Synergistic effects of akt on abrogation of cytokine-dependency induced by raf and mek

Abstract

The Raf/MEK/ERK kinase cascade is pivotal in transmitting signals from membrane receptors to transcription factors which regulate gene expression. This cascade is also involved in the prevention of apoptosis as ERK2 can phosphorylate Bcl-2 and Bad. The Akt kinase is involved in the regulation of apoptosis and can phosphorylate Bad. To determine whether Akt could interact with the Raf/MEK/ERK cascade and result in a significant oncological response, the ability of Akt to increase the frequency of abrogation of cytokine-dependency of FL5.12 hematopoietic cells was determined. Abrogation of cytokine-dependency is considered an important step in leukemogenesis. The Akt cDNA either containing or lacking a myristolation sequence was ligated to the hormone binding domain of a mutated estrogen receptor which preferentially responds to tamoxifen. Tamoxifen-induced Akt activity, by itself, did not abrogate the cytokine-dependency of FL5.12 cells. Moreover, hematopoietic cells are not efficiently transformed to cytokine-independence after infection with either an activated conditional Raf (ΔRaf-1:ER) or MEK1 (ΔMEK1:ER) genes. The combination of Akt and either Raf or MEK expression increased the transformation 100-fold. The efficiency of transformation was further enhanced 50-fold when cytokine-dependent cells were subsequently infected with retroviral vectors encoding either Bcl-2 or Bcl-XL. Two different types of cells were isolated in these experiments, cytokine-dependent and Raf or MEK responsive cells. The Raf-responsive, Akt:ER cells expressed higher levels of the ΔRaf-1:ER kinase and downstream MEK kinase activities than the cytokine-dependent cells. These cells should be useful in elucidating the interactions between Akt and Raf/MEK/ERK kinase cascade in the prevention of apoptosis.