Key Roles of the Raf-1 and Akt in Drug Resistance, Cell Cycle Progression and the Prevention of Apoptosis in Hematopoietic Cells.

The Raf/MEK/ERK and PI3K/Akt kinase cascades are pivotal in transmitting signals from membrane receptors to transcription factors which regulate growth, apoptosis and chemotherapeutic drug resistance. Previously we determined that activated Raf-1 or Akt expression by themselves, did not relieve cytokine-dependence of FL5.12 hematopoietic cells. However, when activated forms were introduced and activated in the same cell, cytokine-dependence was relieved. This was determined with conditionally active forms of Raf (Raf-1:AR) and Akt (Akt:ER) that are activated upon testosterone (binds AR) and tamoxifen (binds ER) treatment respectively. Upon hormone deprivation for 24 hrs, FL/Akt:ER+Raf-1:AR cells exited the cell cycle and underwent apoptosis. The effects of Raf, Akt and cytokines on cell cycle progression, prevention of apoptosis, downstream signal transduction pathways and chemotherapeutic drug resistance were examined. Activation of Raf or Akt individually suppressed but did not totally inhibit apoptosis, while their simultaneous activation prevented apoptosis. Activation of Raf was associated with cell cycle progression and prevention of caspase 3 activation. In fact, Raf activation by itself resulted in a more potent induction of cell cycle progression than when Raf and Akt were both activated but Akt activation was required for the long term growth of the cells. Treatment with either MEK or mTOR inhibitors suppressed proliferation and induced apoptosis and their combined treatment had a synergistic effect. Parental FL5.12 cells normally undergo G1 arrest after doxorubicin treatment and G2/M arrest after paclitaxel treament for 24 to 48 hrs. The FL/Akt:ER+Raf-1:AR cells are more resistant to these drugs and did not display as dramatic G1 or G2/M arrests after either doxorubicin or paclitaxel treatment respectively. Stable drug resistant FL/Akt:ER+Raf-1:AR cells were obtained by culturing the cells under limiting dilution conditions in the presence of 10 to 50 nM doxorubicin. These cells also displayed resistance to paclitaxel. Activation of Raf in the drug resistant cells increased the IC50 for doxorubicin 80-fold as compared to when it was not activated or when just Akt was activated. In contrast, activation of Raf in the unselected FL/Akt:ER+Raf-1:AR cells increased the IC50 for doxorubicin 10-fold. The drug resistant and drug sensitive cells displayed activation of p53 phosphorylation at S15 in response to doxorubicin indicating that the drug resistant cells did not become drug resistant by an altered p53 response. Both drug sensitive and resistant cells induced ERK upon Raf activation or doxorubicin treatment. Higher levels of the MRP-1 drug pump and p27Kip1 CDK inhibitor were detected in the drug resistant cells in response to Raf-1 activation than in the drug sensitive cells. The effects of MEK and mTOR inhibitors on the sensitivity to doxorubicin and paclitaxel were examined to determine the efficacy of combining classical chemotherapy with targeted therapy. MEK inhibitors efficiently induced apoptosis in the presence of doxorubicin or paclitaxel while the mTOR inhibitor rapamycin was less effective. Thus the Raf/MEK/ERK pathway is associated with cell cycle progression, prevention of caspase 3 activation and subsequent apoptosis and increase in MRP-1 and p27Kip1 expression in drug resistant cells. The Akt pathway is necessary for long term proliferation in the presence and absence of doxorubicin.