Investigations of antisense oligonucleotides targeted against bcl-2 RNAs

Expression of the bcl-2 gene becomes deregulated in many non-Hodgkin lymphomas as the result of t(14;18) chromosomal translocations. Because bcl-2 regulates the survival of cells, and because its over-expression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation, this gene and its mRNA and protein products represent ideal targets for designing novel therapeutic strategies for the treatment of cancer. Here we describe the effects of an 18-mer phosphodiester oligonucleotide that is complementary to the first 6 codons of the bcl-2 mRNA's open reading frame. When tested for inhibition of in vitro protein synthesis using RNAse-H-supplemented reticulocyte lysates and RNA prepared by in vitro transcription of a human bcl-2 cDNA, the bcl-2 antisense (AS) oligomer completely abolished Bcl-2 protein production at 10 microM, but had no effect on the in vitro translation of a chicken bcl-2 RNA that contained three mismatches relative to the oligomer binding site on the human bcl-2 RNA. A control 18-mer having the same base composition as the AS oligomer but with scrambled order (SC) was not inhibitory. Addition of AS and SC oligomers to cultures of a NIH-3T3 fibroblast cell line that had been stably infected with a recombinant retrovirus containing the same human bcl-2 cDNA used for in vitro transcription/translation experiments revealed concentration-dependent reductions in the relative levels of the 26-kD human Bcl-2 protein (as determined by immunoblotting) by the AS but not by the SC oligomer. Similar results were obtained when AS and SC oligomers were applied to a t(14;18)-containing lymphoma cell line SU-DHL-4 that was cultured in low-serum media. When used at 200 microM, the bcl-2 AS oligomer produced 84-95% reductions in Bcl-2 protein levels in SU-DHL-4 cells but had relatively little effect on the levels of other mitochondrial control proteins, suggesting that the inhibitory effects were specific. Treatment of SU-DHL-4 cells with AS oligomer lead to essentially complete loss of bcl-2 mRNA from cells within 1 day of addition to cultures, but presumably because of the long half-life of the Bcl-2 protein (approximately 14 h), commensurate reductions in Bcl-2 protein levels did not occur until 3 days.(ABSTRACT TRUNCATED AT 400 WORDS)