Inhibition of bcr-abl and/or c-abl gene expression by small interfering, double-stranded RNAs: cross-talk with cell proliferation factors and other oncogenes.
BACKGROUND
Short, 21-mer, double-stranded/small interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia (CML) with a potential also to target c-abl mRNA.


METHODS
ds/siRNAs were transfected into bcr-abl-positive K-562 cells (derived from blast-crisis) or bcr-abl-negative/c-abl-positive Jurkat cells (derived from acute lymphoblastic leukemia) using lipofectamine. ds/siRNAs intracellular uptake was detected by fluorescent confocal microscopy using fluorescein-labeled ds/siRNAs. The treatment was performed over 6 days with repetitive siRNA transfections. Efficiency of the siRNAs was determined 24 hours after single siRNA transfection and 6 days after repetitive siRNA transfections.


RESULTS
Two of the designed ds/siRNAs decreased the target mRNA levels markedly (determined by reverse transcriptase-polymerase chain reaction analysis) and bcr-abl/c-abl oncoproteins (determined by flow cytometry using Fluor-488-labeled, anti-c-abl antibody as well as by Western blot analysis). These sequences also inhibited protein tyrosine kinase activity significantly and suppressed cell proliferation. One of the three selected ds/siRNAs expressed only slight effects on the bcr-abl/c-abl mRNA in K-562 cells (but not on the oncoprotein level), on protein tyrosine kinase activity, and on cell proliferation. The combination of the three ds/siRNA constructs provoked stronger decreases in bcr-abl/c-abl mRNAs and their respective oncoproteins and produced the strongest suppression of cell proliferation.


CONCLUSIONS
The cross-talk between siRNA interference of bcr-abl oncogene and the expression of several apoptotic/antiapoptotic factors, cell proliferation factors, and other oncogenes exists and it was determined by microarray analysis in K-562 cells that were treated over 6 days.
Ohba
Hideki
H
Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology, Takamatsu, Kagawa, Japan.
Zhelev
Zhivko
Z
Bakalova
Rumiana
R
Ewis
Ashraf
A
Omori
Toshiro
T
Ishikawa
Mitsuru
M
Shinohara
Yasuo
Y
Baba
Yoshinobu
Y
D001483:Base Sequence; D002455:Cell Division; D045744:Cell Line, Tumor; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D015870:Gene Expression; D016313:Genes, abl; D006801:Humans; D009857:Oncogenes; D040081:Protein Array Analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl