Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.
RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFect, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.
Department of Medical Genetics, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Box U-2, Knoxville, TN 37920, USA.
Benjamin E
BE
Baker
Daniel P
DP
Kestler
Albert T
AT
Ichiki
D001483:Base Sequence; D001549:Benzamides; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D047468:Immunoprecipitation; D020128:Inhibitory Concentration 50; D008081:Liposomes; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010879:Piperazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection
D001549:Benzamides; D017931:DNA Primers; D008081:Liposomes; D010879:Piperazines; D011743:Pyrimidines; D034741:RNA, Small Interfering; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl