, it is known that the most pri mitive progenitors in the bone marrow require stimulation with multipl e cytokines to promote cell growth, We have determined that c-raf anti sense oligonucleotides inhibit the growth of murine lineage-negative p rogenitors stimulated with two-, three- and four-factor combinations o f growth factors, including GM-CSF + interleukin (IL)-1, IL-3 + steel factor (SLF), IL-3 + IL-11 + SLF and IL-3 + IL-II + SLF + G-CSF, In ad dition, c-raf antisense oligonucleotides inhibit the synergistic respo nse of the MO7e human progenitor cell line induced to proliferate with IL-3 + SLF (99%) or GM-CSF + SLF (99%), In contrast, c-paf antisense oligonucleotides only partially inhibited day 14 colony formation of C D34(+) human progenitors stimulated with IL-3 + SLF (50%) or GM-CSF SLF (55%) but completely inhibited day 7 colony formation, However, pu lsing CD34(+) cells with additional oligonucleotides on day 7 of the c olony assay further inhibited day 14 colony formation (70%-80%), Furth ermore, a comparison of the effect of c-raf antisense oligonucleotides on the synergistic response of normal human fetal liver cells in [H-3 ]thymidine incorporation assays and colony assays showed strong inhibi tion in short-term proliferation assays and partial inhibition in 14-d ay colony assays, Taken together, these results demonstrate that parti al inhibition of colony formation of primitive human progenitors stimu lated with multiple growth factors is a result of the length (14 days) of the human colony assay and does not represent a differential requi rement of primitive progenitors for Raf-l, Thus Raf-l is required for the proliferation and differentiation of primitive hematopoietic proge nitor cells stimulated with synergistic combinations of cytokines.