Raf-1 protein is required for growth factor-induced proliferation of primitive hematopoietic progenitors stimulated with synergistic combinations of cytokines.

Raf-1 is a serine/threonine kinase that has been identified as a component of growth factor-activated signal transduction pathways, and is required for growth factor-induced proliferation of leukemic cell lines and colony formation of hematopoietic progenitors stimulated with single colony-stimulating factors, which promote the growth of committed hematopoietic progenitor cells. However, it is known that the most primitive progenitors in the bone marrow require stimulation with multiple cytokines to promote cell growth. We have determined that c-raf antisense oligonucleotides inhibit the growth of murine lineage-negative progenitors stimulated with two-, three- and four-factor combinations of growth factors, including GM-CSF + interleukin (IL)- 1, IL-3 + steel factor (SLF), IL-3 + IL-11 + SLF and IL-3 + IL-11 + SLF + G-CSF. In addition, c-raf antisense oligonucleotides inhibit the synergistic response of the MO7e human progenitor cell line induced to proliferate with IL-3 + SLF (99%) or GM-CSF + SLF (99%). In contrast, c-raf antisense oligonucleotides only partially inhibited day 14 colony formation of CD34+ human progenitors stimulated with IL-3 + SLF (50%) or GM-CSF + SLF (55%) but completely inhibited day 7 colony formation. However, pulsing CD34+ cells with additional oligonucleotides on day 7 of the colony assay further inhibited day 14 colony formation (70%-80%). Furthermore, a comparison of the effect of c-raf antisense oligonucleotides on the synergistic response of normal human fetal liver cells in [3H]thymidine incorporation assays and colony assays showed strong inhibition in short-term proliferation assays and partial inhibition in 14-day colony assays. Taken together, these results demonstrate that partial inhibition of colony formation of primitive human progenitors stimulated with multiple growth factors is a result of the length (14 days) of the human colony assay and does not represent a differential requirement of primitive progenitors for Raf-1. Thus Raf-1 is required for the proliferation and differentiation of primitive hematopoietic progenitor cells stimulated with synergistic combinations of cytokines.

The effect of c-raf antisense oligonucleotides on growth factor-induced proliferation of hematopoietic cells.

While it is well established that Raf-1 kinase is activated by phosphorylation in growth factor-dependent hematopoietic cell lines stimulated with a variety of hematopoietic growth factors, little is known about the biological effects of Raf-1 activation on normal hematopoietic cells. Therefore, we examined the requirement for Raf-1 in growth factor-regulated proliferation and differentiation of hematopoietic cells using c-faf antisense oligonucleotide. Raf-1 required for the proliferation of growth factor dependent cell lines stimulated by IL-2, IL-3, G-CSF, GM-CSF and EPO that bind to the hematopoietin class of receptors. Raf-1 is also required for the proliferation of cell lines stimulated by growth factors that use the tyrosine kinase containing receptor class, including SLF and CSF-1. In addition, Raf-1 is also required for IL-6, LIF- and OSM-induced proliferation whose receptors share the gp 130 subunit. In contrast to previous results which demonstrated that IL-4 could not activate Raf-1 kinase, c-raf antisense oligonucleotides also inhibited IL-4-induced proliferation of T cell and myeloid cell lines. Using normal hematopoietic cells, c-raf antisense oligonucleotides completely suppressed the colony formation of murine hematopoietic progenitors in response to single growth factors, such as IL-3, CSF-1 or GM-CSF. Further, c-raf antisense oligonucleotides inhibited the growth of murine progenitors stimulated with synergistic combinations of growth factors (required for primitive progenitor growth) including two, three and four factor combinations. In comparison to murine hematopoietic cells, c-raf antisense oligonucleotides also inhibited both IL-3 and GM-CSF-induced colony formation of CD 34+ purified human progenitors. In addition, Raf-1 is required for the synergistic response of CD 34+ human bone marrow progenitors to multiple cytokines; however, this effect was only observed when additional antisense oligonucleotides were added to the cultures at day 7 of a 14 day assay. Finally, Raf-1 is required for the synergistic response of human Mo-7e cells and of normal human fetal liver cells to five factor combinations. Thus, Raf-1 is required to transduce growth factor-induced proliferative signals in factor-dependent progenitor cells lines for all known classes of hematopoietic growth factor receptors, and is required for the growth of normal murine and human bone marrow-derived progenitors.