FLOW CYTOMETRIC AND MICROSCOPIC CHARACTERIZATION OF THE UPTAKE AND DISTRIBUTION OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES IN HUMAN KERATINOCYTES

Gene-specific inhibition by antisense oligonucleotides has been succes sful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studie d oligonucleotide uptake in cultured human keratinocytes. This study r evealed a heterogeneous uptake of fluorescently labeled phosphorothioa te oligonucleotides. Flow cytometric and microscopic analysis showed t wo fluorescent cell populations with differences in intensity: a 'brig ht' population of highly fluorescent small cells and a 'dim' populatio n of less fluorescent but larger cells. The heterogeneity in uptake be tween these two populations was not a result of differences in cell cy cle phases of the keratinocytes, as shown by flow cytometric sorting a nd measurements of relative DNA content. In both populations the oligo nucleotides were transported intracellularly and mere mainly located i n the cytoplasm, A typically speckled localization pattern was demonst rated by confocal laser scanning microscopy, We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus, The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocyt es was dependent on oligonucleotide concentration, incubation time and temperature, This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be conside red when specific oligonucleotides are used against skin disease-speci fic genes.