Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells.
An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, was fluorescently labeled to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry and fluorescence microscopy show the following: within around 0.5-2 h, an apparent steady-state distribution of the oligonucleotide is achieved in which the intracellular oligonucleotide concentration is less than that present in the external medium; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free medium, an oligonucleotide efflux, with a time constant similar to that for uptake, is observed (although a significant fraction of the phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is nonlinear, being more efficient at lower concentrations (less than 2 microM); and a predominant oligonucleotide localization within the cell nucleus and perinuclear organelles is observed.
Marti
G
G
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland.
Egan
W
W
Noguchi
P
P
Zon
G
G
Matsukura
M
M
Broder
S
S
D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000378:metabolism*; D063086:Organothiophosphates / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D016376:Oligonucleotides, Antisense; D063086:Organothiophosphates