Nuclear translocation of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells.
Poly-l-lysine, with 40% of its amino groups substituted with lactose, is an effective vector to transfer the CFTR gene into CF airway epithelial cells and correct the chloride channel dysfunction. The intracellular fate of the lactosylated poly-l-lysine/cDNA complex was studied using confocal microscopy. In the presence of chloroquine the complex remained intact during internalization, intracellular transport, and, most importantly, transport into the nucleus. When cells were transfected in the presence of agents that enhance transfection efficiency such as E5CA peptide, a fusogenic peptide, or glycerol a similar fate of the lactosylated poly-l-lysine/cDNA complex was seen. However, when these agents were omitted from the transfection medium, the complex remained in the perinuclear region. Uncomplexed lactosylated poly-l-lysine reached the nucleus efficiently. In contrast mannosylated poly-l-lysine or unsubstituted poly-l-lysine complexed to plasmid did not. Therefore the nuclear accumulation of the complex may be attributed to the substitution of poly-l-lysine with lactose. It is hypothesized that the lactose residues provide for nuclear localization by means of targeting a potential lectin-like protein with galactose/lactose specificity. This mechanism may be responsible for the nuclear internalization of the complex.
Klink
D T
DT
The Cystic Fibrosis Center, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Chao
S
S
Glick
M C
MC
Scanlin
T F
TF
D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002738:Chloroquine / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D018076:DNA, Complementary / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007124:Immunoenzyme Techniques; D007785:Lactose; D018613:Microscopy, Confocal; D011107:Polylysine / Q000378:metabolism*
D018076:DNA, Complementary; D011107:Polylysine; D002738:Chloroquine; D007785:Lactose