Inhibition of Bcl-2 by Anti-Sense Oligonucleotide and siRNA in Specimens from Patients with Waldenström’s Macroglobulinemia (WM).

We have previously shown that Bcl-2 antisense oligonucleotides (AS-ODN) specifically downregulate protein and message for Bcl-2 in the WSU-WM Waldenström’s Macroglobulinemia cell line ( a gift from Dr. Al-Katib, Wayne State University). This downregulation is associated with decreased cell viability and appeared to have some additive cell killing with chemotherapeutic agents active against this line. In the current study we sought to replicate these findings in patient specimens, and to test the specificity of Bcl-2 as the target, using siRNA specifically designed against Bcl-2. Bone marrow specimens from 2 patients with WM having more than 90% marrow involvement with lymphoplasmacytoid cells, were obtained with informed consent. Lymphoid cells were separated using Histopaque(Sigma). Cells were washed with Opti-MEM media (Gibco) and seeded at 100x10⁴ cells per well and transfected with 15μg/ml Lipofectin (Invitrogen) and 400nm oligonucleotide or 25nM concentration of siRNA and corresponding control molecules. The total incubation time was 72 hours. Cells were lysed and Western blots performed. G3139 (Genta), an 18mer phosphorothioate oligodeoxynucleotide targeted to the initiation codon region of the Bcl-2 mRNA and G4126 (a 2-base mismatch control ODN) were tested. G3139 was able to specifically downregulate Bcl-2 protein expression by 75% with no changes in expression of other apoptotic family proteins including bcl-xL, bax and PKC-α. These findings were similar to those in the WSU-WM cell line and were associated with increased cell death. Levels of tubulin, as a control protein, were not affected. D6, an siRNA designed against Bcl-2 (Dharmacon) also was able to downregulate Bcl-2 protein by Western blotting, while D3, a control siRNA, was not. These results replicated those seen with the WSU-WM cell line. RT-PCR of RNA from these cells using primers for bcl-2 mRNA confirmed that message is also inhibited after 3 days of treatment with specific (but not control) AS-ODN and siRNA. Of interest, expression of p21 (Cip-1), a cyclin-dependent kinase inhibitor, was upregulated >10-fold with the downregulation of Bcl-2 by both AS-ODN and siRNA for Bcl-2. p21 may play a role not only as a tumor suppressor, but also as an inhibitor of cell proliferation and therefore apoptosis. The possibility that p21 acts as an additional inhibitor of apoptosis in WM is currently under study. These findings confirm that in WM patient specimens as well as cell lines, specific downregulation of Bcl-2 is achievable with antisense ODN and siRNA and is associated with inhibition of cell growth. Both of these agents may have clinical applicability in this disease. The WSU-WM cell line appears to be an excellent model for the study of apoptosis in WM, as studies in patient specimens mirror the cell line findings. The role of p21 as a potential secondary inhibitor of apoptosis after inhibition of Bcl-2 in WM is currently under study as a potential target for combined biologic therapy in this disease.