THE EFFECT OF BCR-ABL ANTISENSE OLIGONUCLEOTIDE ON DNA-SYNTHESIS AND APOPTOSIS IN K562 CHRONIC MYELOID-LEUKEMIA CELLS

Mutations in oncogenes have traditionally been viewed as inducing mali gnancy by causing excessive cell division. However, an additional poss ible tumorigenic mechanism is inhibition of normally occurring apoptos is. We have studied the mechanism of action of bcr-abl in chronic myel oid leukemia (CML) by inhibiting its expression using antisense oligon ucleotides. K562 cells, derived initially from a patient with CML, wer e incubated with 16 mu M 3',5'-capped bcr-abl antisense phosphodiester 18mer targeting the bcr-abl junctional sequence, Antisense reduced ce ll number by day 5 by 44% +/- 2.5% S.E. compared to nonsense or no-oli gomer controls. Compared to nonsense oligomer, antisense oligomer redu ced [H-3]thymidine incorporation by only 13%+/-1%. By the more reliabl e bromodeoxyuridine incorporation method, antisense had no inhibiting effect on DNA synthesis. In contrast to its minimal effect on DNA synt hesis, antisense had a large effect on apoptosis. At day 4, after 3 da ys of oligomer treatment, antisense increased the proportion of cells with less than 2 N DNA 2.5+/-0.3-fold compared to nonsense, as reveale d by analysis of DNA distribution following propidium iodide-staining. After 3 days of oligomer treatment and 24 h of serum deprivation, ant isense increased the proportion of cells with less than 2 N DNA even m ore, over 3.1+/-1.1-fold compared to nonsense. Because CML cells are r esistant to the induction of apoptosis (as judged by DNA laddering on electrophoresis, which requires double-stranded breaks), we also assay ed the binding of terminal deoxynucleotidyl transferase (TdT), which r equires only single-stranded DNA breaks. Antisense treatment for 3 day s 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 gr eater 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 develo pment of the blastic phase of the disease. Copyright (C) 1996 Elsevier Science Ltd.