Effects of BCR-ABL antisense oligonucleotides (AS-ODN) on human chronic myeloid leukemic cells: AS-ODN as effective purging agents.
We examined phosphorothioate oligodeoxyribonucleotides (ODNs) directed against bcr in exon 3 or exon 2, which are rearranged with exon 2 of abl (B3A2 and B2A2) at t(9;22) of chronic myelogenous leukemia (CML). Since these ODNs are designed to be CML cell specific, we studied their effects on the human CML cell line K562, which is known to have B3A2 rearrangement, and leukemic cells from patients, as well as normal hematopoietic stem cells in vitro. In vitro experiments were performed to determine a potential role of these two ODNs as ex vivo purging agents. Incubation of B3A2 antisense at 40, 80, and 120 micrograms/ml with K562 CML cells for 72 hours at 37 degrees C resulted in 44%, 56%, and 63% reduction of CFU-L as compared to controls. In contrast, B3A2 sense and B2A2 antisense had no significant growth inhibitory effect on K562 cells. Incubation of B3A2 and B2A2 antisense ODNs at concentration of 80 micrograms/ml at 37 degrees C for 36 hours with normal peripheral blood stem/progenitor cells (PBSC) resulted in 124% and 98% CFU-GM formation as compared to untreated controls, respectively. However, incubation of PBSC with B3A2 and B2A2 sense-ODNs resulted in a 22% and 44% reduction in CFU-GM, respectively. In order to determine the ex vivo purging effects of bcr-abl ODNs, the K562 cells were mixed with PBSC from normal donors at a ratio of 1:20 (CML:PBSC). The mixture of cells was then incubated with B3A2 antisense at 80 micrograms/ml for 36 hrs at 37 degrees C. After incubation, no CML cells were detected by fluorescence in situ hybridization (FISH) as compared to untreated controls. These results were confirmed by RT-PCR using bcr-abl primers and mRNA isolated from the mixture of cells. Further, these results support the hypothesis that bcr-abl antisense ODNs are potentially effective agents for ex vivo purging of autologous stem cells before transplantation to eliminate/reduce the burden of leukemic cells. No significant toxicity to normal hematopoietic stem/progenitor cell population by the bcr-abl antisense ODNs was observed. Although unanticipated reductions in normal hematopoietic progenitor cells (CFU-GM) were observed with sense ODNs, no reduction in CFU-GM was observed with unrelated phosphorothioate ODN controls.
Wu
A G
AG
Department of Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha 68198-3330, USA.
Joshi
S S
SS
Chan
W C
WC
Iversen
P L
PL
Jackson
J D
JD
Kessinger
A
A
Pirruccello
S J
SJ
Sanger
W G
WG
Sharp
J G
JG
Verbik
D J
DJ
D001483:Base Sequence; D016465:Bone Marrow Purging / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003114:Colony-Forming Units Assay; D017931:DNA Primers; D005091:Exons; D016044:Fusion Proteins, bcr-abl / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D004915:Leukemia, Erythroblastic, Acute; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D013873:Thionucleotides; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D016044:Fusion Proteins, bcr-abl