title: In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.
abstract: Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.
authors.affiliation: Centre of Biotechnology, University of Urbino, Fano, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Caprodossi
authors.forename: Luca
authors.initials: L
authors.lastname: Galluzzi
authors.forename: Simona
authors.initials: S
authors.lastname: Biagetti
authors.forename: Giulia
authors.initials: G
authors.lastname: Della Chiara
authors.forename: Pier Giuseppe
authors.initials: PG
authors.lastname: Pelicci
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
authors.forename: Mirco
authors.initials: M
authors.lastname: Fanelli
mesh_terms: D002454:Cell Differentiation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000235:genetics; D008081:Liposomes / Q000737:chemistry; D009696:Nucleic Acids / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D015514:Oncogene Proteins, Fusion / Q000737:chemistry; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014162:Transfection; D014178:Translocation, Genetic; D020298:U937 Cells
chemical_list: D008081:Liposomes; D009696:Nucleic Acids; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein