title: Selective inhibition of inducible cyclo-oxygenase-2 expression by antisense peptide nucleic acids in intact murine macrophages.
abstract: Prostaglandins are important molecules involved in inflammation and immunomodulation. The rate-limiting step in the synthesis of these potent mediators is the expression of the enzyme cyclo-oxygenase (COX). The isoform responsible, COX-2, is encoded by an immediate-early gene induced by various pro-inflammatory agents in macrophages. Selective blockade of COX-2 by the use of an antisense strategy would overcome the undesirable side effects of conventional inhibitors. Here we describe cellular internalization and activity of a novel class of oligonucleotide analogues named peptide nucleic acids (PNAs) as inhibitors of COX-2 translation. In particular, we designed two antisense murine COX-2 PNA molecules, directed against a mRNA region spanning the AUG translation-initiation codon and a homopurinic sequence inside the COX-2 mRNA reading frame. These two PNA sequences, used separately or mixed together, demonstrated the capacity to inhibit the translation of murine COX-2 enzyme in a cell-free translation model using a rabbit retculocyte lysate model. Since PNAs display very low natural permeability across lipids bilayers, the two molecules were also re-synthesized, modified to be used in intact cells by means of linkage to a hydrophobic peptide to obtain membrane-diffusable PNA chimaerae. Finally, stimulated macrophages were found to be affected strongly by these two compounds, used separately or together, monitoring inhibition of COX-2 synthesis by Western blot analysis of total lysates and enzymic activity via radioactive assay on the microsomal fractions.
authors.affiliation: Department of Experimental Medicine, Biochemistry Section, University of Genoa, Viale Benedetto XV 1, 16132 Genoa, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Scarfì
authors.forename: Marco
authors.initials: M
authors.lastname: Giovine
authors.forename: Raffaela
authors.initials: R
authors.lastname: Pintus
authors.forename: Enrico
authors.initials: E
authors.lastname: Millo
authors.forename: Emanuela
authors.initials: E
authors.lastname: Clavarino
authors.forename: Marina
authors.initials: M
authors.lastname: Pozzolini
authors.forename: Laura
authors.initials: L
authors.lastname: Sturla
authors.forename: Roberto Pablo
authors.initials: RP
authors.lastname: Stock
authors.forename: Umberto
authors.initials: U
authors.lastname: Benatti
authors.forename: Gianluca
authors.initials: G
authors.lastname: Damonte
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002474:Cell-Free System; D051546:Cyclooxygenase 2; D004790:Enzyme Induction; D015870:Gene Expression / Q000187:drug effects*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases