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. 1995 Apr;95(4):1814–1823. doi: 10.1172/JCI117860

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

C Beltinger 1, H U Saragovi 1, R M Smith 1, L LeSauteur 1, N Shah 1, L DeDionisio 1, L Christensen 1, A Raible 1, L Jarett 1, A M Gewirtz 1
PMCID: PMC295714  PMID: 7706488

Abstract

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agutter P. S., Prochnow D. Nucleocytoplasmic transport. Biochem J. 1994 Jun 15;300(Pt 3):609–618. doi: 10.1042/bj3000609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayever E., Iversen P. Oligonucleotides in the treatment of leukemia. Hematol Oncol. 1994 Jan-Feb;12(1):9–14. doi: 10.1002/hon.2900120103. [DOI] [PubMed] [Google Scholar]
  3. Bennett R. M. As nature intended? The uptake of DNA and oligonucleotides by eukaryotic cells. Antisense Res Dev. 1993 Fall;3(3):235–241. doi: 10.1089/ard.1993.3.235. [DOI] [PubMed] [Google Scholar]
  4. Bennett R. M., Gabor G. T., Merritt M. M. DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA. J Clin Invest. 1985 Dec;76(6):2182–2190. doi: 10.1172/JCI112226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Besterman J. M., Airhart J. A., Woodworth R. C., Low R. B. Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation. J Cell Biol. 1981 Dec;91(3 Pt 1):716–727. doi: 10.1083/jcb.91.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowers-Komro D. M., McCormick D. B. Biotin uptake by isolated rat liver hepatocytes. Ann N Y Acad Sci. 1985;447:350–358. doi: 10.1111/j.1749-6632.1985.tb18450.x. [DOI] [PubMed] [Google Scholar]
  7. Burton J., Goldman C. K., Rao P., Moos M., Waldmann T. A. Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7329–7333. doi: 10.1073/pnas.87.18.7329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chalifour L. E., Dakshinamurti K. The characterization of the uptake of avidin-biotin complex by HeLa cells. Biochim Biophys Acta. 1982 Sep 13;721(1):64–69. doi: 10.1016/0167-4889(82)90024-6. [DOI] [PubMed] [Google Scholar]
  9. Chin D. J., Green G. A., Zon G., Szoka F. C., Jr, Straubinger R. M. Rapid nuclear accumulation of injected oligodeoxyribonucleotides. New Biol. 1990 Dec;2(12):1091–1100. [PubMed] [Google Scholar]
  10. Cohen N. D., Thomas M. Biotin transport into fully differentiated 3T3-L1 cells. Biochem Biophys Res Commun. 1982 Oct 29;108(4):1508–1516. doi: 10.1016/s0006-291x(82)80078-8. [DOI] [PubMed] [Google Scholar]
  11. Finlay D. R., Newmeyer D. D., Price T. M., Forbes D. J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. doi: 10.1083/jcb.104.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gao W. Y., Jaroszewski J. W., Cohen J. S., Cheng Y. C. Mechanisms of inhibition of herpes simplex virus type 2 growth by 28-mer phosphorothioate oligodeoxycytidine. J Biol Chem. 1990 Nov 25;265(33):20172–20178. [PubMed] [Google Scholar]
  13. Gao W. Y., Storm C., Egan W., Cheng Y. C. Cellular pharmacology of phosphorothioate homooligodeoxynucleotides in human cells. Mol Pharmacol. 1993 Jan;43(1):45–50. [PubMed] [Google Scholar]
  14. Geselowitz D. A., Neckers L. M. Analysis of oligonucleotide binding, internalization, and intracellular trafficking utilizing a novel radiolabeled crosslinker. Antisense Res Dev. 1992 Spring;2(1):17–25. doi: 10.1089/ard.1992.2.17. [DOI] [PubMed] [Google Scholar]
  15. Gewirtz A. M., Xu W. Y., Mangan K. F. Role of natural killer cells, in comparison with T lymphocytes and monocytes, in the regulation of normal human megakaryocytopoiesis in vitro. J Immunol. 1987 Nov 1;139(9):2915–2924. [PubMed] [Google Scholar]
  16. Hijiya N., Zhang J., Ratajczak M. Z., Kant J. A., DeRiel K., Herlyn M., Zon G., Gewirtz A. M. Biologic and therapeutic significance of MYB expression in human melanoma. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4499–4503. doi: 10.1073/pnas.91.10.4499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iversen P. L., Zhu S., Meyer A., Zon G. Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res Dev. 1992 Fall;2(3):211–222. doi: 10.1089/ard.1992.2.211. [DOI] [PubMed] [Google Scholar]
