Nitric oxide donors inhibit luciferase expression in a promoter-independent fashion.
Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 microm NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at approximately 300 microm. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.
Fan
Xian
X
Department of Medicine, Emory University Medical School and Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA. xfan@emory.edu
Roy
Eileen
E
Zhu
Liping
L
Murphy
Tamara C
TC
Kozlowski
Mirek
M
Nanes
Mark S
MS
Rubin
Janet
J
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006207:Half-Life; D006801:Humans; D008156:Luciferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D009569:Nitric Oxide; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase