Dominant Role of Raf/MEK/ERK Signaling in Hematopoietic Cell Cycle Progression, Prevention of Apoptosis and Drug Resistance.

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades and p53 play critical roles in the transmission of signals from growth factor receptors to regulate gene expression, growth, malignant transformation and drug resistance. We have investigated the roles these two pathways play in cell cycle progression, prevention of apoptosis and drug resistance in cytokine-dependent FL5.12 hematopoietic cells and derivative FL/Akt:ER+Raf-1:AR cells conditionally transformed to grow in response to Akt and Raf activation. The effects of these two pathways on cell cycle progression and sensitivity to chemotherapeutic drugs could be more definitively examined in FL/Akt:ER+Raf-1:AR cells where it is possible to induce either Akt or Raf by themselves or together by the addition of tamoxifen or testosterone. Raf-1 was approximately 8-fold more effective than Akt in maintaining cell cycle progression and preventing apoptosis induced by the chemotherapeutic drugs. However, growth in the presence of chemotherapeutic drugs was enhanced significantly when both Akt and Raf were activated, documenting roles for both Raf and Akt in drug resistance. Drug resistant cells were isolated from both parental and derivative lines in the presence of 10 to 100 nM doxorubicin. Doxorubicin resistant cells were also cross resistant to paclitaxel and daunomycin but not to cisplatin and 5-fluoruracil which are transported by different drug pumps, however, drug resistance did not appear to be mediated by MDR-1/MRP-1 as neither elevated drug pump activity nor higher levels of proteins or mRNAs encoding these proteins were detected, while they were detected in appropriate controls. Drug resistant FL5.12 cells (which lacked any introduced gene) displayed elevated expression of activated ERK, hypersensitivity to Raf and MEK inhibitors and decreased caspase 3 cleavage following doxorubicin treatment. The drug resistance of FL5.12 cells could be further increased 5-fold by introduction of an activated MEK1 gene into the cells and likewise suppressed 8-fold by introduction of a dominant negative (DN) MEK1 gene. Introduction of activated MEK1 also resulted in a further 10-fold decrease in sensitivity to the induction of apoptosis stimulated by doxorubicin treatment as measured by Annexin V/PI binding and Caspase 3 cleavage. These results provide evidence for roles of MEK/ERK in hematopoietic drug resistance. p53 was also involved in drug sensitivity as introduction of DN p53 into FL5.12 cells, which normally express wild-type p53, increased their doxorubicin resistance approximately 3-fold and importantly sensitivity to MEK inhibitors was also determined to be p53-dependent. In summary, our results indicate that the Raf/MEK/ERK pathway is critical in chemotherapy-induced drug resistance and provide rationale for combinations of chemotherapy and Raf/MEK/ERK inhibitors.