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 highdensity 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 fluidphase 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.

Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery

“…(i) an initial, exponential phase describing a rapid receptor-mediated internalization of liposomes; (ii) a plateau, typically indicative of saturation of uptake-mediating receptors [34];…”

Intracellular Distribution of TiO2−DNA Oligonucleotide Nanoconjugates Directed to Nucleolus and Mitochondria Indicates Sequence Specificity

“…Natural uptake of DNA can be either enocytosis or receptor mediated, depending on the concentration of DNA 10 . In both cases a certain amount of transfected DNA does end up inside the nucleus.…”

Endocytosis of the somatostatin analogue, octreotide, by the proximal tubule-derived opossum kidney (OK) cell line

“…Recombinant rat receptor-associated protein (RAP) was obtained from RDI (Flanders, NJ, USA). Iron-saturated transferrin (Sigma Chemical Co., St. Louis, MO, USA) was conjugated to Alexa Fluor 568 (Molecular Probes, Eugene, OR, USA), as recommended by the manufacturer [21].…”

“…To validate a fluorescent tracer for further morphologic studies, 0.1 nmol fluorescein isothiocyanate (FITC)-D-Phe 1 -octreotide, a kind gift of Novartis Pharma (Basel, Switzerland), was labeled with Na 125 I (37 MBq) using iodogen-precoated tubes ([1,3,4,6-tetrachloro3a-6a-diphenylglycoluril]-precoated iodination tubes) (Iodo-Gen ) (Pierce Polylab, Antwerp, Belgium) [21]. Radiolabeled peptide extraction was performed using a Sep-Pack C18 column as described by Bakker et al [22].…”