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.

  • Paper
  • Published:

Cellular redox state alters recombinant adeno-associated virus transduction through tyrosine phosphatase pathways

Gene Therapy volume 6, pages 1427–1437 (1999)Cite this article

Abstract

Several types of environmental damage including UV, hydroxyurea and ionizing irradiation have been shown to augment rAAV transduction. Current hypotheses suggest that these environmental stimuli lead to the enhanced production and/or activation of cellular factors important in the conversion of single-stranded DNA genomes to expressible forms. However, the mechanisms of action are currently unknown. We hypothesized that reactive oxygen intermediates (ROI) may play a common role in the augmentation of rAAV transduction by these environmental stimuli. Our results demonstrate that treatment with hydrogen peroxide can give equivalent or greater levels of augmentation in rAAV transduction as that seen by hydroxyurea or UV irradiation. For all environmental stimuli, pretreatment with the hydroxyl radical (H0.) scavenger, N-acetyl-L-cysteine (NAC), completely blocked augmentation of rAAV transduction. Furthermore, using electron spin resonance spectroscopy (ESR), we demonstrated that both UV and H2O2 treatment of cell lines lead to the induction of H0. radicals. Our results demonstrating that NaOV inhibits the augmentation of rAAV transduction following UV and H2O2 treatment, implicate H0. radicals as modulators of tyrosine phosphatase pathways involved in rAAV transduction. Alterations in the cellular redox state and subsequent activation of tyrosine phosphatase pathways appear to alter the phosphorylation status of the previously identified single-stranded sequence binding protein (ssD-BP), with reduced phosphorylation correlating with an enhancement in rAAV transduction. In summary, we conclude that the cellular redox state may play an important role in regulating rAAV transduction.

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
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Bennett J, Duan D, Engelhardt JF, Maguire AM . Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction Invest Ophthalmol Vis Sci 1997 38: 2857–2863.

    CAS  PubMed  Google Scholar 

  2. Ali RR et al. Gene transfer into the mouse retina mediated by an adeno-associated viral vector Hum Mol Genet 1996 5: 591–594

    Article  CAS  PubMed  Google Scholar 

  3. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector J Virol 1996 70: 8098–8108

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy Nature Med 1997 3: 306–312

    Article  CAS  PubMed  Google Scholar 

  5. Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain Nat Genet 1994 8: 148–154

    Article  CAS  PubMed  Google Scholar 

  6. Summerford C, Samulski RJ . Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions J Virol 1998 72: 1438–1445

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Qing K et al. Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression Proc Natl Acad Sci USA 1997 94: 10879–10884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Qing K et al. Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo J Virol 1998 72: 1593–1599

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Musonda CA, Chipman JK . Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-kappa B DNA binding activity and DNA damage in HepG2 cells Carcinogenesis 1998 19: 1583–1589

    Article  CAS  PubMed  Google Scholar 

  10. Zhang X et al. Identification of possible reactive oxygen species involved in ultraviolet radiation-induced oxidative DNA damage Free Radic Biol Med 1997 23: 980–985

    Article  CAS  PubMed  Google Scholar 

  11. Devary Y, Rosette C, DiDonato JA, Karin M . NF-kappa B activation by ultraviolet light not dependent on a nuclear signal Science 1993 261: 1442–1445

    Article  CAS  PubMed  Google Scholar 

  12. Schreck R, Rieber P, Baeuerle PA . Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1 EMBO J 1991 10: 2247–2258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sasaki K, Bannai S, Makino N . Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture Biochim Biophys Acta 1998 1380: 275–288

    Article  CAS  PubMed  Google Scholar 

  14. Oredsson S, Plate G, Qvarfordt P . Experimental evaluation of oxygen free radical scavengers in the prevention of reperfusion injury in skeletal muscle Eur J Surg 1994 160: 97–103

    CAS  PubMed  Google Scholar 

  15. Miller SC et al. Regulation of NF-kappa B and HIV-1 LTR activity in mouse L cells by ultraviolet radiation: LTR trans-activation in a nonirradiated genome in heterokaryons Exp Cell Res 1997 230: 9–21

    Article  CAS  PubMed  Google Scholar 

  16. Huang RP, Wu JX, Fan Y, Adamson ED . UV activates growth factor receptors via reactive oxygen intermediates J Cell Biol 1996 133: 211–220

