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.