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.

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/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.

• Authors: Hideki Ohba, Zhivko Zhelev, Rumiana Bakalova, Ashraf Ewis, Toshiro Omori, Mitsuru Ishikawa, Yasuo Shinohara, Yoshinobu Baba
• Journal: Cancer (Cancer) Vol. 101 Issue 6 Pg. 1390-403 (Sep 15 2004) ISSN: 0008-543X [Print] United States
• PMID: 15368327 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2004 American Cancer Society.

Chemical References

• RNA, Double-Stranded
• RNA, Messenger
• RNA, Small Interfering
• Protein-Tyrosine Kinases
• Fusion Proteins, bcr-abl

Topics

• Base Sequence
• Cell Division
• Cell Line, Tumor
• Fusion Proteins, bcr-abl (metabolism)
• Gene Expression
• Genes, abl
• Humans
• Oncogenes
• Protein Array Analysis
• Protein-Tyrosine Kinases (metabolism)
• RNA, Double-Stranded (pharmacology)
• RNA, Messenger (metabolism)
• RNA, Small Interfering (pharmacology)
• Reverse Transcriptase Polymerase Chain Reaction
• Transfection