Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes.
Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D014407:Tumor Cells, Cultured