Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.
We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations 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 colony 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 comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.
de Fabritiis
P
P
Haematology, University La Sapienza, Rome, Italy.
Skorski
T
T
De Propris
M S
MS
Paggi
M G
MG
Nieborowska-Skorska
M
M
Lisci
A
A
Buffolino
S
S
Campbell
K
K
Geiser
T
T
Calabretta
B
B
D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001483:Base Sequence; D001692:Biological Transport; D001853:Bone Marrow / Q000473:pathology; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects*; D002999:Clone Cells; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D007700:Kinetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D013873:Thionucleotides; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018952:Antigens, CD34; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D016044:Fusion Proteins, bcr-abl