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Intracellular Compartmentalization of DNA Fragments in Cultured Airway Epithelial Cells Mediated by Cationic Lipids

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Abstract

Purpose. The amount and intracellular distribution of DNA fragments (491 -bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection.

Methods. Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine® at various charge ratios.

Results. A 5/1 (+/−) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 ± 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 ± 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA.

Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.

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REFERENCES

  1. K. K. Goncz, K. K. Kunzelmann, Z. Xu, and D. C. Gruenert. Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragment. Hum. Mol. Genet. 7:1913–1919 (1998).

    Google Scholar 

  2. K. Kunzelmann, J.-Y. Legendre, D. L. Knoell, L. C. Escobar, Z. Xu, and D. C. Gruenert. Gene targeting of CFTR DNA in CF epithelial cells. Gene Ther. 3:859–867 (1996).

    Google Scholar 

  3. T. L. Fisher, T. Terhorst, X. Cao, and R. W. Wagner. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 21:3857–3865 (1993).

    Google Scholar 

  4. J. E. Hagstrom, J. J. Ludtke, M. C. Bassik, M. G. Sebestyen, S. A. Adam, and J. A. Wolff. Nuclear import of DNA in digitonin-permeabilized cells. J. Cell Sci. 110:2323–2331 (1997).

    Google Scholar 

  5. S. L. Loke, C. A. Stein, X. H. Zhang, K. Mori, M. Nakanishi, C. Subasinghe, J. S. Cohen, and L. M. Neckers. Characterization of oligonucleotide transport into living cells. Proc. Natl. Acad. Sci. USA 86:3474–3478 (1989).

    Google Scholar 

  6. D. F. Ma and A. Q. Wei. Enhanced delivery of synthetic oligonu-cleotides to human leukemia cells by liposomes and immunoliposomes. Leukemia Res. 20:925–930 (1996).

    Google Scholar 

  7. S. B. Noonberg, M. R. Garovoy, and A. Hunt. Characteristics of oligonucleotide uptake in human keratinocyte cultures. J. Invest. Dermatol. 101:727–731 (1993).

    Google Scholar 

  8. F. M. Orson, D. W. Thomas, W. M. McShan, D. J. Kessler, and M. E. Hogan. Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes. Nucleic Acids Res. 19:3435–3441 (1991).

    Google Scholar 

  9. J. G. Pickering, J. M. Isner, C. Ford, L. Weir, A. Lazarovits, E. F. Rocnick, and L. H. Chow. Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Circulation 93:772–780 (1996).

    Google Scholar 

  10. A. Quattrone, L. Papucci, N. Schiavone, E. Mini, and S. Capaccioli. Intracellular enhancement of intact antisense oligonucleotide steady-state levels by cationic lipids. Anticancer Drug Des. 9:549–553 (1994).

    Google Scholar 

  11. A. Tari, M. Khodadadian, D. Ellerson, A. Deisseroth, and G. Lopez-Berestein. Liposomal delivery of oligodeoxynucleotides. Leukemia Lymphoma 21:93–97 (1996).

    Google Scholar 

  12. K. Lappalainen, A. Urtti, I. Jaaskelainen, K. Syrjanen, and S. Syrjanen. Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells. Antiviral Res. 23:119–130 (1994).

    Google Scholar 

  13. C. F. Bennett, M. Y. Chiang, H. Chan, J. E. Shoemaker, and C. K. Mirabelli. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41:1023–1033 (1992).

    Google Scholar 

  14. S. Capaccioli, G. Di Pasquale, E. Mini, T. Mazzei, and A. Quattrone. Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum [published erratum appears in Biochem Biophys. Res. Commun. 200:1769 (1994)]. Biochem. Biophys. Res. Commun. 197:818–825 (1993).

    Google Scholar 

  15. O. Zelphati and F. C. Szoka, Jr. Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharmacol. Res. 13:1367–1372 (1996).

    Google Scholar 

  16. X. Gao and L. Huang. Cationic liposome-mediated gene transfer. Gene Then. 2:710–722 (1995).

    Google Scholar 

  17. H. Schreier. The new frontier: gene and oligonucleotide therapy. Pharm. Acta Helv. 68:145–159 (1994).

    Google Scholar 

  18. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987).

