Effects of deregulated Raf and MEK 1 expression on the cytokine- dependency of hematopoietic cells

The effects of inducible Raf or MEK activity on signal transduction pathways associated with the proliferation of hematopoietic cells were investigated. Cytokine-dependent murine FDC-P1 and human TF-1 cells were infected with conditionally-activated (ΔRaf:ER or ΔMEK1:ER) genes which contained the activated Raf and MEK1 cDNAs ligated to the hormone binding domain of the human estrogen receptor which rendered the activities of the introduced Raf or MEK1 proteins dependent upon β-estradiol. Deregulated Raf or MEK1 expression permitted growth of some infected FDC-P1 or TF-1 cells in the absence of exogenous cytokines. The ability of the three different Raf genes to abrogate cytokine-dependency of FDC-P1 and TF-1 cells varied. ΔA- Raf:ER was more efficient than ΔRaf-1:ER or ΔMEK1:ER and cytokine- independent cells were very infrequently isolated after infection with ΔB- Raf:ER. The ability of the anti-apoptotic Bcl-2 protein to synergize with Raf or MEK1 expression was examined by determining the frequency of Raf and MEK1 responsive cells obtained after Bcl-2 infection. Bcl-2 infection increased the frequency of cytokine-independent cells approximately 10-fold. The ΔRaf:ER and ΔMEK1:ER oncoproteins abrogated the cytokine-dependency of FDC- P1 and TF-1 cells by an autocrine mechanism as autocrine GM-CSF expression was detected in the ΔRaf:ER and ΔMEK1:ER responsive cells. The interaction of the Raf/MEK/MAPK cascade with other signaling pathways was examined. Stimulation of the Raf and IL-3 signal transduction pathways synergized and resulted in increased levels of cell proliferation. Activation of protein kinase C after treatment with phorbol esters synergized with IL-3 and also resulted in increased [³H]-thymidine incorporation. In contrast, PMA did not augment the response to Raf indicating that they induced overlapping patterns of signal transduction. By use of kinase inhibitors, the interactions of other signal transduction pathways were determined. The Jak kinase pathway interacted with ΔRaf:ER mediated growth. Thus it is likely that a Jak kinase is necessary for full activation of the signal transduction cascade induced by Raf. PI3 K inhibitors strongly suppressed both IL-3 and Raf mediated growth, indicating the importance of this enzyme in the stimulation of growth and the prevention of apoptosis. Recently it has been observed that PI3 K can activate the kinase Akt that can in turn phosphorylate the pro-apoptotic Bad protein. Inhibition of Akt activity by the Ly294002 compound may result in induction of apoptosis. The results presented in our study document that deregulated Raf and MEK1 expression could affect downstream signal transduction pathways and result in the abnormal proliferation of hematopoietic cells. Furthermore, the signal cascades induced by Raf and MEK1 can interact with other signal transduction pathways, which shows how complicated cytokine signal transduction is.