Combination of Bcr-Abl-specific RNA Interference with lmatinib Treatment in the K-562 Cell Line

Author

Benjamin Eugene Baker, University of Tennessee, Knoxville

Date of Award

5-2004

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

Albert T. Ichiki

Committee Members

Robert L. Donnell, Daniel P. Kestler, Karla J. Matteson

Abstract

RNA interference (RNAi) involves the specific repression of the translation of a gene through mRNA degradation. Its application has been extended to a variety of studies both in vitro and in vivo. The Bcr-Abl translocation is the cytogenetic marker for chronic myelogenous leukemia (CML) and has been studied extensively. The K-562 cell line possesses the Bcr-Abl fusion gene and has been established as a model for RNAi. Imatinib mesylate (Gleeved) is a proven specific inhibitor of the Bcr-Abl tyrosine kinase. The aim of this study was to combine K-562 cells primes with short interfering RNA (siRNA) targeting the Bcr-Abl fusion site with treatment with Gleevec. Two different preperations of siRNA: homogenous-synthetic Bcr-Abl and heterogeneous-transcribed-digested Bcr-Abl were used to silence the Bcr-Abl fusion gene. The synthetic siRNA consisted of a homogenous mixture of 21nt long double stranded RNA duplexes specific for the Bcr-Abl fusion site. The transcribed-digested Bcr-Abl siRNA were generated using an in vitro transcription method producing a 450 bp cloned fragment with the Bcr-Abl fusion site in the center of the cloned region. This cloned fragment was further digested with RNAse III to produce a heterogeneous mizture of Bcr-Abl 19-21nt siRNA duplexes. We demonstrated a 70% down-regulation of the Bcr-Abl mRNA through real time PCR and RT-PCR as well as a 75% down-regulation of the Bcl-Abl and Bcl-_XL proteins in K-562 cells transfected with synthetic Bcr-Abl siRNA. The IC₅₀ of Gleevec alone in the K-562 subline F₁ was lowered from 0.2μM to 0.06μM in cells transfected with both preparations of Bcr-Abl siRNA, while no effect was observed in an irrelevant siRNA control. This suggests an additive relationship between Gleevec and Bcr-Abl-specific-siRNA treated cells. An increase in apoptosis was also seen in K-562 cells primes with Bcr-Abl siRNA and treated with Gleevec indicating the additive relationship between Gleevec and Bcr-Abl siRNA. These results indicate that priming K-562 cells with Bcr-Abl siRNA correlate with a decrease in the effective dose of Gleevec required to inhibit the Bcr-Abl protein.

Recommended Citation

Baker, Benjamin Eugene, "Combination of Bcr-Abl-specific RNA Interference with lmatinib Treatment in the K-562 Cell Line. " Master's Thesis, University of Tennessee, 2004.
https://trace.tennessee.edu/utk_gradthes/4657