Am. J. Respir. Cell Mol. Biol., Vol 16, No. 5, 05 1997, 501-509.
Modulation of adenovirus-mediated gene transfer by nitric oxide
IY Haddad, EJ Sorscher, RI Garver Jr, J Hong, E Tzeng and S Matalon
Department of Pediatrics, University of Alabama at Birmingham, 35233- 6810, USA.
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 microM), 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 of NIH3T3/iNOS with 8-bromo-cGMP (400 microM)
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 transriptional
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 microl 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 > or = 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.
