An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, has been fluorescently- and radio-labeled in order to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry, fluorescence microscopy, and autoradiography show the following: oligonucleotide localization within the cell nucleus and per-nuclear organelles; within ca. 0.5 to 5 hours, depending on the cell line, an apparent steady-state distribution of the oligonucleotide is achieved, wherein the intracellular oligonucleotide concentration is significantly below that present in the external media; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free media, an oligonucleotide efflux, with a time constant similar to that for uptake, from the cells is observed (although a significant fraction of phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is non-linear, being considerably more efficient at lower concentrations (less than 2 micro-M). In a separate series of experiments, unlabeled PS-ODN was used to ascertain the inhibition of gpl2O expression in the cell line 8E5 which constitutively secretes a non-infectious HIV particle. In a two-color analysis, using a fluoresceinated anti-transferrin receptor monoclonal antibody and anti-gpl2O detected using a PE-conjugated sub-isotype specific goat antimouse immunoglobulin, a significant decrease in the expression of surface gpl2O was seen. Limited studies comparing alpha-PO, alpha-PS and beta-PS are underway.
