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

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.