Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells.

Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.

Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells.

Antisense Res Dev. 1992 Fall;2(3):211-22. Unique Identifier : AIDSLINE MED/93144892
Iversen PL; Zhu S; Meyer A; Zon G; Department of Pharmacology, University of Nebraska Medical; Center, Omaha.

Abstract: A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for cellular uptake and subcellular distribution. The relationship between extracellular oligonucleotide concentration and the distribution and accumulation into subcellular organelles is important to the design, potential side effects, and understanding of a therapeutically useful antisense oligonucleotide. These studies employed uptake of both 35S- and fluorescence-labeled phosphorothioate oligonucleotides. Experiments with V79, HeLa, H9, and fresh human peripheral blood monocytes indicate that accumulations of oligonucleotide inside cells exceeds the concentration of oligonucleotide in culture media by over 100 times following 1 h of exposure at 37 degrees C. Uptake is more efficient at low concentrations, suggesting a saturable process. The total oligonucleotide that remains in cells begins to reach a plateau after 45-60 min, indicating either that efflux pathways exist or that uptake is saturable. Subcellular fractionation studies with 35S-labeled phosphorothioate demonstrate the oligonucleotide is sequestered into both the nuclei and the mitochondria of cultured HeLa cells in a time-dependent manner. The subcellular fractionation was examined with fluorescence-labeled phosphorothioate by both confocal and fluorescence microscopy, which confirmed the rate and localization of oligonucleotide into cultured cells. Finally, cellular uptake is not uniform for all cells in a nonsynchronous culture.

Keywords: Animal Base Sequence Binding Sites Biological Availability Cricetulus Fluoresceins Genes, rev Hamsters Hela Cells Human Molecular Sequence Data Oligonucleotides/*PHARMACOKINETICS Subcellular Fractions/*METABOLISM Sulfur Radioisotopes Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Thionucleotides/*PHARMACOKINETICS Tissue Distribution Tumor Cells, Cultured JOURNAL ARTICLE $KWDpharmacokineticssubcellularfractions/$
930530
M9350949

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