Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with streptolysin O.
Most mammalian cell types appear to take up antisense oligonucleotides and oligonucleotide analogs from the bathing medium by highly inefficient endocytic mechanisms, and most if not all intracellular oligomer is sequestered in vesicles, still separated by a membrane from the target mRNA. On the other hand, oligonucleotides introduced directly into the cytoplasm by microinjection rapidly accumulate in the cell nucleus. Poor delivery to the designated site of action of antisense oligonucleotides is a major problem limiting their routine use in genetic research and their development as potential therapeutic agents. In view of this difficulty, various means of membrane permeabilization were applied to cultured human leukemia cells in an attempt to enhance intracytoplasmic delivery of fluorescein-tagged oligodeoxynucleotides. The outcome of the manipulations was monitored by flow cytometry and fluorescence microscopy. This work has directly confirmed the conclusion suggested by reported antisense effects, that streptolysin O reversibly permeabilizes the plasma membrane toward oligonucleotides and may be utilized to effect biochemical "microinjection" of these molecules directly into the cytoplasm. KY01 myelogenous leukemia cells treated in this way accumulated over 100-fold higher intracellular levels of oligodeoxynucleotides than in the absence of streptolysin O and, in contrast to the latter case, were observed to concentrate internalized molecules in their nuclei.
Spiller
D G
DG
Department of Biochemistry, University of Liverpool, UK.
Tidd
D M
DM
D001426:Bacterial Proteins; D001483:Base Sequence; D002460:Cell Line; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000378:metabolism*; D013301:Streptolysins; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D005452:Fluoresceins; D016376:Oligonucleotides, Antisense; D013301:Streptolysins; C025964:streptolysin O