title: N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Respiratory Division, Geneva University Hospitals, 1211 Geneva 14, Switzerland. Lan.H.Jornot@hcuge.ch
authors.forename: L
authors.initials: L
authors.lastname: Jornot
authors.forename: M A
authors.initials: MA
authors.lastname: Morris
authors.forename: H
authors.initials: H
authors.lastname: Petersen
authors.forename: I
authors.initials: I
authors.lastname: Moix
authors.forename: T
authors.initials: T
authors.lastname: Rochat
mesh_terms: D000111:Acetylcysteine; D000256:Adenoviridae / Q000502:physiology*; D002478:Cells, Cultured; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D004730:Endothelium, Vascular / Q000502:physiology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D011991:Receptors, Virus / Q000502:physiology; D008063:Thioctic Acid / Q000502:physiology; D014158:Transcription, Genetic; D019076:Transgenes; D014471:Umbilical Veins; D015854:Up-Regulation; D001616:beta-Galactosidase
chemical_list: C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus; D008063:Thioctic Acid; D001616:beta-Galactosidase; D000111:Acetylcysteine