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
✍ Scribed by Hideki Ohba; Zhivko Zhelev; Rumiana Bakalova; Ashraf Ewis; Toshiro Omori; Mitsuru Ishikawa; Yasuo Shinohara; Yoshinobu Baba
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 789 KB
- Volume
- 101
- Category
- Article
- ISSN
- 0008-543X
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✦ Synopsis
Abstract
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/__siRNA__s. The treatment was performed over 6 days with repetitive siRNA transfections. Efficiency of the __siRNA__s was determined 24 hours after single siRNA transfection and 6 days after repetitive siRNA transfections.
RESULTS
Two of the designed ds/__siRNA__s 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/__siRNA__s 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. Cancer 2004. © 2004 American Cancer Society.