title: Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking.
abstract: 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.
authors.affiliation: Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: de Diesbach
authors.forename: Francisca
authors.initials: F
authors.lastname: N'Kuli
authors.forename: Catherine
authors.initials: C
authors.lastname: Berens
authors.forename: Etienne
authors.initials: E
authors.lastname: Sonveaux
authors.forename: Michel
authors.initials: M
authors.lastname: Monsigny
authors.forename: Annie-Claude
authors.initials: AC
authors.lastname: Roche
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Courtoy
mesh_terms: D001692:Biological Transport; D005591:Chemical Fractionation; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D009841:Oligonucleotides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D011956:Receptors, Cell Surface