title: Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells.
abstract: Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA)-modified oligonucleotides to silence BCR/ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79 degrees C and 75 degrees C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts ( approximately 20% of control) and protein p210(BCR/ABL) ( approximately 30% of control). Moreover, the antisense oligonucleotides suppressed cell growth up to 40% of control and induced apoptosis, as indicated by the increase of caspase-3/7 activity in the treated cells. Finally, the b2a2-specific antisense gapmer used in combination with STI571 (imatinib mesylate), a tyrosine kinase inhibitor of p210(BCR/ABL), produced an enhanced antiproliferative effect in KYO-1 cells, which compared with K562 cells are refractory to STI571. The data of this study support the application of BCR/ABL antisense LNA-DNA gapmers, used either alone or in combination with STI571, as potential antileukemic agents.
authors.affiliation: Department of Biomedical Sciences and Technologies, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Rapozzi
authors.forename: Susanna
authors.initials: S
authors.lastname: Cogoi
authors.forename: Luigi E
authors.initials: LE
authors.lastname: Xodo
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000737:chemistry / Q000627:therapeutic use*; D017209:Apoptosis / Q000235:genetics; D001549:Benzamides; D049109:Cell Proliferation / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000627:therapeutic use*; D010879:Piperazines / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000627:therapeutic use; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D016914:Ribonuclease H / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D001549:Benzamides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; C477371:locked nucleic acid; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D016914:Ribonuclease H