Cationic lipid is not required for uptake and selective inhibitory activity of ICAM-1 phosphorothioate antisense oligonucleotides in keratinocytes.
Keratinocyte intercellular adhesion molecule-1 (ICAM-1) is important in mediating retention of T cells within the epidermal compartment. To determine if antisense oligonucleotides designed to hybridize to various ICAM-1 mRNA regions could selectively influence cultured keratinocyte ICAM-1 expression following gamma interferon (IFN-gamma), cells were exposed to several antisense compounds, in the absence and presence of cationic lipid (lipofectin). Keratinocytes rapidly internalized sense and antisense compounds (within 30-60 min), even in the absence of lipofectin with approximately 30% of the cell possessing positive nuclei. Such nuclear accumulation was not observed in the absence of lipofectin in cultured fibroblasts, smooth muscle cells, or endothelial cells, even though total cellular uptake within the cytoplasm was significantly increased in all these cell types. Using flow cytometry, IFN-gamma-inducible ICAM-1 expression was reduced 50% by antisense compounds with lipofectin, and by 30% without lipofectin. This inhibition was specific as no change was observed for HLA-DR or tumor necrosis factor-alpha receptor expression. Northern blot hybridization studies confirmed that ICAM-1 antisense oligonucleotides selectively and significantly inhibited ICAM-1 expression. These results suggest that such antisense compounds interact with keratinocytes differently than other cell types, and provide the in vitro basis for clinical trials in which reduction (or elimination) of ICAM-1 expression by epidermal keratinocytes could be selectively accomplished without necessarily influencing dermal cell types such as fibroblasts, endothelial cells, or smooth muscle cells.
Nestle
F O
FO
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
Mitra
R S
RS
Bennett
C F
CF
Chan
H
H
Nickoloff
B J
BJ
D001483:Base Sequence; D015152:Blotting, Northern; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense / Q000032:analysis* / Q000493:pharmacokinetics / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry; D013873:Thionucleotides / Q000032:analysis* / Q000493:pharmacokinetics / Q000494:pharmacology
D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; D013873:Thionucleotides; D018799:Intercellular Adhesion Molecule-1; C020888:1,2-dielaidoylphosphatidylethanolamine; D016650:Fluorescein-5-isothiocyanate; C089753:alicaforsen