    Google Scholar 

  19. N. J. Caplen, E. W. Alton, P. G. Middleton, J. R. Stevenson, X. Gao, S. R. Durham, P. K. Jeffery, M. E. Hodson, C. Coutelle, L. Huang, D. J. Porteous, R. Williamson, and D. M. Geddes. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis [published erratum appears in Nat. Med 3:272 (1995)]. Nat. Med. 1:39–46 (1995).

    Google Scholar 

  20. F. Ruiz et el. Gene transfer of CFTR to lower airways of cystic fibrosis patients and description of a clincial syndrome attributable to lipid/DNA administration. Ped. Pulm. Suppl. 17:266 (1998).

    Google Scholar 

  21. C. M. Gorman, M. Aikawa, B. Fox, E. Fox, C. Lapuz, B. Michaud, H. Nguyen, E. Roche, T. Sawa, and J. P. Wiener Kronish. Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC. Gene Ther. 4:483–492 (1997).

    Google Scholar 

  22. D. C. Gruenert, W. E. Finkbeiner, and J. H. Widdicombe. Culture and transformation of airway epithelial cells. Am. J. Physiol. 268:L347–L360 (1995).

    Google Scholar 

  23. K. Kunzelmann, D. C. Lei, K. Eng, L. C. Escobar, T. Koslowsky, and D. C. Gruenert. Epithelial cell specific properties and genetic complementation in a ΔF508 cystic fibrosis nasal polyp cell line. In Vitro Cell Dev. Biol. 31A:617–624 (1995).

    Google Scholar 

  24. K. Kunzelmann, E. Schwiebert, W.-L. Kuo, B. A. Stanton, and D. C. Gruenert. An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the ΔF508 CFTR mutation. Am. J. Respir. Cell. Mol. Biol. 8:522–529 (1993).

    Google Scholar 

  25. A. L. Cozens, M. J. Yezzi, L. Chin, E. M. Simon, W. E. Finkbeiner, J. A. Wagner, and D. C. Gruenert. Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells [published erratum appears in Proc. Natl. Acad. Sci. USA 89:7849 (1992)]. Proc. Natl. Acad. Sci. USA 89:5171–5175 (1992).

    Google Scholar 

  26. D. C. Gruenert, C. B. Basbaum, and J. H. Widdicombe. Longterm culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions. In Vitro Cell Dev. Biol. 26:411–418 (1990).

    Google Scholar 

  27. D. J. Chin, G. A. Green, G. Zon, F. C. Szoka, Jr., and R. M. Straubinger. Rapid nuclear accumulation of injected oligodeoxyri-bonucleotides. New Biol. 2:1091–1100 (1990).

    Google Scholar 

  28. D. R. Roop, J. L. Nordstrom, S. Y. Tsai, M. J. Tsai, and B. W. O'Malley. Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors. Cell 15:671–685 (1978).

    Google Scholar 

  29. J. P. Shaw, K. Kent, J. Fishback, and B. Froehler. Modified deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Res. 19:747–750 (1991).

    Google Scholar 

  30. J. P. Leonetti, N. Mechti, G. Degols, C. Gagnor, and B. Lebleu. Intracellular distribution of microinjected antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 88:2702–2706 (1991).

    Google Scholar 

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Authors and Affiliations

  1. Cardiovascular Research Institute, University of California, Gene Therapy Core Center, San Francisco, California, 94143

    Adam R. Holmes, Austin F. Dohrman, Kaarin K. Goncz & Dieter C. Gruenert

  2. F.I. Proctor Foundation, University of California, San Francisco, California, 94143

    Aaron R. Ellison

  3. Departments of Laboratory Medicine and Stomatology, University of California, San Francisco, California, 94143

    Dieter C. Gruenert

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  1. Adam R. Holmes
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  2. Austin F. Dohrman
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  4. Kaarin K. Goncz
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  5. Dieter C. Gruenert
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Correspondence to Dieter C. Gruenert.

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Holmes, A.R., Dohrman, A.F., Ellison, A.R. et al. Intracellular Compartmentalization of DNA Fragments in Cultured Airway Epithelial Cells Mediated by Cationic Lipids. Pharm Res 16, 1020–1025 (1999). https://doi.org/10.1023/A:1018927531003

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