Bcl-x_S Is an Important Pro-Apoptotic Bcl-2 Family Member in Chronic Lymphocytic Leukaemia and Its Expression Is Regulated by SAM68, hnRNP-F and hnRNP-H.

We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-x_S is induced in chronic lymphocytic leukaemia cells during spontaneous and drug induced apoptosis to a greater extent than other pro-apoptotic Bcl-2 family members including Bim, Puma and Noxa. Puma, Noxa and also Bid were induced strongly by stimulation with CD40 ligand, in culture conditions causing CLL cell proliferation and survival, and associated with strong induction of the pro-survival Bcl-x isoform, Bcl-x_L. Immunohistochemistry demonstrates Bcl-x_L expression in lymph node proliferation centres. We, therefore, conclude that splicing of Bcl-x mRNA is important for regulation of CLL survival such that Bcl-x_L is required during proliferation in the lymph node or bone marrow microenvironment, but Bcl-x_S is induced during apoptosis. Splicing of Bcl-x mRNA in HeLa cells is known to be controlled by RNA binding proteins including SAM68, heterogeneous ribonucleoprotein-F (hnRNP-F) and hnRNP-H, but expression and regulation of these molecules has not been investigated in CLL. We have demonstrated that expression of hnRNP-F and hnRNP-H decline in association with an increase in Bcl-xS expression. Conversely expression of the ribonucleoproteins is maintained (relative to expression in freshly isolated peripheral blood CLL cells) or increases on CD40 stimulation. SAM68 showed a similar pattern of expression with increased expression on CD40 stimulation and decreased under conditions of apoptosis. Phosphorylation of SAM68 can control the RNA binding activity of this molecule. We immunoprecipitated SAM68 from freshly isolated CLL cells and cells that had been cultured with CD40 ligand (to promote proliferation) or on tissue culture plastic (to promote apoptosis) for 2 days. Westerns of the immunoprecipitated protein were probed with an anti-phosphotyrosine antibody. We found variable patterns: phosphorylation was uniformly present in cells cultured on plastic (4/4 cases) but was only detectable in freshly isolated cells in 1 case and on CD40 stimulation in 2 cases. We conclude that the RNA binding proteins hnRNP-F, hnRNP-H and SAM68 have important roles in controlling Bcl-x splicing in CLL cells, and that their effects are largely due to variation in expression level rather than phosphorylation. Strategies to bias production of pro-apoptotic Bcl-x_S using siRNA either directed against Bcl-x_L mRNA or against RNA binding proteins may be therapeutically useful.