Adenovirus-mediated inducible nitric oxide synthase gene transfer inhibits hepatocyte apoptosis.
BACKGROUND
Apoptosis limits hepatocyte viability in bioartificial livers in vitro and may contribute to liver dysfunction in vivo. Nitric oxide (NO) inhibits hepatocyte apoptosis; however, methods to deliver NO in a sustained manner to hepatocytes are limited. Here, we tested the feasibility of inducible NO synthase (iNOS) gene transfer as an approach to deliver an intracellular source of NO to inhibit spontaneous and tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in cultured hepatocytes.


METHODS
An adenoviral vector carrying the human iNOS gene (AdiNOS) was used to overexpress iNOS in cultured rat hepatocytes. Spontaneous apoptosis was induced by prolonged culture (4 days), and stimulated apoptosis was induced by exposure to TNF-alpha + actinomycin D (TNF-alpha ActD). Nitrite (NO2-), cell viability, and cellular caspase-3-like protease activity were measured.


RESULTS
AdiNOS gene transfer resulted in sustained NO production and protected hepatocytes from spontaneous and TNF-alpha + ActD-induced apoptosis. Apoptosis was associated with increases in caspase-3-like protease activity, which was suppressed by iNOS gene transfer in an NO-dependent manner. Dithiothreitol partially reversed the NO-induced suppression of caspase-3-like activity, which is consistent with S-nitrosylation of caspase-3.


CONCLUSIONS
Adenovirus-mediated iNOS gene transfer effectively blocks spontaneous and TNF-alpha + ActD-induced cell killing in hepatocytes. iNOS gene transfer could be used to suppress apoptotic hepatocyte death in vitro and possibly in vivo.
Department of Surgery, University of Pittsburgh, Pa 15261, USA.
E
E
Tzeng
T R
TR
Billiar
D L
DL
Williams
J
J
Li
A
A
Lizonova
I
I
Kovesdi
Y M
YM
Kim
D000256:Adenoviridae; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008297:Male; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004792:Enzyme Precursors; D011500:Protein Synthesis Inhibitors; D013439:Sulfhydryl Reagents; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D004229:Dithiothreitol