INTERNALIZATION OF OLIGODEOXYNUCLEOTIDE ANTISENSE TO TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR MESSENGER-RNA IN ENDOTHELIAL-CELLS - A 3-DIMENSIONAL RECONSTRUCTION BY CONFOCAL MICROSCOPY

A three-dimensional reconstruction analysis of localization of phospho diester and phosphorothioate oligonucleotide antisense to type-1 plasm inogen activator inhibitor (PAI-I) mRNA within endothelial cells is de scribed. When EA.hy 926 cells were incubated with fluorescently labell ed phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness compo sition of the images of the particular samples was much higher for PS- 16 than PO-16 and dependent upon the extracellular concentration and t he incubation time. The 3-D reconstructions based on the series of opt ical sections of the samples, spaced every 1.5 mu m, showed the punctu ate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. E ven after 24 h incubation of endothelial cells with 2.5 mu M of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide lo calization within the cytoplasm and only traces of oligonucleotides co uld be seen in the cell nucleus and/or perinuclear organelles.