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.

  • Original Article
  • Published:

Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo

Cancer Gene Therapy volume 17, pages 86–96 (2010)Cite this article

Abstract

Herpes simplex virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We described earlier two strategies to overcome this limitation: (1) generation of HSVTK mutants with improved GCV activation potential and (2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants, mutant 30 and SR39, were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, shows significant reduction in IC50 values of approximately 12 500-fold, 100-fold, and 125-fold compared with HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses show that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1 mg kg−1) that has no effect on wild-type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2500-fold or 25-fold compared with HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC50 values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 seems to elicit stronger bystander killing effects in which 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1 mg kg−1 GCV. However, at a lower dose of GCV (0.1 mg kg−1), MGMK/SR39 seems to have slightly greater tumor growth inhibition properties compared with SR39 (P⩽0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1 mg kg−1 GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Kokoris MS, Black ME . Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity. Protein Sci 2002; 11: 2267–2272.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Moolten FL . Tumor sensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 1986; 46: 5276–5281.

    CAS  PubMed  Google Scholar 

  3. Edelstein ML, Abedi MR, Wixon J, Edelstein RM . Gene therapy clinical trials worldwide 1989–2004—an overview. J Gen Med 2004; 6: 597–602.

    Article  Google Scholar 

  4. Miller WH, Miller RL . Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase. J Biol Chem 1980; 255: 7204–7207.

    CAS  PubMed  Google Scholar 

  5. Reardon JE . Herpes simplex virus type 1 and human DNA polymerase interactions with 2′-deoxyguanosine 5′-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition. J Biol Chem 1989; 264: 19039–19044.

    CAS  PubMed  Google Scholar 

  6. Field AK, Davies ME, DeWitt C, Perry HC, Liou R, Germershausen J et al. 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci USA 1983; 80: 4139–4143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Greco O, Dachs G . Gene directed enzyme/prodrug therapy of cancer: historical appraisal and future prospective. J Cell Physiol 2001; 187: 22–36.

    Article  CAS  PubMed  Google Scholar 

  8. Freeman SM, Ramesh R, Marrogi AJ . Immune system in suicide-gene therapy. Lancet 1997; 349: 2–3.

    Article  CAS  PubMed  Google Scholar 

  9. Kokoris MS, Sabo P, Black ME . Enhancement of tumor ablation by a selected HSV-thymidine kinase mutant. Gene Ther 1999; 6: 1415–1426.

    Article  CAS  PubMed  Google Scholar 

  10. Brady WA, Kokoris MS, Fitzgibbon M, Black M . Cloning, characterization, and modeling of mouse and human guanylate kinases. J Biol Chem 1996; 271: 16734–16740.

    Article  CAS  PubMed  Google Scholar 

  11. Boehme R . Phosphorylation of 9-b-D-arabinofuranosylguanine monophosphate by Drosophila melanogaster guanylate kinase. J Biol Chem 1984; 258: 12346–12349.

    Google Scholar 

  12. Ostermann N, Lavie A, Padiyar S, Brundiers R, Veit T, Reinstein J et al. Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants’ improved kinetics with the HIV prodrug metabolite AZTMP. J Mol Biol 2000; 304: 43–53.

    Article  CAS  PubMed  Google Scholar 

  13. Fridland A, Connelly MC, Ashmun R . Relationship of deoxynucleotide changes to inhibition of DNA synthesis induced by the antiretroviral agent 3′-azido-3′-deoxythymidine and release of its monophosphate by human lymphoid cells (CCRF-CEM). Mol Pharmacol 1990; 37: 665–670.

    CAS  PubMed  Google Scholar 

  14. Black ME, Newcomb TG, Wilson HM, Loeb LA . Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc Natl Acad Sci USA 1996; 93: 3525–3529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Black ME, Kokoris MS, Sabo P . Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing. Cancer Res 2001; 61: 3022–3026.

    CAS  PubMed  Google Scholar 

  16. Willmon CL, Krabbenhoft E, Black ME . A guanylate kinase//HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. Gene Ther 2006; 13: 1309–1312.

    Article  CAS  PubMed  Google Scholar 

  17. Stolworthy TS, Krabbenhoft E, Black ME . A novel Escherichia coli strain allows functional analysis of guanylate kinase drug resistance and sensitivity. Anal Biochem 2003; 322: 40–47.

    Article  CAS  PubMed  Google Scholar 

  18. Dilber MS, Abedi MR, Christensson B, Bjorkstrand B, Kidder GM, Naus CCG et al. Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo. Cancer Res 1997; 57: 1523–1528.

    CAS  PubMed  Google Scholar 

  19. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM . In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992; 256: 1550–1552.

    Article  CAS  PubMed  Google Scholar 

  20. Kaldor J, Day N, Pettersson F, Clarke E, Pederson D, Mehnest W et al. Leukemia following chemotherapy for ovarian cancer. N Engl J Med 1990; 322: 1–6.

    Article  CAS  PubMed  Google Scholar 

  21. Relling MV, Rubnitz JE, Rivera GK, Boyett JM, Hancock ML, Felix CA et al. High incidence of secondary brain tumours after radiotherapy and antimetabolites. Lancet 1999; 354: 34–39.

    Article  CAS  PubMed  Google Scholar 

  22. Dachs GU, Tupper J, Tozer G . From bench to bedside for gene-directed enzyme prodrug therapy of cancer. Anticancer Drugs 2005; 16: 349–359.

    Article  CAS  PubMed  Google Scholar 

  23. Norris JS, Norris KL, Holman DH, El-Zawahry A, Keane TE, Dong Jy et al. The present and future for gene and viral therapy of directly accessible prostate and squamous cell cancers of the head and neck. Future Oncol 2005; 1: 115–123.

    Article  CAS  PubMed  Google Scholar 

  24. Pope IM, Poston GJ, Kinsella AR . The role of the bystander effect in suicide gene therapy. Eur J Cancer 1997; 33: 1005–1016.

    Article  CAS  PubMed  Google Scholar 

  25. Vrionis FD, Wu JK, Qi P, Waltzman M, Cherington V, Spray DC . The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions. Gene Ther 1997; 4: 577–585.

    Article  CAS  PubMed  Google Scholar 

  26. Akyürek L, Nallamshetty S, Aoki K, San H, Yang Z, Nabel G et al. Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol Ther 2001; 3: 779–786.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health through Grants R01CA85939 (to MEB) and T32GM008336 (to MS).

Author information

Authors and Affiliations

  1. School of Molecular Biosciences, Washington State University, Pullman, WA, USA

    A Ardiani & M E Black

  2. Department of Pharmaceutical Sciences, Washington State University, Pullman, WA, USA

    M Sanchez-Bonilla & M E Black

Authors
  1. A Ardiani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Sanchez-Bonilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M E Black
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M E Black.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ardiani, A., Sanchez-Bonilla, M. & Black, M. Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. Cancer Gene Ther 17, 86–96 (2010). https://doi.org/10.1038/cgt.2009.60

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2009.60

Keywords

This article is cited by

Search

Advanced search

Quick links