Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD

Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival.

Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry.

Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases.

Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.