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Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression
Benjamin E Baker1, Daniel P Kestler, Albert T Ichiki
1Department of Medical Genetics, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Box U-2, Knoxville, TN 37920, USA.
Abstract:
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.
Insights
Small interfering RNA (siRNA) effectively targets Bcr-Abl transcripts in K-562 cells. This RNA interference approach enhances Gleevec
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Cancer Therapeutics
Background:
- RNA interference (RNAi) offers a mechanism for gene expression control via mRNA degradation.
- RNAi has been applied to modulate various human transcripts in cellular models.
Purpose of the Study:
- To deliver Bcr-Abl specific small interfering RNA (siRNA) using a liposome-based method.
- To demonstrate the specificity of Bcr-Abl siRNA in K-562 cells, alone and combined with Gleevec.
Main Methods:
- Utilized both synthetic and recombinant Bcr-Abl siRNA targeting the fusion site.
- Transfected K-562 cells with siRNA using RNAiFect, achieving >90% efficiency.
- Quantified Bcr-Abl transcript inhibition via RT-PCR and real-time PCR.
Main Results:
- Bcr-Abl transcript levels were significantly reduced by both siRNA types.
- Transfection with Bcr-Abl siRNA reduced Gleevec's IC50 by over 3-fold in K-562 cells.
- Control siRNA showed no effect, confirming Bcr-Abl siRNA specificity.
Conclusions:
- RNAiFect efficiently delivers Bcr-Abl siRNA into K-562 cells.
- Bcr-Abl siRNA enhances Gleevec efficacy in inhibiting Bcr-Abl protein in K-562 cells.
