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bcl-2 Antisense Induces Apoptosis and Potentiates Activity of Both Cytotoxic Chemotherapy and Rituximab in Primary Chronic Lymphocytic Leukemia Cells

bcl-2 Antisense Induces Apoptosis and Potentiates Activity of Both Cytotoxic Chemotherapy and Rituximab in Primary Chronic Lymphocytic Leukemia Cells

Failure of treatment in chronic lymphocytic leukemia (CLL) is often characterized by increased expression of the antiapoptosis protein bcl-2. High levels of bcl-2 protein block the apoptotic death machinery at the mitochondrial level by maintaining the permeability transition pore (PTP) in the closed position. This study aimed to investigate the response of primary CLL cells to down-regulation of the bcl-2 protein by bcl-2 phosphorothioate antisense oligonucleotide (ASO) G3139 (Genasense).

Primary CLL cells were obtained from peripheral blood of 18 patients and were selected by Ficoll separation followed by CD19 magnetic bead selection. bcl-2 expression was confirmed by immunohistochemistry. Short-term liquid cultures in RPMI and 10% fetal calf serum were set up in microtiter format. All data sets were carried out in triplicate. Cells were treated optimally for 72 hours with ASO (0.5-5 µM), or control sense or nonsense oligonucleotides. Consistent bcl-2 down-regulation could be confirmed at 72 hours (41% ® 85%, P < .0001). Various doses of rituximab (Rituxan) (10-50 µg/mL) and dexamethasone were added to the culture. Assessment of the mitochondrial PTP response (JC-1, DiOC6) and apoptosis (cell membrane MC540) were then performed by flow analysis at 4, 24, and 48 hours.

The cells treated with bcl-2 antisense alone showed marked and highly significant apoptotic responses maximal at 5 µM (P < .0001, MC540 and P = .005, DiOC6 24 hours). This was more marked than responses seen in lymphoma cell lines (DoHH2 and SUD4). Control oligonucleotide-treated cells remained unaffected. The single-agent activity of bcl-2 antisense (P < .001) in this system was greater than fludarabine (Fludara) (P = .098) or cyclophosphamide (Cytoxan, Neosar) (P = .022). Cells subsequently treated in combination with rituximab (1, 1.5, 2.5, 5 µg/mL) showed enhanced response in combination with ASO in a dose-response relationship. Similar potentiation was seen when ASO was added to dexamethasone (1 µM) or fludarabine (50 µM) (P < .001).

CONCLUSION: In summary, down-regulation of bcl-2 protein by bcl-2 antisense ASO alone shows considerable apoptotic effect in primary CLL cells at a concentration easily achieved in vivo without toxicity. bcl-2 antisense was synergistic with rituximab. bcl-2 antisense ASO alone or in combination with current therapies should produce an enhanced clinical response in patients with CLL.

Click here to read Dr. Bruce Cheson's commentary on this abstract.

 
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