Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571)

Abstract

Bcr-Abl proteins are effective inducers of the leukemic phenotype in chronic myeloid leukemia (CML) and distinct variants of acute lymphoblastic leukemia (ALL). Targeting bcr-abl by treatment with the selective tyrosine kinase inhibitor imatinib has proved to be highly efficient for controlling leukemic growth. However, it is unclear whether imatinib is sufficient to eradicate the disease because of primary or secondary resistance of leukemic cells. Therefore, targeting BcrAbl with an alternative approach is of great interest. We demonstrate that RNA interference (RNAi) with a breakpointspecific short-interfering RNA (

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression

“…This suggests that the number of unique Bcr-Abl interfering motifs are limited and, perhaps, localized. Downregulation of the Bcr-Abl mRNA and protein required multiple transfections, as previously reported [6], and, as suggested, was likely due to siRNA dilution through cell divisions and the long half-life of the Bcr-Abl protein (>48 h). No detectable decrease of the Bcr-Abl protein was found in cells transfected with the rBcr-Abl siRNA preparations.…”

“…Two transfections were reported to be required to down-regulate the Bcr-Abl protein in K-562 cells [6]. Western blot analysis showed that synBcr-Abl siRNA lowered the Bcr-Abl protein compared to the irrelevant siRNA control ( Figure 3A).…”

“…This suggests that the Bcr-Abl anti-apoptotic pathway had been abrogated. Determination of Bcr-Abl protein modulation by Western analysis is difficult in K-562 cells since the protein has been shown to have a long half-life [3,[6][7]. Thus, the ability to visualize significant modulation of Bcr-Abl polypeptide could be buffered by the longevity of the Bcr-Abl protein in K-562 cells.…”

“…Since synthesis methods of siRNA have steadily improved, recent focus has been placed on the delivery of RNAi for both in vitro and in vivo applications. The Bcr-Abl translocation present in chronic myelogenous leukemia (CML) has been a target for RNAi studies and has been silenced using lipid-base amine liposome [3][4][5], electroporation [6,7], and vector-based RNAi constructs [8,9]. The K-562 cell line expresses high levels of the Bcr-Abl fusion transcript and expresses high amounts of Bcr-Abl protein, much greater than primary CML cells, which makes it a rigorous model for testing in vitro silencing of Bcr-Abl [10].…”

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition

“…Several recent studies have demonstrated the ability of small interfering RNAs to reduce Bcr-Abl expression in CD34 þ cells from CML patients (Scherr et al, 2003;Rangatia and Bonnet, 2005) and different established cell lines including the K562 CML cell line (Wohlbold et al, 2003;Rangatia and Bonnet, 2005;Withey et al, 2005), conferring increased sensitivity to apoptosis. So we thought to determine, using inducible shRNA interference, whether Bcr-Abl silencing could mimic the effect of imatinib and PD166326 on K562 cell death and differentiation.…”