Current evidence suggests that the stimulation of growth factor receptors by their respective ligands affects lineage commitment in hemopoietic cells. It has also been shown that the Raf-I kinase is phosphorylated in response to stimulation by a variety of hemopoietic growth factors including interleukin-2, interleukin-3, macrophage colony stimulating factor (CSF-1), and granulocyte-macrophage colony stimulating factor. This suggests a role for the raf gene product as a signal transduction molecule in pathways known to affect the growth and differentiation of hemopoietic cells. In addition, it has been shown that raf family genes are differentially expressed in cells committed to the macrophage and B-cell lineages. Experiments infecting mouse bone marrow cells with a v-myc/v-raf containing retrovirus generated B-cells and macrophages in which the presence of identical heavy chain gene rearrangements in both cell types suggested that cells previously committed to the B-cell lineage had subsequently differentiated into mature macrophages. Experiments were undertaken to analyze the role of each oncogene in the alteration of lineage commitment in these cells. Preliminary results suggest that the v-myc gene inhibits the expression of markers associated with commitment to the B-cell lineage while v-raf induces these cells to either differentiate into sIg+ B-cells or to switch their lineage commitment and differentiate along the macrophage lineage. In addition, the appearance of a cell surface marker associated with hemopoietic stem cells suggests that cells revert to a stem cell phenotype prior to differentiation along the macrophage lineage. To establish the extent to which these effects are related to raf gene function, reagents have been designed which will permit conditional expression of the v-raf gene in v-myc/vraf-transformed bone marrow cells. A retroviral construct expressing a temperature-sensitive v-raf gene and a dexamethasone inducible dominant negative Raf-1 mutant will be used to evaluate the association between raf gene expression, lineage commitment and differentiation in hemopoietic cells.
