Nucleic Acids Research, 2002, Vol. 30, No. 7 1512-1521
© 2002 Oxford University Press
Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking
1Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium, 2Laboratoire de Chimie Thérapeutique et de Radiopharmacie, Université catholique de Louvain, UCL 7340, 73 Avenue E. Mounier, B-1200 Brussels, Belgium and 3Glycobiologie, Centre de Biophysique Moléculaire and Université d’Orléans, CNRS UPR 4301, rue Charles-Sadron, F-45071 Orléans Cedex 02, France
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 µM 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.
* To whom correspondence should be addressed. Tel: +32 2 764 75 69; Fax: +32 2 764 75 43; Email: courtoy{at}cell.ucl.ac.be
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