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 oftencharacterized by increased expression of the antiapoptosis protein bcl-2. Highlevels of bcl-2 protein block the apoptotic death machinery at the mitochondriallevel by maintaining the permeability transition pore (PTP) in the closedposition. This study aimed to investigate the response of primary CLL cells todown-regulation of the bcl-2 protein by bcl-2 phosphorothioate antisenseoligonucleotide (ASO) G3139 (Genasense).

Primary CLL cells were obtained from peripheral blood of 18 patients and wereselected by Ficoll separation followed by CD19 magnetic bead selection. bcl-2expression was confirmed by immunohistochemistry. Short-term liquid cultures inRPMI and 10% fetal calf serum were set up in microtiter format. All data setswere carried out in triplicate. Cells were treated optimally for 72 hours withASO (0.5-5 µM), or control sense or nonsense oligonucleotides. Consistentbcl-2 down-regulation could be confirmed at 72 hours (41% ®85%, P < .0001).Various doses of rituximab (Rituxan) (10-50 µg/mL) and dexamethasone wereadded 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 highlysignificant apoptotic responses maximal at 5 µM (P < .0001, MC540 and P =.005, DiOC6 24 hours). This was more marked than responses seen in lymphoma celllines (DoHH2 and SUD4). Control oligonucleotide-treated cells remainedunaffected. The single-agent activity of bcl-2 antisense (P < .001) in thissystem was greater than fludarabine (Fludara) (P = .098) or cyclophosphamide(Cytoxan, Neosar) (P = .022). Cells subsequently treated in combination withrituximab (1, 1.5, 2.5, 5 µg/mL) showed enhanced response in combination withASO in a dose-response relationship. Similar potentiation was seen when ASO wasadded to dexamethasone (1 µM) or fludarabine (50 µM) (P < .001).

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

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