Specific Antisense Oligomer Anti Bcr-abl Junctions in Chronic Myeloid Leukemia: A Cell Cycle Analysis and CFU-GM Study

Abstract

Antisense oligonucleotides were used to determine the role of the BCR-ABL gene in the proliferation of chronic myeloid leukaemia (CML) clonogenic cells. Peripheral blood Philadelphia chromosome positive cells were obtained from eight CML patients at diagnosis (chronic phase = 7; accelerared phase = 1). Mononuclear cells were incubated with synthetic antisense 18-mer oligonucleotides complementary to the two different junctions b2a2 or b3a2. The type of junction (b2a2 or b3a2) was previously determined by RT-PCR techniques. Cells incubated for 12 to 14 hours with or without sense oligonucleotides served as controls. After incubation with oligonucleotides, the cell DNA synthesis was analysed by flow cytometry using the BrdUrd/DNA method and, the cell plating efficiency in methylcellulose was determined. In six of the seven patients in chronic phase, there was a significant inhibition of CFU-GM production which was only 68.4 ± 19%; (p <.01) of that found in controls. The S phase index, which depends upon the percentage of S phase cells ax well as the fluorescence intensity, was 48 ± 29% (p <.01) of the control values for the seven patients in chronic phase. Interestingly, for the only CML patient in accelerated phase, antisense oligomers had no inhibitory effect on either the production of CFU-GM or the number of S phase cells. In improving the specificity of oligomers, it might be usefull for gene-targeted anti-leukemic therapy and/or bone marrow purging.

Key Words:

• antisense oligonucleotide chronic myeloid leukaemia