Synergistic effects of PI3K/Akt on abrogation of cytokine-dependency induced by oncogenic Raf

The effects of inducible Raf and PI3K/Akt activity on signal transduction pathways associated with the proliferation and prevention of apoptosis of hematopoietic cells were investigated. Cytokine dependent murine FL5.12 cells were infected with conditionally-activated GFPΔRaf:ER and Akt:ER genes ligated to the hormone binding domain of the human estrogen receptor which rendered the activities of the introduced Raf or Akt proteins dependent upon the estrogen receptor antagonist, 4HT. In addition, some cytokine-dependent FL/GFPΔRaf:ER cells were infected with retro-viruses encoding constitutively-active PI3K or AKT oncogenes. Deregulated Raf or PI3K/Akt expression by themselves did not permit the growth of FL5.12 cells in the absence of exogenous cytokines. However, the combination of a growth stimulus (Raf) and an anti-apoptotic signal (PI3K/ Akt) synergized and led to the ability of the doubly-infected cells to grow m the absence of exogenous cytokines. Recently it has been shown that Akt can phosphorylate Raf and result in its inhibition (71, 72). However this inhibition is dependent on the differentiation state of the cell and may be specific for certain types of cells (myotubules, kidney cells) as this observation has not been observed in other cells. The activated Raf, PI3K and Akt oncoproteins abrogated the cytokine -dependency of FL5.12 cells by an autocrine mechanism as autocrine GMCSF expression was detected in the cells which grew in response to Raf and Akt but not in the cells which remained cytokine-dependent. The ability of the anti-apoptotic Bcl-2 protein to synergize with Raf and Akt expression was examined by determining the frequency of Raf and Akt infected cells obtained after Bcl-2 infection. Bcl-2 infection increased the frequency of cytokine-independent cells approximately 25-fold in those cells which had functional activated Raf and Akt. The results presented in our study document that deregulated Raf and Akt expression could affect downstream signal transduction pathways and result in the abnormal proliferation of hematopoietic cells. The ERK 1,2 and p38^Map kinases were constitutively detected in the cells which grew in response to Raf and PI3K/Akt activation. Thus the signal cascades induced by Raf and PI3K/Akt can interact with other signal transduction pathways. A model for the effects of the activated Raf and PI3K/Akt oncoproteins on the autocrine growth of hematopoietic cells is presented in Fig. 14. In this model, we have attempted to illustrate how the Raf and PI3K/Akt pathways can interact and result in the prevention of apoptosis. Further studies will elucidate how these kinases activate transcription factors and stimulate autocrine growth.