EFFECT OF BCR-ABL OLIGODEOXYNUCLEOTIDES ON THE CLONOGENIC GROWTH OF CHRONIC MYELOGENOUS LEUKEMIA-CELLS

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]OD Ns) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukae mia (CML). Experiments included the evaluation of the anti-leukaemic e ffect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34 (+) cells, evaluation of incubation time and correlation of colony gro wth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34(+) cell p opulations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colo ny recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34(+) cells was compa rable to that observed on mononuclear cells in terms of colony inhibit ion; however, a higher proportion of cases showed a significant inhibi tion of colony formation. In comparison with the 26-mer antisense [S]O DNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was les s evident on mononuclear cells and comparable on CD34(+) cells; howeve r, a more specific effect was evident on both target cells. Hybridizat ion experiments confirmed a partial cross-reactivity when the 26-mer O DNs were hybridized with their complementary sequence; this did not oc cur when 16-mer ODNs were similarly tested. Experiments aimed at evalu ating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to t hat measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs f rom day 1 to day 5. The accumulation of [S]ODNs correlated with a mark ed down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The downregulation of p210(bcr-abl) levels fo llowing treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expres sion. These studies confirm that, under optimal conditions of target c ell culture and ODN size, antisense [S]ODNs complementary to the bcr-a bl junction have specific anti-leukaemic effects.