Specific inhibition of bcr-abl gene expression by small interfering RNA

Abstract

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34 ؉ cells, respectively. Finally, antibcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells. IntroductionRNA interference (RNAi) describes a highly conserved regulatory mechanism that mediates sequence-specific posttranscriptional gene silencing initiated by double-stranded RNA (dsRNA). 1-3 The RNase III enzyme Dicer processes dsRNA into approximately 22-nucleotide (nt) small interfering RNAs (siRNAs) 4 that serve as guide sequences to induce targetspecific mRNA cleavage by the RNA-induced silencing complex RISC. 5 In plants and Caenorhabditis elegans, RNAi may involve the amplification of dsRNA by an RNA-dependent RNA polymerase (RdRP), 6 and it enables systemic, long-term, and heritable gene silencing. In contrast, RNAi in Drosophila and mammals seems cell autonomous, transient, and nonheritable. Because exogenous application of siRNAs can efficiently trigger RNAi in mammalian cells, 7,8 siRNAs are increasingly used in transient (co)transfection assays to modulate gene expression in mammalian cells, including human cells. [9][10][11][12] Fusion transcripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi approach. The Philadelphia (Ph) translocation t(9;22)(q34;q11) generates the bcr-abl fusion gene characteristic for chronic myelogenous leukemia (CML) and Phϩ acute lymphoblastic leukemia (ALL). 13 Bcr-abl encodes a constitutively active cytoplasmic tyrosine kinase that is necessary and sufficient to induce and maintain leukemic transformation. [14][15][16] We demonstrate that anti-bcr-abl siRNAs specifically inhibit bcr-abl mRNA expression in hematopoietic cell lines and primary CML cells. They reduce BCR-ABL protein expression and inhibit BCR-ABL-dependent, but not cytokine-dependent, cell proliferation. Therefore, anti-bcr-abl siRNAs may allow further analysis of BCR-ABL functions and, eventually, may lead to RNAi-based therapeutics. Study design siRNAsTwenty-one-nucleotide single-stranded RNAs directed against the fusion sequence of bcr-abl were chemically synthesized (BioSpring, Frankfurt, Germany) ( Figure 1A). The sense and antisense sequences were: b3a2_1, 5Ј-GCAGAGUUCAAAAGCCCUUdTdT-3Ј; b3a2_3, 5Ј-AGCAGAGUUCAAAAGCCCUdTdT-3Ј; and b3a2_1, 5Ј-AAGGGCU...

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.…”

“…Caspase 3 and Bcr-Abl shRNAi siRNA-expressing plasmids were constructed targeting a specific region of human caspase 3 (5 0 -AGTGAAGCAAAT-CAGAAAC-3 0 ) (Carlile et al, 2004) or human BCR-ABL (5 0 -AGCAGAGTTCAAAAGCCCT-3 0 ) (Scherr et al, 2003). The oligonucleotides purchased from Eurogentec s.a. (Seraing, Belgium) were annealed and cloned into the pTER vector (a generous gift from Dr Hans Clevers at the Hubrecht Laboratory, Utrecht, The Netherlands) using the BglII and HindIII restriction sites (van de Wetering et al, 2003).…”

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells

“…12 Finally, the successful use of small interfering RNAs (siRNAs) to downregulate gene expression in several model systems [13][14][15][16][17][18] has led to many attempts to explore this methodology in potentially therapeutic settings. 19,20 Along this line, several recent studies have documented: (a) successful downregulation of BCR-ABL expression, resulting in increased apoptosis and decreased proliferation, in chronic myeloid leukemia cells; 21,22 (b) the MDR1 downregulation followed by chemosensitization of human pancreatic and gastric cell lines; 23 (c) combined inhibition of Bcl-2 and Raf-1 in human myeloid leukemia cells resulting in induction of apoptosis and chemosensitization. 24 Bearing in mind that RNAi is feasible in MCF-7 cell line [25][26][27] and that these cells are known to be relatively resistant to etoposide and doxorubicin, 28,29 the purpose of this study was to investigate if specific downregulation of bcl-2 or xIAP gene expression by RNAi sensitized MCF-7 cells to those chemotherapeutic drugs.…”

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

“…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].…”