Differential effect of an antisense oligonucleotide to the transferrin receptor in hematopoietic cell lines correlates to subcellular distribution of the oligonucleotide

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There are several factors that may hamper the effect of antisense oligonucleotides (ASOs), e.g., cellular uptake and distribution to the cytoplasm and the nucleus, ASO degradation, lack of specificity resulting in defective hybrid formation or deficient triggering of target mRNA digestion. We observed different effects of an ASO complementary to the AUG region of the transferrin receptor (TfR) mRNA in various cell lines tested, regarding both growth inhibition and decrease in protein expression. Namely, the K562 cell line was unaffected by the TfR ASO either in respect to protein expression or proliferation, unlike other cell lines as BV173. This finding prompted us to examine the cellular uptake of the TfR ASO in these two cell lines by fluorescence and confocal microscopy using a FITC-labelled oligonucleotide. In BV173, the ASO was retained in vesicle scattered throughout the cytoplasm with some diffusion to the cytoplasm and nucleus, while in K562 the oligonucleotide containing vesicles seemed to be sequestered in a region corresponding to the Golgi area with apparently no cytoplasmic or nuclear diffusion. This distribution was partially reverted by coupling the ASO to the transfection reagent lipofectin, increasing the ligonucleotide diffusion to the nucleus and cytoplasm and this correlated with a sharp decrease in TfR protein content in K562 cells.