Nucleic Acids Research, Vol 27, Issue 11 2315-2324, Copyright © 1999 by Oxford University Press
PP Laktionov, JE Dazard, E Vives, EY Rykova, J Piette, VV Vlassov and B Lebleu
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.
ARTICLES
Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes
Institute of Bioorganic Chemistry, Academy of Sciences Siberian Division, Novosibirsk 630090, Russia and Institute of Molecular Genetics, UMR 5535, IFR 24, CNRS, 34293 Montpellier Cedex 5, France.
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