N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry

J Gene Med. Jan-Feb 2002;4(1):54-65. doi: 10.1002/jgm.232.


Background: It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated.

Methods: HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis.

Results: Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA.

Conclusion: Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine*
  • Adenoviridae / physiology*
  • Cells, Cultured
  • Coxsackie and Adenovirus Receptor-Like Membrane Protein
  • Endothelium, Vascular / physiology*
  • Gene Expression
  • Gene Transfer Techniques*
  • Genetic Vectors*
  • Humans
  • Lac Operon
  • Receptors, Virus / physiology
  • Thioctic Acid / physiology
  • Transcription, Genetic
  • Transgenes
  • Umbilical Veins
  • Up-Regulation
  • beta-Galactosidase


  • CLMP protein, human
  • Coxsackie and Adenovirus Receptor-Like Membrane Protein
  • Receptors, Virus
  • Thioctic Acid
  • beta-Galactosidase
  • Acetylcysteine