Characteristics of Oligonucleotide Uptake in Human Keratinocyte Cultures

Abstract

Oligodeoxyribonucleotides have the potential to interfere selectivity with cellular protein synthesis by sequence-specific hybridization to DNA or RNA molecules. We have, investigated the properties of uptake and intracellular localization of fluorescently labeled oligonucleotides in cultured human keratinocytes using confocal laser scanning microscopy. Unlike many other cell types studied, keratinocytes can internalize oligonucleotides without apparent sequestration in endosomes or cell surface accumulation. Uptake is primarily nuclear and unaltered by sodium azide, monensin, or chloroquin pretreatment. We have verified our results with two different fluorophores, fluorescein and Bodipy, and found similar uptake and distribution patterns in both live and fixed cell populations. Surprisingly, we have found uptake to be heterogeneous within a population, with 15-30% of cells internalizing the oligonucleotides. This percentage is drastically increased to roughly 80% at cell population margins, and after release from M phase arrest. These results on uptake and intracellular localization suggest that keratinocytes may have increased sensitivity as target cells for oligonucleotide based gene regulation strategies