title: Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer.
abstract: Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.
authors.affiliation: Program in Cell and Lung Biology and Lung Gene Therapy, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Lechardeur
authors.forename: K J
authors.initials: KJ
authors.lastname: Sohn
authors.forename: M
authors.initials: M
authors.lastname: Haardt
authors.forename: P B
authors.initials: PB
authors.lastname: Joshi
authors.forename: M
authors.initials: M
authors.lastname: Monck
authors.forename: R W
authors.initials: RW
authors.lastname: Graham
authors.forename: B
authors.initials: B
authors.lastname: Beatty
authors.forename: J
authors.initials: J
authors.lastname: Squire
authors.forename: H
authors.initials: H
authors.lastname: O'Brodovich
authors.forename: G L
authors.initials: GL
authors.lastname: Lukacs
mesh_terms: D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D004072:Digitonin / Q000378:metabolism; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008845:Microinjections; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism*; D014162:Transfection
chemical_list: D003851:Deoxyribonucleases; D004072:Digitonin; D002118:Calcium