Electron micrographic studies of transport of oligodeoxynucleotides across eukaryotic cell membranes
Department of Cell Biology
Animals; Base Sequence; Biological Transport; Cell Membrane; Cell Nucleus; Cells, Cultured; Chick Embryo; Hela Cells; Humans; Mice; Microscopy, Electron; Molecular Sequence Data; Oligodeoxyribonucleotides; Time Factors
Unmodified oligodeoxynucleotides (ODNs) were synthesized and tested for their ability to cross external eukaryotic cell membranes and to enter the cytosol and nucleus in tissue cultures. The ODNs were labeled with high-specific-activity [3H]thymidine (> or = 100 Ci/mmol), or [ alpha-32P]ATP or [ gamma-32P]ATP (300-1000 Ci/mmol; 1 Ci = 37 GBq), and the label was either in the central portion of the molecule or at the 3' or 5' end. The cells employed were for the most part 3T6 murine fibroblasts, grown in monolayers, either semiconfluent or confluent, but some experiments were carried out with chicken embryo fibroblasts or human HeLa cells. Parallel wells in the same experiment were prepared for electron microscopy or for cell fractionation and radioactivity assays. Electron microscopic autoradiography indicated that ODNs cross the external cell membrane, traverse the cytosol, and begin to enter the cell nucleus within a few seconds to 5 min at 37 degrees C in Dulbecco's medium without added serum. After 30-60 min of incubation with ODNs, abundant silver grains were observed at or just inside the nuclear membrane or well distributed across the nucleus, particularly in association with euchromatin. There was a paucity of silver grains associated with nucleoli. Cell entry of oligomer was related to cell cycling events and was energy dependent. Degradation of oligomer to monomers, with reincorporation into DNA, does not appear to explain these results. No sequestration of labeled oligomer in cytoplasmic vesicles en route from the exterior of the cell to the nucleus was observed. The observations are more suggestive of internalization of oligonucleotide by a mechanism as yet unclear or, alternatively, by a caveolar, potocytotic mechanism rather than by endocytosis.
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Citation: Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3156-60.
Proceedings of the National Academy of Sciences of the United States of America
Zamecnik, Paul; Aghajanian, John; Zamecnik, Mary; Goodchild, John; and Witman, George B., "Electron micrographic studies of transport of oligodeoxynucleotides across eukaryotic cell membranes" (1994). Cell and Developmental Biology Publications and Presentations. 8.