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:

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses

Gene Therapy volume 13, pages 893–905 (2006)Cite this article

Abstract

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdΔEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Δ19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122–129 deletion (Δ24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to αv integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

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

Similar content being viewed by others

References

  1. Kirn D, Martuza RL, Zwiebel J . Replication-selective virotherapy for cancer: biological principles, risk management and future directions. Nat Med 2001; 7: 781–787.

    CAS  PubMed  Google Scholar 

  2. Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, Henderson DR . Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 1997; 57: 2559–2563.

    CAS  PubMed  Google Scholar 

  3. Hallenbeck PL, Chang YN, Hay C, Golightly D, Stewart D, Lin J et al. A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma. Hum Gene Ther 1999; 10: 1721–1733.

    CAS  PubMed  Google Scholar 

  4. Nettelbeck DM, Rivera AA, Balague C, Alemany R, Curiel DT . Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter. Cancer Res 2002; 62: 4663–4670.

    CAS  PubMed  Google Scholar 

  5. Zhang L, Akbulut H, Tang Y, Peng X, Pizzorno G, Sapi E et al. Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma. Mol Ther 2002; 6: 386–393.

    CAS  PubMed  Google Scholar 

  6. Peter I, Graf C, Dummer R, Schaffner W, Greber UF, Hemmi S . A novel attenuated replication-competent adenovirus for melanoma therapy. Gene Therapy 2003; 10: 530–539.

    CAS  PubMed  Google Scholar 

  7. Liu Y, Ye T, Sun D, Maynard J, Deisseroth A . Conditionally replication-competent adenoviral vectors with enhanced infectivity for use in gene therapy of melanoma. Hum Gene Ther 2004; 15: 637–647.

    CAS  PubMed  Google Scholar 

  8. Everts B, van der Poel HG . Replication-selective oncolytic viruses in the treatment of cancer. Cancer Gene Ther 2005; 12: 141–161.

    CAS  PubMed  Google Scholar 

  9. Heise CC, Williams A, Olesch J, Kirn DH . Efficacy of a replication-competent adenovirus (ONYX-015) following intratumoral injection: intratumoral spread and distribution effects. Cancer Gene Ther 1999; 6: 499–504.

    CAS  PubMed  Google Scholar 

  10. Bilbao R, Bustos M, Alzuguren P, Pajares MJ, Drozdzik M, Qian C et al. A blood–tumor barrier limits gene transfer to experimental liver cancer: the effect of vasoactive compounds. Gene Therapy 2000; 7: 1824–1832.

    CAS  PubMed  Google Scholar 

  11. Harrison D, Sauthoff H, Heitner S, Jagirdar J, Rom WN, Hay JG . Wild-type adenovirus decreases tumor xenograft growth, but despite viral persistence complete tumor responses are rarely achieved—deletion of the viral E1b-19-kD gene increases the viral oncolytic effect. Hum Gene Ther 2001; 12: 1323–1332.

    CAS  PubMed  Google Scholar 

  12. Barker DD, Berk AJ . Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology 1987; 156: 107–121.

    CAS  PubMed  Google Scholar 

  13. Sauthoff H, Heitner S, Rom WN, Hay JG . Deletion of the adenoviral E1b-19kD gene enhances tumor cell killing of a replicating adenoviral vector. Hum Gene Ther 2000; 11: 379–388.

    CAS  PubMed  Google Scholar 

  14. Kim J, Cho JY, Kim JH, Jung KC, Yun CO . Evaluation of E1B gene-attenuated replicating adenoviruses for cancer gene therapy. Cancer Gene Ther 2002; 9: 725–736.

    CAS  PubMed  Google Scholar 

  15. Liu TC, Hallden G, Wang Y, Brooks G, Francis J, Lemoine N et al. An E1B-19kDa gene deletion mutant adenovirus demonstrates tumor necrosis factor-enhanced cancer selectivity and enhanced oncolytic potency. Mol Ther 2004; 9: 786–803.

    CAS  PubMed  Google Scholar 

  16. Degenhardt K, Perez D, White E . Pathways used by adenovirus E1B 19K to inhibit apoptosis. Symp Soc Exp Biol 2000; 52: 241–251.

    CAS  PubMed  Google Scholar 

  17. Sherr CJ . The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000; 60: 3689–3695.

    CAS  PubMed  Google Scholar 

  18. Piepkorn M . Melanoma genetics: an update with focus on the CDKN2A(p16)/ARF tumor suppressors. J Am Acad Dermatol 2000; 42: 705–722.

    CAS  PubMed  Google Scholar 

  19. Fueyo J, Gomez-Manzano C, Alemany R, Lee PS, McDonnell TJ, Mitlianga P et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000; 19: 2–12.

    CAS  PubMed  Google Scholar 

  20. Heise C, Hermiston T, Johnson L, Brooks G, Sampson-Johannes A, Williams A et al. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med 2000; 6: 1134–1139.

    CAS  PubMed  Google Scholar 

  21. Doronin K, Toth K, Kuppuswamy M, Ward P, Tollefson AE, Wold WS . Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein. J Virol 2000; 74: 6147–6155.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Howe JA, Demers GW, Johnson DE, Neugebauer SE, Perry ST, Vaillancourt MT et al. Evaluation of E1-mutant adenoviruses as conditionally replicating agents for cancer therapy. Mol Ther 2000; 2: 485–495.

    CAS  PubMed  Google Scholar 

  23. Cripe TP, Dunphy EJ, Holub AD, Saini A, Vasi NH, Mahller YY et al. Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells. Cancer Res 2001; 61: 2953–2960.

    CAS  PubMed  Google Scholar 

  24. Mizuguchi H, Hayakawa T . Targeted adenovirus vectors. Hum Gene Ther 2004; 15: 1034–1044.

    CAS  PubMed  Google Scholar 

  25. Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998; 72: 9706–9713.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Koizumi N, Mizuguchi H, Hosono T, Ishii-Watabe A, Uchida E, Utoguchi N et al. Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide. Biochim Biophys Acta 2001; 1568: 13–20.

    CAS  PubMed  Google Scholar 

  27. Garcia-Castro J, Segovia JC, Garcia-Sanchez F, Lillo R, Gomez-Navarro J, Curiel DT et al. Selective transduction of murine myelomonocytic leukemia cells (WEHI-3B) with regular and RGD-adenoviral vectors. Mol Ther 2001; 3: 70–77.

    CAS  PubMed  Google Scholar 

  28. Nakamura T, Sato K, Hamada H . Effective gene transfer to human melanomas via integrin-targeted adenoviral vectors. Hum Gene Ther 2002; 13: 613–626.

    CAS  PubMed  Google Scholar 

  29. Nagel H, Maag S, Tassis A, Nestle FO, Greber UF, Hemmi S . The alphavbeta5 integrin of hematopoietic and nonhematopoietic cells is a transduction receptor of RGD-4C fiber-modified adenoviruses. Gene Therapy 2003; 10: 1643–1653.

    CAS  PubMed  Google Scholar 

  30. Volk AL, Rivera AA, Kanerva A, Bauerschmitz G, Dmitriev I, Nettelbeck DM et al. Enhanced adenovirus infection of melanoma cells by fiber-modification: incorporation of RGD peptide or Ad5/3 chimerism. Cancer Biol Ther 2003; 2: 511–515.

    CAS  PubMed  Google Scholar 

  31. Rivera AA, Davydova J, Schierer S, Wang M, Krasnykh V, Yamamoto M et al. Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells. Gene Therapy 2004; 11: 1694–1702.

    CAS  PubMed  Google Scholar 

  32. Shayakhmetov DM, Papayannopoulou T, Stamatoyannopoulos G, Lieber A . Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector. J Virol 2000; 74: 2567–2583.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Havenga MJ, Lemckert AA, Grimbergen JM, Vogels R, Huisman LG, Valerio D et al. Improved adenovirus vectors for infection of cardiovascular tissues. J Virol 2001; 75: 3335–3342.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Havenga MJ, Lemckert AA, Ophorst OJ, van Meijer M, Germeraad WT, Grimbergen J et al. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J Virol 2002; 76: 4612–4620.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Segerman A, Atkinson JP, Marttila M, Dennerquist V, Wadell G, Arnberg N . Adenovirus type 11 uses CD46 as a cellular receptor. J Virol 2003; 77: 9183–9191.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Gaggar A, Shayakhmetov DM, Lieber A . CD46 is a cellular receptor for group B adenoviruses. Nat Med 2003; 9: 1408–1412.

    CAS  PubMed  Google Scholar 

  37. Sirena D, Lilienfeld B, Eisenhut M, Kalin S, Boucke K, Beerli RR et al. The human membrane cofactor CD46 is a receptor for species B adenovirus Serotype 3. J Virol 2004; 78: 4454–4462.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Pilder S, Logan J, Shenk T . Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA. J Virol 1984; 52: 664–671.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Fleischli C, Verhaagh S, Havenga M, Sirena D, Schaffner W, Cattaneo R et al. The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35. J Virol 2005; 79: 10013–10022.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Stephens C, Harlow E . Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins. EMBO J 1987; 6: 2027–2035.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. White E, Cipriani R, Sabbatini P, Denton A . Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J Virol 1991; 65: 2968–2978.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Subramanian T, Tarodi B, Chinnadurai G . p53-independent apoptotic and necrotic cell deaths induced by adenovirus infection: suppression by E1B 19K and Bcl-2 proteins. Cell Growth Differ 1995; 6: 131–137.

    CAS  PubMed  Google Scholar 

  43. Hemmi S, Geertsen R, Mezzacasa A, Peter I, Dummer R . The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures. Hum Gene Ther 1998; 9: 2363–2373.

    CAS  PubMed  Google Scholar 

  44. Short JJ, Pereboev AV, Kawakami Y, Vasu C, Holterman MJ, Curiel DT . Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology 2004; 322: 349–359.

    CAS  PubMed  Google Scholar 

  45. Zhang J, Ramesh N, Chen Y, Li Y, Dilley J, Working P et al. Identification of human uroplakin II promoter and its use in the construction of CG8840, a urothelium-specific adenovirus variant that eliminates established bladder tumors in combination with docetaxel. Cancer Res 2002; 62: 3743–3750.

    CAS  PubMed  Google Scholar 

  46. Subramanian T, Kuppuswamy M, Gysbers J, Mak S, Chinnadurai G . 19-kDa tumor antigen coded by early region E1b of adenovirus 2 is required for efficient synthesis and for protection of viral DNA. J Biol Chem 1984; 259: 11777–11783.

    CAS  PubMed  Google Scholar 

  47. Subramanian T, Kuppuswamy M, Mak S, Chinnadurai G . Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen. J Virol 1984; 52: 336–343.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Hu MC, Hsu MT . Adenovirus E1B 19K protein is required for efficient DNA replication in U937 cells. Virology 1997; 227: 295–304.

    CAS  PubMed  Google Scholar 

  49. Chiou SK, White E . Inhibition of ICE-like proteases inhibits apoptosis and increases virus production during adenovirus infection. Virology 1998; 244: 108–118.

    CAS  PubMed  Google Scholar 

  50. Tollefson AE, Ryerse JS, Scaria A, Hermiston TW, Wold WS . The E3-11.6-kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants. Virology 1996; 220: 152–162.

    CAS  PubMed  Google Scholar 

  51. Sauthoff H, Pipiya T, Heitner S, Chen S, Norman RG, Rom WN et al. Late expression of p53 from a replicating adenovirus improves tumor cell killing and is more tumor cell specific than expression of the adenoviral death protein. Hum Gene Ther 2002; 13: 1859–1871.

