Modulation of adenovirus-mediated gene transfer by nitric oxide

We assessed the role of . NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 mu M), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N-G-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin mediated induction of . NO and restored gene transfer to baseline values. Incubation bf NIH3T3/iNOS with 8-bromo-cGMP (400 mu M) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of . NO, did not alter the efficacy of gene transfer. . NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting . NO inhibited gene expression at either the transcriptional or posttranscriptional levels. To investigate the effects of inhaled . NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 mu l of saline) and exposed to . NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n greater than or equal to 8). We concluded that overproduction of . NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.