    Article  CAS  PubMed  Google Scholar 

  17. Gamou S, Shimizu N . Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor FEBS Lett 1995 357: 161–164

    Article  CAS  PubMed  Google Scholar 

  18. Russell DW, Alexander IE, Miller AD . DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors Proc Natl Acad Sci USA 1995 92: 5719–5723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kawada N, Seki S, Inoue M, Kuroki T . Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells Hepatology 1998 27: 1265–1274

    Article  CAS  PubMed  Google Scholar 

  20. Duan D et al. Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4-ORF6 and E2a gene expression J Virol 1999 73: 161–169

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Blackwell TS et al. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation J Immunol 1996 157: 1630–1637

    CAS  PubMed  Google Scholar 

  22. Kikumori T et al. Activation of transcriptionally active nuclear factor-kappa B by tumor necrosis factor-alpha and its inhibition by antioxidants in rat thyroid FRTL-5 cells Endocrinology 1998 139: 1715–1722

    Article  CAS  PubMed  Google Scholar 

  23. Sprong RC et al. Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality Am J Respir Crit Care Med 1998 157: 1283–1293

    Article  CAS  PubMed  Google Scholar 

  24. Fisher KJ et al. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis J Virol 1996 70: 520–532

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors J Virol 1996 70: 3227–3234

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Flohe L et al. Redox regulation of NF-kappa B activation Free Radic Biol Med 1997 22: 1115–1126

    Article  CAS  PubMed  Google Scholar 

  27. Hirota K et al. AP-I transcriptional activity is regulated by a direct association between thioredoxin and Ref-1 Proc Natl Acad Sci USA 1997 94: 3633–3638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lo YYC, Wong JMS, Cruz TF . Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases J Biol Chem 1996 271: 15703–15707

    Article  CAS  PubMed  Google Scholar 

  29. Milne DM, Campbell LE, Campbell DG, Meek DW . p53 is phosphorylated in vitro and in vivo by an ultraviolet radiation-induced protein kinase characteristic of the c-Jun kinase, JNK1 J Biol Chem 1995 270: 5511–5518

    Article  CAS  PubMed  Google Scholar 

  30. Irani K et al. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts (see comments) Science 1997 275: 1649–1652

    Article  CAS  PubMed  Google Scholar 

  31. Jayaraman L et al. Identification of redox/repair protein Ref-1 as a potent activator of p53 Genes Dev 1997 11: 558–570

    Article  CAS  PubMed  Google Scholar 

  32. Samulski RJ, Chang LS, Shenk T . A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication J Virol 1987 61: 3096–3101

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Duan D, Fisher KJ, Burda JF, Engelhardt JF . Structural and functional heterogeneity of integrated recombinant AAV genomes Virus Res 1997 48: 41–56

    Article  CAS  PubMed  Google Scholar 

  34. Iimuro Y et al. NF-kappa B prevents apoptosis and liver dysfunction during liver regeneration J Clin Invest 1998 101: 802–811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Engelhardt JF et al. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses Nat Genet 1993 4: 27–34

    Article  CAS  PubMed  Google Scholar 

  36. Andrews NC, Faller DV . A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells Nucleic Acids Res 1991 19: 2499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The work described here was supported by NIH R01 DK/HL58340 (JFE) and the Gene Therapy Center supported by NIDDK (DK54759) and the Cystic Fibrosis Foundation. We also gratefully acknowledge Dr Larry Oberley for his helpful discussions on redox mechanisms and Dr Gary R Buettner, ESR facility at the University of Iowa.

Author information

Authors and Affiliations

  1. Department of Anatomy and Cell Biology and Department of Internal Medicine at the University of Iowa College of Medicine, Iowa City, Iowa, USA

    S Sanlioglu & J F Engelhardt

Authors
  1. S Sanlioglu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J F Engelhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sanlioglu, S., Engelhardt, J. Cellular redox state alters recombinant adeno-associated virus transduction through tyrosine phosphatase pathways. Gene Ther 6, 1427–1437 (1999). https://doi.org/10.1038/sj.gt.3300967

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3300967

Keywords

This article is cited by

Search

Advanced search

Quick links