Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA–dependent protein kinase PKR

Nature Biotechnology volume 20, pages 895–900 (2002)Cite this article

Abstract

Activated double-stranded RNA (dsRNA)–dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MGΔEGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Δ(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mechanism of specific activation of PKR in U87MGΔEGFR cells.
Figure 2: Effect of ΔEGFR AS RNA on the growth of glioma cells in vitro.
Figure 3: Elimination of the U87MGΔEGFR colonies by ΔEGFR AS RNA.
Figure 4: Inhibition of proliferation of U87MGΔEGFR cells in vivo by ΔEGFR AS RNA.
Figure 5: Involvement of PKR in death of U87MGΔEGFR cells.
Figure 6: Rescue of the U87MGΔEGFR cells by PKR inhibitors.
Figure 7: Induction of apoptosis in U87MGΔEGFR cells.

Similar content being viewed by others

References

  1. Farrell, P.J. et al. Interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded RNA. Proc. Natl. Acad. Sci. USA 75, 5893–5897 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jagus, R., Joshi, B. & Barber, G.N. PKR, apoptosis and cancer. Int. J. Biochem. Cell Biol. 31, 123–138 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Wu, S. & Kaufman, R.J. A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR. J. Biol. Chem. 272, 1291–1296 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Manche, L., Green, S.R., Schmedt, C. & Mathews, M.B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol. Cell. Biol. 12, 5238–5248 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nishikawa, R. et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA 91, 7727–7731 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nagane, M., Levitzki, A., Gazit, A., Cavenee, W.K. & Huang, H.J. Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc. Natl. Acad. Sci. USA 95, 5724–5729 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. He, Y. et al. Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter. J. Natl. Cancer Inst. 90, 1080–1087 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Naldini, L. & Verma, I.M. Lentiviral vectors. Adv. Virus Res. 55, 599–609 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Hu, Y. & Conway, T.W. 2-Aminopurine inhibits the double-stranded RNA-dependent protein kinase both in vitro and in vivo. J. Interferon Res. 13, 323–328 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Koromilas, A.E., Roy, S., Barber, G.N., Katze, M.G. & Sonenberg, N. Malignant transformation by a mutant of the IFNα-inducible dsRNA-dependent protein kinase. Science 257, 1685–1689 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Davies, M.V., Chang, H.W., Jacobs, B.L. & Kaufman, R.J. The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms. J. Virol. 67, 1688–1692 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Denhardt, D.T. Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation. Ann. NY Acad. Sci. 660, 70–76 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Player, M.R. & Torrence, P.F. The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation. Pharmacol. Ther. 78, 55–113 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Iordanov, M.S. et al. Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)- terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways. Mol. Cell. Biol. 20, 617–627 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li, S., Nagai, K. & Koromilas, A.E. A diminished activation capacity of the interferon-inducible protein kinase PKR in human T lymphocytes. Eur. J. Biochem. 267, 1598–1606 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Haroun, R.I. & Brem, H. Local drug delivery. Curr. Opin. Oncol. 12, 187–193 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. He, Y. & Huang, L. Growth inhibition of human papillomavirus 16 DNA-positive mouse tumor by antisense RNA transcribed from U6 promoter. Cancer. Res. 5, 3993–3999 (1997).

    Google Scholar 

  19. Bevilacqua, P.C., George, C.X., Samuel, C.E. & Cech, T.R. Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes. Biochemistry 37, 6303–6316 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L. & Trono, D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15, 871–875 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Hansen, M.B., Nielsen, S.E. & Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119, 203–210 (1989).

    Article  CAS  PubMed  Google Scholar 

  23. Sasaki, M., Wizigmann-Voos, S., Risau, W. & Plate, K.H. Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas. Int. J. Dev. Neurosci. 17, 579–591 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Read, T.A. et al. Local endostatin treatment of gliomas administered by microencapsulated producer cells. Nat. Biotechnol. 19, 29–34 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Sood, R., Porter, A.C., Olsen, D.A., Cavener, D.R. & Wek, R.C. A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2α. Genetics 154, 787–801 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge N. Sonenberg for constructs coding for K3L, E3L, and PKRΔ6 and J. Grandis for U6 expression plasmid. We also thank S. Klein for critical reading of the manuscript. This work was partially supported by Algen Pharmaceuticals.

Author information

Authors and Affiliations

  1. Department of Biological Chemistry, Unit of Cellular Signaling, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, 91904, Jerusalem, Israel

    Alexei Shir & Alexander Levitzki

Authors
  1. Alexei Shir
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexander Levitzki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander Levitzki.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shir, A., Levitzki, A. Inhibition of glioma growth by tumor-specific activation of double-stranded RNA–dependent protein kinase PKR. Nat Biotechnol 20, 895–900 (2002). https://doi.org/10.1038/nbt730

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt730

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing