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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 [ 3 H]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.

stem cells

Raf‐1 Protein is Required for Growth Factor‐Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines