SV40 Enhancer Research Articles

Protection from acute cellular injury using Sleeping Beauty mediated telomerase gene transfer

We developed a Sleeping Beauty (SB) transposon mediated hTERT gene delivery system for in vitro use. We have constructed telomerase or luciferase gene expressing SB-transposons with a SV40 enhancer (pT3.hTERT.Con and pT3.Con, respectively) or without an enhancer (pT3.Pro). Using the SB transposon system in vitro hTERT gene overexpression has protective effects from acute cellular injury by tert-butyl hydroperoxide ( t-BH), carbon tetrachloride (CCl 4), and d-galactosamine ( d-GalN) in normal human cells IMR-90. pT3.hTERT.Con vector and helper plasmid co-transfection resulted in a ∼3-fold increase in telomerase activity which was maintained for 14 days. Trypan blue and Cell Death Detection Assays showed the protective effects of the telomerase gene against toxic agents. Fourteen days after co-transfection with pT3.hTERT.Con vector and helper plasmid, IMR-90 cells were incubated with 1.2 mM t-BH for 50 min, 5 mM CCl 4 for 1.5 h or 30 mM d-GalN for 24 h. Cell viability of SB-mediated telomerase overexpressing cells significantly increased by 48% ( t-BH), 43% (CCl 4), and 25% ( d-GalN) in comparison to mock treated cells. Cell Death Detection ELISA showed a decrease in the rate of apoptosis by 47%. In summary, SB transposon mediated telomerase gene transfer may have a protective effect against t-BH, CCl 4, or d-GalN induced acute cellular injury, and this results suggested SB-mediated telomerase therapy for tissue engineering.

Functional characterization of putative promoter elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1 kb with three putative open reading frames (ORFs) on the same DNA strand designated, the Left, Middle, and Right ORFs. The Left ORF codes for non-structural protein and the Right ORF codes for capsid protein, whereas the role of the Middle ORF is still unknown. Two putative promoters, designated P2 and P61, were detected upstream of the Left ORF and Right ORF, respectively. We evaluated the activities of these two promoters with or without a transcriptional enhancer element via the use of firefly luciferase reporter constructs in insect and fish cells, and in shrimp tail muscle. In insect and fish cells, the P2 promoter was stronger than the P61 promoter. The presence of the SV40 enhancer element negatively affected P2 but not P61 promoter activity in insect cells. However, in fish cells, the SV40 enhancer element dramatically increased the activities of both promoters. In shrimp, there was no significant difference in luciferase expression driven by these two promoters. In shrimp tail muscle, the presence of SV40 enhancer element in the construct had no significant effect on the P2 promoter and a negative effect on the P61 promoter. The IHHNV P2 and P61 promoters were found to be constitutive promoters that can drive gene expression in both invertebrate and vertebrate hosts.

Analysis of transcriptional regulation of mouse FoxP3 gene expression in CD4+ CD25+ regulatory T cells (35.21)

Abstract FoxP3 is expressed at high level in CD4+CD25+ regulatory T cells (Treg) and functions as a key transcriptional factor that determines cell phenotype. Despite of the inherent interest, little is known concerning process of transcriptional regulation of FoxP3 due to lack of experimentally feasible to analyze promoter activity using transfection-based methods in natural Treg cells. We have, therefore, identified a model cell line that expresses FoxP3 at high levels upon stimulation. To identify the promoter region of the mouse FoxP3 gene, we determined the transcription initiation site by 5′RACE, using RNAs from CD4+CD25+ Treg cells and the cell line. The major start site mapped to the same position in the both type of cells, indicating that this cell line can be used as a model system. Luciferase reporter assays were performed using a 1.7kb 5′ franking fragment of the FoxpP3 gene as promoter. Although the cell line expressed FoxP3 mRNA at high levels, we could not detect any promoter activity in this region, suggesting that enhancers might be essential for this promoter to function. Indeed, we elicited that FoxP3 promoter activity by coupling it to the SV40 enhancer. We have now identified an enhancer region in the vicinity of the FoxP3 gene, that upon analysis have revealed important and unexpected insights into the transcriptional control of the FoxP3 gene.

Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer

Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed. Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice. It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner. The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

Improvement of transcriptional activity of hTERT promoter by SV40 enhancer

Improvement of transcriptional activity of hTERT promoter by SV40 enhancer

Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus

To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy.

Assessing IRES activity in the HIF-1α and other cellular 5′ UTRs

Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

Activation of a Methylated Promoter Mediated by a Sequence-specific DNA-binding Protein, RFX

The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation.

Transcriptional Activity of the Paired-like Homeodomain Proteins CHX10 and VSX1

CHX10 and VSX1 are homeodomain (HD) proteins essential for normal retinal development. CHX10 is required first for retinal progenitor cell proliferation and later for bipolar cell differentiation, whereas VSX1 is important in the terminal differentiation of a subset of bipolar cells. Elucidating the transcriptional activity of CHX10 and VSX1 is required to understand how these factors control retinal development. We show that CHX10 and Vsx1 can function as transcriptional repressors. When tethered to a promoter by a heterologous LexA DNA-binding domain or its HD, CHX10 repressed multiple classes of activators in different immortalized cell lines. CHX10 blocked TATA-containing and TATA-less promoters, repressed at a distance, and inhibited a complex enhancer positioned upstream or downstream of the reporter gene, whereas retinoblastoma protein (RB) inhibited the downstream enhancer only. Interestingly, CHX10 mildly potentiated a subset of activators in chick neuronal cultures. Thus, CHX10 is both a versatile repressor and a context-specific weak activator. The CHX10 HD and CVC domains were sufficient for DNA binding and repression. VSX1 contains closely related homeo and CVC domains and, like CHX10, also repressed transcription. A VSX1 HD mutation, R166W, that impairs DNA binding and causes keratoconus in humans, hindered repressor function. Therefore, CHX10 and VSX1 may control retinal bipolar cell specification or differentiation by repressing genes required for the development of other cell types.

Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer

It is well established that gene expression in eukaryotes is controlled by sequence-dependent binding of trans-acting proteins to regulatory elements like promoters, enhancers or silencers. A less well understood level of gene regulation is governed by the various structural and functional states of chromatin, which have been ascribed to changes in covalent modification of core histone proteins. And, much on how topological domains in the genome take part in establishing and maintaining distinct gene expression patterns is still unknown. Here we present a set of regulatory proteins that allow to reversibly alter the DNA structure in vivo and in vitro by adding low molecular weight effectors that control their oligomerization and DNA binding. Using this approach, we completely regulate the activity of an SV40 enhancer in HeLa cells by reversible loop formation to topologically separate it from the promoter. This result establishes a new mechanism for DNA-structure-dependent gene regulation in vivo and provides evidence supporting the structural model of insulator function.

Adenovirus-Mediated Suicide SCLC Gene Therapy Using the Increased Activity of the hTERT Promoter by the MMRE and SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc-Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60 approximately 80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.

Enhanced Expression of Apoptin by the Myc–Max Binding Motif and SV40 Enhancer for SCLC Gene Therapy

Apoptin is derived from chicken anemia virus (CAV) and known to induce tumor specific apoptosis but not normal cells. The aim of this study was to use increased expression of apoptin by the Myc-Max response element (MMRE) and SV40 enhancer in small-cell lung cancer (SCLC) gene therapy. To investigate the possibility of the utilization of the MMRE, apoptin, and SV40 promoter/enhancer in targeted cancer gene therapy, adenovirus vector expressing apoptin controlled by the MMRE, and SV40 promoter/enhancer was constructed. Ad-MMRE-apoptin-enh infected SCLC cells were significantly suppressed and induced apoptosis more than those of Ad-apoptin or Ad-apoptin-enh. Infection with Ad-MMRE-apoptin-enh of normal cells did not increase apoptosis. About 85% of SCLC tumors show overexpression of the myc family, so the increased expression of apoptin by MMRE and SV40 enhancer can be used in targeted SCLC gene therapy. These results indicate that apoptin expression was increased by the MMRE and SV40 promoter/enhancer, and that this strategy can be used in SCLC targeted cancer gene therapy.

The –1438A/G polymorphism in the 5-hydroxytryptamine type 2A receptor gene affects promoter activity

The -1438A/G single nucleotide polymorphism (SNP) lies just upstream of two alternative promoters for the 5-hydroxytryptamine type 2A (5-HT2A) receptor gene (HTR2A) and is in strong linkage disequilibrium with the 102T/C SNP. Both SNPs are associated with numerous psychiatric disorders and related phenotypes. A possible functional affect of the -1438A/G SNP might underlie associations of both linked SNPs with these neuropsychiatric disorders. A prior investigation into affects of this SNP on promoter function, lacking the more downstream promoter, found no significant difference with a reporter gene assay. To investigate possible functional effects of -1438A/G on either promoter, two different reporter gene assays were used in three cell lines. Promoter activity was consistently detected that, in the presence of the SV40 enhancer, was significantly greater in the presence of the A allele relative to the G allele but only in cell lines that express endogenous HTR2A, suggesting that transcriptional factor(s) and the presence of both promoters might be necessary to elicit this effect. These findings show that the -1438A/G SNP has the potential to modulate HTR2A promoter activity and might be the functional variant responsible for the associations of both SNPs with many neuropsychiatric phenotypes.

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer.

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.

343NUCLEAR LOCALIZATION SIGNAL AND CELL SYNCHRONY ENHANCES GENE TARGETING EFFICIENCY IN FETAL BOVINE FIBROBLASTS

The use of primary somatic cells for nuclear transfer has facilitated the manipulation of the domestic animal genome via homologous recombination. Yet, the absolute frequency of homologous recombination (HR) in somatic cells is two orders of magnitude lower than in ES cells whereas frequencies of non-homologous end joining are higher. While a few loci have been targeted in somatic cells using enrichment strategies similar to those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells, and difficulties in cloning the cell after targeting. We present evidence that the use of a nuclear localization signal (nls) and S-phase cell cycle synchronization by thymidine block enhances targeting efficiency at the hypoxanthine phosphoribosyl transferase locus in primary fetal bovine fibroblasts. We designed two hypoxanthine phosphoribosyl transferase (HPRT)-targeting constructs, HPRT-DEx6 and HPRT-DEx6-nls. Both constructs have a 31-bp deletion and a PGK-puro insertion in exon 6 to ensure inactivation of the HPRT locus. Additionally, the HPRT-DEx6-nls construct contains a 180-bp cassette comprised of two 72-bp tandem repeats from SV40 enhancer known to act as nuclear localization signal. Diploid male cells that undergo targeted gene disruption at the single copy X-linked HPRT locus can be selected with 8-Azaguanine (8-AG) as HPRT cells are resistant to 8-AG; all transformants, random and targeted, can be selected in puromycin. Male primary bovine fibroblasts were electroporated with linearized targeting constructs, and plated in media containing puromycin or puromycin plus 8AG. All experiments were done in triplicate and data were analyzed by two-way ANOVA with NLS and cell synchrony as the two factors. Significance was set at P<0.01. While the total number of insertions (random plus targeted) with both constructs were equivalent, the HPRT-DEx6-nls construct produced a significantly higher number of targeted colonies (1–2 8AG-resistant colonies per 9.5×106 cells) than HPRT-DEx6 where no targeted events were seen. Cells were synchronized in the S-phase of the cell cycle by a 2mM thymidine treatment for 24 hours and electroporated with the linearized targeting constructs. Compared to non-synchronous cells, the total number of insertions (random plus targeted) was reduced by 59-fold in constructs with or without nls (P<0.01), while targeted insertions increased 6-fold in the HPRT-DEx6-nls construct, from an average of 10 per million cells without nls to 7.6 per 10 million with nls (P<0.01). All 8AG-resistant colonies were verified by Long Range-PCR, and PCR products confirmed by end sequencing. This finding has important implications for targeting in somatic cells, as with a drastic reduction in the number of random insertions, and increased targeting due to the presence of the nls, identification of a targeted colony is greatly facilitated even in cases where no enrichment protocols are available.

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression

The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.

Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature.

Although the use of nonviral vectors for gene therapy offers distinct advantages including the lack of significant inflammatory and immune responses, the levels of expression in vivo remain much lower than those obtained with their viral counterparts. One reason for such low expression is that unlike many viruses, plasmids have not evolved mechanisms to target to the nucleus of the nondividing cell. In the absence of mitosis, plasmids are imported into the nucleus in a sequence-specific manner, and we have shown in cultured cells by transfection and microinjection experiments that the SV40 enhancer mediates plasmid nuclear import in all cell types tested (Dean et al., 1999, Exp Cell Res 253: 713-722). To test the effect of this import sequence on gene transfer in the intact animal, we have recently developed an electroporation method for DNA delivery to the intact mesenteric vasculature of the rat. Plasmids expressing luciferase or GFP from the CMV immediate-early promoter/enhancer and either containing or lacking the SV40 enhancer downstream of the reporter gene were transferred to the vasculature by electroporation. When transfected into actively dividing populations of smooth muscle or epithelial cells, the plasmids gave similar levels of expression. By contrast, the presence of the SV40 sequence greatly enhanced gene expression of both reporters in the target tissue. At 2 days post-transfer, plasmids with the SV40 sequence gave 10-fold higher levels of luciferase expression, and at 3 days the difference was over 40-fold. The presence of the SV40 sequence did not simply increase the rate of nuclear import and expression, since expression from the SV40-lacking plasmid did not increase beyond that seen at day 2, the time of maximum expression for either plasmid. In situ hybridization experiments confirmed that the increased gene transfer and expression was indeed due to increased nuclear localization of the delivered SV40 sequence-containing plasmid. Based on these findings, the ability to target DNA to the nucleus can increase gene transfer in vivo and inclusion of the SV40 sequence into plasmids will enhance nonviral gene delivery.

Isolation and functional analysis of the melanoma specific promoter region of human GD3 synthase gene

Human GD3 synthase gene consisted of five exons and span about 135 kilobases. The 5′-flanking region lacked canonical TATA and CAAT boxes, but contained SP1 binding site(s) as in rat and mouse. The promoter activity in the 5′-flanking region (−2262∼+1) became definite when SV40 enhancer was added to the reporter plasmid. Luciferase assay with deletion mutants suggested the existence of a silencer region between −2262 and −978 nt similarly with those in mouse and rat. They also commonly contained a GT/CG repeat sequence at upstream of −1200∼−1300 nt, suggesting that they form Z-type DNA, and are involved in the gene regulation.

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.