Phosphorothioate BCR-ABL antisense oligonucleotides induce cell death, but fail to reduce cellular bcr-abl protein levels.
The bcr-abl oncogene is a fusion gene resulting from a reciprocal translocation which forms the hallmark of chronic myeloid leukemia (CML). Antisense oligonucleotides complementary to the two possible mRNA breakpoints were found to inhibit cell growth of CML patient cells and cell lines, but doubt exists about their specificity. In order to test the specificity, phosphorothioate and 3' phosphorothioate capped antisense BCR-ABL oligonucleotides of different length were used. Stability, cellular uptake of oligonucleotides and effect on cell growth were studied in two CML cell lines, BV173 and LAMA-84. Phosphorothioate antisense BCR-ABL oligonucleotides were most stable, showed the highest uptake and induced cell death in BV173 but not in LAMA-84 cells. We selected the most effective antisense oligonucleotide for further analysis. The BV173 and LAMA-84 cell lines do not express the normal c-abl protein, we therefore used a c-abl specific monoclonal antibody for the detection of p210bcr-abl expression by flow cytometry. Dead cells found after treatment were gated out of analysis. Although BCR-ABL antisense oligonucleotides can induce apoptosis, no reduction of p210bcr-abl levels could be detected in living cells after treatment with antisense oligonucleotides. We conclude that antisense mediated inhibition of translation of mRNA into p210bcr-abl is not the mechanism responsible for the induction of apoptosis in cell line BV173.
Division of Hematology, University Hospital St. Radboud, Nijmegen, The Netherlands.
T F
TF
Smetsers
L T
LT
van de Locht
A H
AH
Pennings
H M
HM
Wessels
T M
TM
de Witte
E J
EJ
Mensink
D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl