Antisense-mediated Inhibition of BCL2 Protooncogene Expression and Leukemic Cell Growth and Survival: Comparisons of Phosphodiester and Phosphorothioate Oligodeoxynucleotides

Abstract

Antisense oligodeoxynucleotides specific for sequences in mRNAs from the B-cell lymphoma/leukemia-2 (BCL2) gene were used to inhibit the growth in culture of a human leukemia cell line, 697. Normal phosphodiester (PO) and nuclease-resistant phosphorothioate (PS) oligodeoxynucleotides were compared with regard to specificity, potency, and kinetics. Both PO and PS antisense BCL2 oligodeoxynucleotides were specific inhibitors of cellular proliferation, since sense versions of these synthetic DNAs were inactive at similar concentrations. Specificity was further confirmed by quantitative immunofluorescence studies, showing that PO and PS antisense BCL2 oligodeoxynucleotides (when used at appropriate concentrations) reduced levels of BCL2 protein without influencing expression of HLA-DR and other control antigens. The onset of inhibition by PO oligodeoxynucleotides was faster, with reductions in cell numbers occurring within 1 day of addition to cultures, in contrast to phosphorothioates, which were ineffective until 3–4 days. Phosphorothioates were more potent that phosphodiesters, however, with half-maximal inhibition of leukemic cell growth occurring at concentrations 5–10 times lower. As expected from previous studies demonstrating the importance of BCL2 for regulating lymphoid cell survival, BCL2 antisense oligodeoxynucleotides also led to 697 leukemic cell death through sequence-specific mechanisms, with reductions in cellular viability generally lagging the inhibitory effects on cellular growth by about 2 days. Taken together, these data indicate that PO and PS oligodeoxynucleotides targeted against the human BCL2 protooncogene can be sequencespecific inhibitors of leukemic cell growth and survival.