  18. Kitajima I., Shinohara T., Bilakovics J., Brown D. A., Xu X., Nerenberg M. Ablation of transplanted HTLV-I tax-transformed tumors in mice by antisense inhibition of NF-kappa B. Science. 1993 Mar 12;259(5101):1523–1523. doi: 10.1126/science.8456277. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Loke S. L., Stein C. A., Zhang X. H., Mori K., Nakanishi M., Subasinghe C., Cohen J. S., Neckers L. M. Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci U S A. 1989 May;86(10):3474–3478. doi: 10.1073/pnas.86.10.3474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ma D. D., Doan T. L. Antisense oligonucleotide therapies: are they the "magic bullets"? Ann Intern Med. 1994 Jan 15;120(2):161–163. doi: 10.7326/0003-4819-120-2-199401150-00010. [DOI] [PubMed] [Google Scholar]
  22. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  23. Morishita R., Gibbons G. H., Ellison K. E., Nakajima M., von der Leyen H., Zhang L., Kaneda Y., Ogihara T., Dzau V. J. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest. 1994 Apr;93(4):1458–1464. doi: 10.1172/JCI117123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pardridge W. M., Boado R. J. Enhanced cellular uptake of biotinylated antisense oligonucleotide or peptide mediated by avidin, a cationic protein. FEBS Lett. 1991 Aug 19;288(1-2):30–32. doi: 10.1016/0014-5793(91)80996-g. [DOI] [PubMed] [Google Scholar]
  25. Ratajczak M. Z., Kant J. A., Luger S. M., Hijiya N., Zhang J., Zon G., Gewirtz A. M. In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11823–11827. doi: 10.1073/pnas.89.24.11823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roghani M., Mansukhani A., Dell'Era P., Bellosta P., Basilico C., Rifkin D. B., Moscatelli D. Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding. J Biol Chem. 1994 Feb 11;269(6):3976–3984. [PubMed] [Google Scholar]
  27. Simons M., Edelman E. R., DeKeyser J. L., Langer R., Rosenberg R. D. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992 Sep 3;359(6390):67–70. doi: 10.1038/359067a0. [DOI] [PubMed] [Google Scholar]
  28. Skorski T., Nieborowska-Skorska M., Nicolaides N. C., Szczylik C., Iversen P., Iozzo R. V., Zon G., Calabretta B. Suppression of Philadelphia1 leukemia cell growth in mice by BCR-ABL antisense oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4504–4508. doi: 10.1073/pnas.91.10.4504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stein A., Iversen P. L., Subasinghe C., Cohen J. S., Stec W. J., Zon G. Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry. Anal Biochem. 1990 Jul;188(1):11–16. doi: 10.1016/0003-2697(90)90521-a. [DOI] [PubMed] [Google Scholar]
  30. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  31. Stein C. A., Tonkinson J. L., Zhang L. M., Yakubov L., Gervasoni J., Taub R., Rotenberg S. A. Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells. Biochemistry. 1993 May 11;32(18):4855–4861. doi: 10.1021/bi00069a022. [DOI] [PubMed] [Google Scholar]
  32. Yakubov L. A., Deeva E. A., Zarytova V. F., Ivanova E. M., Ryte A. S., Yurchenko L. V., Vlassov V. V. Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc Natl Acad Sci U S A. 1989 Sep;86(17):6454–6458. doi: 10.1073/pnas.86.17.6454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zamecnik P., Aghajanian J., Zamecnik M., Goodchild J., Witman G. Electron micrographic studies of transport of oligodeoxynucleotides across eukaryotic cell membranes. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3156–3160. doi: 10.1073/pnas.91.8.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]

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