title: Facilitation of the cellular uptake of a triplex-forming oligonucleotide by novel polyamine analogues: structure-activity relationships.
abstract: The inefficient uptake of oligodeoxynucleotides, including that of TFO, through the cell membrane is a limiting factor in developing gene therapy approaches for cancer and other diseases. To develop a new strategy for oligonucleotide delivery into the nucleus, we synthesized a series of novel polyamine analogues and examined their effects on the uptake of a 37-mer [32P]-labeled TFO, targeted to the promoter region of c-myc oncogene. We used MCF-7 breast cancer cells to investigate the efficacy of polyamines on the internalization of the TFO. The uptake of TFO was enhanced by complexing it with several unsubstituted polyamine analogues at 0. 1-5 microM concentrations, with up to 6-fold increase in TFO uptake in the presence of a hexamine, 1,21-diamino-4,9,13, 18-tetraazahenicosane (H2N(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(2) or 3-4-3-4-3). TFO uptake increased with the cationicity of the polyamines; however, bis(ethyl) substitution and structural features of the methylene bridging region had significant effects on TFO uptake. The majority of labeled TFO was recovered from the nuclear fraction containing genomic DNA. Electrophoretic mobility shift assay revealed enhanced binding of TFO to a target duplex containing promoter region sequence of c-myc oncogene. Treatment of MCF-7 cells with the TFO complexed with 0.5 microM 3-4-3-4-3 suppressed c-myc mRNA level by 65%, as determined by Northern blot analysis. These data indicate a novel approach to deliver oligodeoxynucleotides to the cell nucleus, and suppress the expression of target genes, and provide new insights into the mechanism of oligonucleotide transport in living cells.
authors.affiliation: Department of Medicine, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick 08903, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Thomas
authors.forename: T
authors.initials: T
authors.lastname: Thomas
authors.forename: M
authors.initials: M
authors.lastname: Wada
authors.forename: L H
authors.initials: LH
authors.lastname: Sigal
authors.forename: A
authors.initials: A
authors.lastname: Shirahata
authors.forename: T J
authors.initials: TJ
authors.lastname: Thomas
mesh_terms: D001943:Breast Neoplasms; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D006801:Humans; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011073:Polyamines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: C122152:1,21-diamino-4,9,13,18-tetraazahenicosane; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011073:Polyamines; C051802:triplex DNA; D004247:DNA