The objective of this project is to gain a better understanding of the molecular basis for leukemia development by utilizing a retrovirus-based mouse model system. 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 its normal regulator 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. Two pathways are known to be activated by Epo: the Jak-Stat pathway and the Raf-1/MAP kinase (MAPK) signaling pathway. Our previous studies indicated that infection of erythroid cells with SFFV resulted in the constitutive activation of the same Stat proteins that are transiently activated by Epo. Although Epo-induced activation of Stat proteins is mediated by the tyrosine kinase Jak2, our recent studies indicate that SFFV-induced activation of Stat proteins is Jak2-independent, as well as independent of other Jak family tyrosine kinases. In order to determine if infection of erythroid cells with SFFV could also lead to the constitutive activation of the Raf-1/MAPK signaling pathway, we analyzed the effects of SFFV infection on the activation of Raf-1; MAP kinase kinase (MEK), a direct substrate of Raf-1; and MAPK, a downstream effector molecule. Our results indicate that unlike uninfected erythroid cells, erythroid cells infected with SFFV show constitutive activation of Raf-1, MEK and MAPK. Inhibition of Raf-1 expression using c-raf antisense oligonucleotides indicated that while Raf-1 was absolutely required for Epo-induced proliferation of uninfected erythroid cells, it was only partially required for proliferation of SFFV-infected cells. Our data demonstrates that SFFV exerts its effects on more than one signal transduction pathway in erythroid cells and suggests that both pathways must be activated by the virus to achieve maximum proliferation of the cells in the absence of Epo.