    CAS  PubMed  Google Scholar 

  52. Banerjee NS, Rivera AA, Wang M, Chow LT, Broker TR, Curiel DT et al. Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures. Mol Cancer Ther 2004; 3: 437–449.

    CAS  PubMed  Google Scholar 

  53. Philipson L, Pettersson RF . The coxsackie-adenovirus receptor—a new receptor in the immunoglobulin family involved in cell adhesion. Curr Top Microbiol Immunol 2004; 273: 87–111.

    CAS  PubMed  Google Scholar 

  54. Kanerva A, Wang M, Bauerschmitz GJ, Lam JT, Desmond RA, Bhoola SM et al. Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses. Mol Ther 2002; 5: 695–704.

    CAS  PubMed  Google Scholar 

  55. Okada N, Masunaga Y, Okada Y, Mizuguchi H, Iiyama S, Mori N et al. Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice. Gene Therapy 2003; 10: 1891–1902.

    CAS  PubMed  Google Scholar 

  56. Yotnda P, Zompeta C, Heslop HE, Andreeff M, Brenner MK, Marini F . Comparison of the efficiency of transduction of leukemic cells by fiber-modified adenoviruses. Hum Gene Ther 2004; 15: 1229–1242.

    CAS  PubMed  Google Scholar 

  57. Nemerow GR, Stewart PL . Role of alpha(v) integrins in adenovirus cell entry and gene delivery. Microbiol Mol Biol Rev 1999; 63: 725–734.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF . Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus. EMBO J 2001; 20: 1310–1319.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Greber UF . Signalling in viral entry. Cell Mol Life Sci 2002; 59: 608–626.

    CAS  PubMed  Google Scholar 

  60. Maizel Jr JV, White DO, Scharff MD . The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 1968; 36: 115–125.

    CAS  PubMed  Google Scholar 

  61. Sarnow P, Sullivan CA, Levine AJ . A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells. Virology 1982; 120: 510–517.

    CAS  PubMed  Google Scholar 

  62. Harlow E, Franza Jr BR, Schley C . Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J Virol 1985; 55: 533–546.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Ebbinghaus C, Al-Jaibaji A, Operschall E, Schoffel A, Peter I, Greber UF et al. Functional and selective targeting of adenovirus to high-affinity Fcgamma receptor I-positive cells by using a bispecific hybrid adapter. J Virol 2001; 75: 480–489.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L Fuchs, J Willers and P Selvam for excellent technical assistance, F Ochsenbein for artwork, S Rusconi (University of Fribourg, Switzerland) for pTG-H5dl324 plasmid and M Havenga (Crucell Holland BC, The Netherlands) for wt Ad35 and AdCMV-eGFP-F35 (rAd5Fib35 eGFP). Isabelle Peter was supported by the Julius Klaus Foundation, Silvio Hemmi was supported by the Cancer Society of the Kanton Zürich and the University of Zürich and Reinhard Dummer and Urs Greber were supported by the Swiss National Science Foundation (3200-063704.00/1 and 31-67002.01, respectively).

Author information

Author notes

  1. M Schmitz, C Graf and T Gut: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biology, University of Zürich, Zürich, Switzerland

    M Schmitz, C Graf, T Gut, D Sirena, I Peter & S Hemmi

  2. Department of Dermatology, University Hospital, Zürich, Switzerland

    R Dummer

  3. Institute of Zoology, University of Zürich, Zürich, Switzerland

    U F Greber

Authors
  1. M Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C Graf
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T Gut
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Sirena
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. I Peter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R Dummer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. U F Greber
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S Hemmi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S Hemmi.

Additional information

Supplementary Information accompanies the paper on the Gene Therapy website (http://www.nature.com/gt).

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmitz, M., Graf, C., Gut, T. et al. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses. Gene Ther 13, 893–905 (2006). https://doi.org/10.1038/sj.gt.3302739

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302739

Keywords

This article is cited by

Search

Advanced search

Quick links