Antisense inhibition of IGF receptor expression in HaCaT keratinocytes: a model for antisense strategies in keratinocytes.
Antisense strategies targeting skin conditions are attractive in concept, with a number of possible pathologic conditions, such a psoriasis, apparently suitable for such an approach. Because in vitro screening of candidate sequences is usually desirable, we have attempted to use a range of new generation cationic lipids to produce significant antisense oligodeoxynucleotide (ODN) uptake in an immortalized keratinocyte cell line (HaCaT). A large number of commercially available lipids were screened for the ability to induce nuclear ODN localization: Tfx-50, Tfx-20, Tfx-10, Superfect, Cytofectin GSV, Perfect lipids 1-8, Lipofectin, and Lipofectamine. All lipids were used at a range of concentrations (1-20 microg/ml) and with a range of ODN concentrations (1-1000) nM). Of all lipids used, only Cytofectin GSV and Superfect produced significant (>30% of cells) levels of nuclear positive cells, with Superfect also producing significant toxicity at the effective concentration used. Only two treatments produced a significant reduction in target mRNA: insulin-like growth factor-1 receptor (IGF-1R)-ODN 64 complexed with Cytofectin GSV (27.1% +/- 3.5% of IGF-1R mRNA in untreated cells,p < 0.01) and ODN 64 complexed with 10 microg/ml Lipofectin (62.2% +/- 3.4% of IGF-1R mRNA in untreated cells, p < 0.05). Only one treatment, ODN 64 complexed with Cytofectin GSV, produced a reduction in cell growth and survival as assessed by amido black assay. These results demonstrate that in HaCaT keratinocytes, Cytofectin GSV alone of all commercially available cationic lipids was effective in delivering antisense ODN into cell nuclei such that a profound antisense effect could be demonstrated.
White
P J
PJ
Department of Pharmaceutical Biology & Pharmacology, Victorian College of Pharmacy, Monash University, Parkville, Australia.
Fogarty
R D
RD
Werther
G A
GA
Wraight
C J
CJ
D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004337:Drug Carriers; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics; D014343:Trypan Blue
D004337:Drug Carriers; D006131:Growth Inhibitors; D020319:Oligodeoxyribonucleotides, Antisense; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger; C020888:1,2-dielaidoylphosphatidylethanolamine; D017526:Receptor, IGF Type 1; D014343:Trypan Blue