PHOSPHOROTHIOATE BCR-ABL ANTISENSE OLIGONUCLEOTIDES INDUCE CELL-DEATH, BUT FAIL TO REDUCE CELLULAR BCR-ABL PROTEIN-LEVELS

The bcr-abl oncogene is a fusion gene resulting from a reciprocal tran slocation which forms the hallmark of chronic myeloid leukemia (CML). Antisense oligonucleotides complementary to the two possible mRNA brea kpoints were found to inhibit cell growth of CML patient cells and cel l lines, but doubt exists about their specificity. In order to test th e specificity, phosphorothioate and 3'phosphorothioate capped antisens e BCR-ABL oligonucleotides of different length were used. Stability, c ellular uptake of oligonucleotides and effect on cell growth were stud ied in two CML cell lines, BV173 and LAMA-84. Phosphorothioate antisen se BCR-ABL oligonucleotides were most stable, showed the highest uptak e and induced cell death in BV173 but not in LAMA-84 cells. We selecte d the most effective antisense oligonucleotide for further analysis. T he BV173 and LAMA-84 cell lines do not express the normal c-abl protei n, we therefore used a c-abl specific monoclonal antibody for the dete ction of p210(bcr-abl) expression by flow cytometry. Dead cells found after treatment were gated out of analysis. Although BCR-ABL antisense oligonucleotides can induce apoptosis, no reduction of p210(bcr-abl) levels could be detected in living cells after treatment with antisens e oligonucleotides. We conclude that antisense mediated inhibition of translation of mRNA into p210(bcr-abl) is not the mechanism responsibl e for the induction of apoptosis in cell line BV173.