Abstract
Transfection of cells with short double-stranded synthetic DNA molecules that contain a transcription factor binding site, known as decoy oligodeoxynucleotides (ODNs), has been proposed as a novel approach in vitro and in vivo for the study of gene regulation and for gene therapy. Once delivered into cells, decoy ODNs are predicted to bind to nuclear transcription factors, preventing their binding to consensus sequences in target genes. Using a fluorescein-labeled decoy ODN containing a consensus sequence for the AP-1 transcription factor, we show that lipid-complexed decoys were readily transfectable into cells, but were consistently detectable in the cytoplasm and not in the nucleus. The same phenomenon was observed in three different cell lines including KB-3, CHO and MDA-MB-231. The AP-1 decoy ODNs failed to inhibit the transcriptional activity of an AP-1-dependent luciferase reporter. The effect of cytoplasmic AP-1 decoy ODNs on the subcellular localization and function of c-Jun induced by the microtubule inhibitor vinblastine, which strongly induced c-Jun expression, was assessed. No difference in protein level or nuclear localization of vinblastine-induced c-Jun, or of one of its target genes, p53, was noted when cells were transfected with wild-type or mutated forms of the decoy ODNs. We suggest that subcellular localization is an unappreciated and key limiting factor for the use of transcription factor decoy ODNs that must be addressed before meaningful data interpretation can be made.