Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.
A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.
Wyroba
E
E
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Pawlowska
Z
Z
Kobylanska
A
A
Pluskota
E
E
Maszewska
M
M
Stec
W J
WJ
Cierniewski
C S
CS
D016375:Antisense Elements (Genetics) / Q000493:pharmacokinetics*; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line / Q000166:cytology / Q000378:metabolism; D004727:Endothelium / Q000166:cytology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D015345:Oligonucleotide Probes / Q000493:pharmacokinetics*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D012333:RNA, Messenger / Q000493:pharmacokinetics; D015842:Serine Proteinase Inhibitors / Q000235:genetics*
D016375:Antisense Elements (Genetics); D015345:Oligonucleotide Probes; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D015842:Serine Proteinase Inhibitors