Abstract
Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis-disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.
Highlights
The most straightforward strategy for gene delivery is represented by free DNA [3]
Cells were detached by trypsin treatment and extensively rinsed prior to flow cytometry analysis; it could not be ruled out that fluorophore-labeled DNA was associated with the cell exterior, especially after short incubation periods
We present evidence that DNA uptake by mammalian cells is mediated by secreted proteins via an endocytotic mechanism that strictly requires cell surface PGs
Summary
Materials—Fluorophores, labeled markers, secondary antibodies, and DNase I were from Invitrogen. In the studies using HA-dynamin-WT/K44A, GFP-Eps15E⌬95/295, and Cav1-YFP construct, 2– 4 ϫ 106 CHO-K1 cells were electroporated at 340 V in 700 l of phosphate-buffered saline containing 50 g of plasmid DNA using a BTX ECM399 (Genetronics) electroporator. Cells were incubated with DNA-AF647 (5 g/ml) and heparin binding CM components (20 g/ml) for 3 h or with transferrin-AF647 (150 g/ml) for 30 min in F12K without serum. Fluorescence Quenching and Gel Retardation—For fluorescence quenching analysis, a mixture of DNA (5 g/ml) and ethidium bromide (DNA:EtBr 20:1, w/w) in 1 mM Tris, 50 mM NaCl, and 1 mM EDTA (or 1 mM CaCl2 and 0.5 mM MgCl2 for samples with divalent cations) was supplemented with varying concentrations of heparin binding CM components (0 –250 g/ml) and/or highly sulfated HS (0 –250 g/ml). In some cases the error bars were smaller than the drawn symbols
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