The effect of bcr-abl antisense oligonucleotide on DNA synthesis and apoptosis in K562 chronic myeloid leukemia cells

Abstract

Mutations in oncogenes have traditionally been viewed as inducing malignancy by causing excessive cell division. However, an additional possible tumorigenic mechanism is inhibition of normally occurring apoptosis. We have studied the mechanism of action of bcr-abl in chronic myeloid leukemia (CML) by inhibiting its expression using antisense oligonucleotides. K562 cells, derived initially from a patient with CML, were incubated with 16 microM 3',5'-capped bcr-abl antisense phosphodiester 18mer targeting the bcr-abl junctional sequence. Antisense reduced cell number by day 5 by 44% +/- 2.5% S.E. compared to nonsense or no-oligomer controls. Compared to nonsense oligomer, antisense oligomer reduced [3H]thymidine incorporation by only 13% +/- 1%. By the more reliable bromodeoxyuridine incorporation method, antisense had no inhibiting effect on DNA synthesis. In contrast to its minimal effect on DNA synthesis, antisense had a large effect on apoptosis. At day 4, after 3 days of oligomer treatment, antisense increased the proportion of cells with less than 2 N DNA 2.5 +/- 0.3-fold compared to nonsense, as revealed by analysis of DNA distribution following propidium iodide-staining. After 3 days of oligomer treatment and 24 h of serum deprivation, antisense increased the proportion of cells with less than 2 N DNA even more, over 3.1 +/- 1.1-fold compared to nonsense. Because CML cells are resistant to the induction of apoptosis (as judged by DNA laddering on electrophoresis, which requires double-stranded breaks), we also assayed the binding of terminal deoxynucleotidyl transferase (TdT), which requires only single-stranded DNA breaks. Antisense treatment for 3 days increased TdT binding at day 4 by 16.4 +/- 8.7-fold. We conclude that, in CML, bcr-abl may lead to the accumulation of myeloid cells to a greater extent by inhibiting apoptosis than by increasing cell division. This bcr-abl induced inhibition of apoptosis may thwart chemotherapy and foster the accumulation of further mutations leading to the development of the blastic phase of the disease.