Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the B2A2 junction.
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.
Laboratoire de Greffe de Moelle, URA CNRS 1456, Université Bordeaux 2, France.
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Mahon
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Ripoche
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Pigeonnier
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Jazwiec
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Pigneux
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Moreau
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Reiffers
D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D017077:Culture Media, Conditioned; D004355:Drug Stability; D005091:Exons; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics* / Q000473:pathology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl