N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.
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.
Jornot
L
L
Respiratory Division, Geneva University Hospitals, 1211 Geneva 14, Switzerland. Lan.H.Jornot@hcuge.ch
Morris
M A
MA
Petersen
H
H
Moix
I
I
Rochat
T
T
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
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus; D008063:Thioctic Acid; D001616:beta-Galactosidase; D000111:Acetylcysteine