Growth factor-independent proliferation of erythroid cells infected with Friend spleen focus-forming virus is protein kinase C dependent but does not require Ras-GTP.

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.

Both the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus induce constitutive activation of the Raf-1/mitogen-activated protein kinase signal transduction pathway.

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an attempt to understand how the virus causes Epo independence, we have been studying signal transduction pathways activated by Epo to determine if SFFV exerts its biological effects by constitutively activating any of these pathways in the absence of Epo. We previously demonstrated that Stat proteins, the downstream components of the Epo-induced Jak-Stat pathway, are constitutively activated in SFFV-infected cells. In this study, we demonstrate that SFFV also activates Raf-1, MEK and mitogen-activated protein (MAP) kinase, the downstream components of the Raf-1/MAP kinase pathway. This pathway was activated in cells infected with the polycythemia-inducing strain of SFFV, which induces both proliferation and differentiation of erythroid cells in the absence of Epo, as well as in cells infected with the anemia-inducing strain of the virus, which still require Epo for differentiation. Inhibition of Raf-1 by using antisense oligonucleotides led to a partial inhibition of the Epo-independent proliferation of SFFV-infected cells. Expression of the transcription factors c-Jun and JunB, but not c-Fos, was induced in SFFV-infected cells in the absence of Epo, suggesting that constitutive activation of the Raf-1/MAP kinase pathway by the virus may result in deregulation of AP-1 activity. We conclude from our studies that infection of erythroid cells with SFFV leads to the constitutive activation of signal transduction molecules in both the Jak-Stat and Raf-1/MAP kinase pathways and that both of these pathways must be activated to achieve maximum proliferation and differentiation of erythroid cells in the absence of Epo.

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.

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 variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage 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 receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.