Nucleic Acids Res  2002 Apr 1;30(7):1512-21 



Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2

cell line: evidence for non-conventional intracellular trafficking.



de Diesbach P, N'Kuli F, Berens C, Sonveaux E, Monsigny M, Roche AC, Courtoy PJ.



Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and

Universite catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200

Brussels, Belgium.



Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we

have re-examined here the kinetics of ON uptake, subcellular distribution and

intracellular localisation in these cells, at concentrations relevant for the

study of a receptor-dependent process. Kinetic parameters of ON endocytosis were

comparable with those of the receptor-mediated endocytosis tracer, transferrin

(uptake equilibrium, saturation with concentration, specific competition and

rapid efflux) and were clearly distinct from those of fluid-phase endocytosis.

By analytical subcellular fractionation, particulate ON showed a bimodal

distribution after 2 h of uptake, with a low-density peak superimposed on the

distribution of endosomes, and a high-density peak overlapping lysosomes. After

an overnight chase, only the high-density peak remained, but it could be

dissociated from lysosomes, based on its refractoriness to displacement upon

chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate

pattern was resolved, by confocal microscopy, from those of transferrin, of a

fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3

microM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and

LysoTracker. Taken together, these data suggest that ON may be internalised at

low concentrations by receptor-mediated endocytosis into unique endosomes, then

to dense structures that are distinct from lysosomes. The nature of these two

compartments and their significance for ON effect deserve further investigation.



PMID: 11917011 [PubMed - indexed for MEDLINE]