Induction of human NAD(P)H:quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin.

Flavonoids are plant polyphenolic compounds ubiquitous in fruits, vegetables and herbs. The flavonol quercetin is one of the most abundant dietary flavonoids. It has diverse biological properties in cultured cells, including cytoprotection, and exhibits antitumorigenic effects in animal models. The mechanism(s) for the protective properties of flavonoids are currently unknown but may involve modulation of phase II detoxifying enzymes. We have investigated the effect of quercetin on expression and enzymatic activity of one of the major phase II detoxification systems, NAD(P)H:quinone oxidoreductase (NQO1) in the MCF-7 human breast carcinoma cell line. We show that treatment of MCF-7 cells for 24 h with 15 microM quercetin results in a twofold increase in NQO1 protein levels and enzyme activity, and a three- to fourfold increase in NQO1 mRNA expression. We found that when these cells were transiently transfected with a luciferase (Luc) reporter plasmid containing two copies of the antioxidant response element (ARE) of the human NQO1 gene linked to a minimal viral promoter, quercetin caused an approximately twofold increase in Luc activity. Quercetin failed to increase Luc activity in cells transfected with a reporter vector containing a mutated ARE. The increase in NQO1 transcription in response to quercetin suggests that dietary plant polyphenols can stimulate transcription of phase II detoxifying systems, potentially through an ARE-dependent mechanism. Induction of the human NQO1 gene by dietary polyphenolics could afford protection against carcinogenic chemicals in molecular pathways utilizing the ARE.

Rapid polyubiquitination and proteasomal degradation of a mutant form of NAD(P)H:quinone oxidoreductase 1.

The NAD(P)H:quinone oxidoreductase 1 (NQO1)*2 polymorphism is characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.

NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed.

DT-diaphorase activity in NSCLC and SCLC cell lines: a role for fos/jun regulation.

To assess the potential differential lung tumour expression of NAD(P)H:quinone reductase (NQO1), the human (h) NQO1 promoter was characterized in gene transfer studies. A deletion panel of 5' flanking hNQO1 promoter constructs was made and tested in transient transfection assays in NSCLC and SCLC cell lines. The largest hNQO1 construct (-1539/+115) containing the antioxidant response element (ARE), exhibited robust levels of reporter activity in the NSCLC (H460, H520, and A549) cell lines and expression was over 12 to 77-fold higher than the minimal (-259/+115) promoter construct. In contrast, there was little difference in promoter activity between the largest and minimal promoter construct in the SCLC (H146, H82 and H187) cell lines. Deletion of the sites for NFkappaB and AP-2 and the XRE did not significantly affect hNQO1 promoter activity in either the NSCLC or SCLC cell lines. Robust promoter activity in NSCLC lines was mediated by a 359 bp segment of the proximal promoter that contained a canonical AP-1 binding site, TGACTCAG, within the ARE. Gel supershift assays with various specific Fos/Jun antibodies identified Fra1, Fra2 and Jun B binding activity in NSCLC cells to a promoter fragment (-477 to -438) spanning the AP-1 site, whereas SCLC do not appear to express functional Fra or Jun B. These results suggest a possible role for AP-1 activity in the differential expression of hNQO1 in NSCLC.

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells.

To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.

Differential expression of the expression site-associated gene I family in African trypanosomes.

A minimum of 20 different mRNA species encoding related members of the expression site-associated gene I (ESAG-I) family occur in metacyclic variant antigen type 4 bloodstream trypanosomes. None of these ESAG-I mRNAs are derived from the metacyclic variant antigen type 4 variant surface glycoprotein (VSG) gene expression site, and some appear to come from pseudogenes. The ESAG-Is are transcribed in both procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a detectable steady state level only in bloodstream trypanosomes. At least five different groups of 3'-untranslated regions (3'-UTRs) are represented among these ESAG-I mRNAs, suggesting that the 3'-UTR does not contribute to their differential expression. Some ESAG-I mRNAs completely lack a 3'-UTR or have only a single nucleotide as a 3'-UTR. Transcription of the ESAG-Is is sensitive to alpha-amanitin, indicating that they are transcribed by a different RNA polymerase than the VSG genes. These results collectively demonstrate that ESAG-I's are a heterogeneous population that can be expressed independently of VSG genes, but like the VSG genes, their mRNAs are present in the bloodstream stage of the parasite and not in the procyclic stage.

GnRH gene expression in neuronal cell lines.

Little is known about the mechanisms that subserve GnRH synthesis. The recent availability of clonal populations of GnRH neurons enables us for the first time to examine the structural organization of the GnRH locus. Studies are underway to dissect the manner in which neural specific expression and developmental regulation of GnRH are programmed. These studies will complement and provide direction to future animal studies designed to better understand the control of GnRH gene expression.

Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH.

The mechanisms by which steroid receptors repress gene expression are not well understood. In this report, we show that progesterone receptor (PR), in the presence of progesterone (P) directly represses rat gonadotropin releasing hormone (rGnRH) gene transcription. Deletion analysis studies using transient transfection assays in GT1-7 neuronal cells mapped the effects of P to sequences in the proximal rGnRH promoter between -171 and -73. This DNA sequence lacks any consensus steroid response element binding sites. Cotransfection of a mutant progesterone receptor that lacks a functional DNA binding region (hPRcys) abolished repression of the rGnRH promoter by P. Gel mobility shift assays confirmed that PR directly binds to the DNA fragments -171/-126, -126/-73, and -111/-73, which encompass the negative progesterone response element (nPRE) of the rGnRH promoter. Mutagenesis of the rGnRH nPRE -171/-126 DNA fragment resulted in a loss of PR binding. Thus, direct DNA binding of PR to nonconsensus elements in the proximal rGnRH promoter inhibits rGnRH gene expression.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells.

1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence. 2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis. 3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression. 4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.

Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells.

To dissect the functional architecture of the rat gonadotropin releasing hormone (GnRH) gene promoter and its regulation by estrogen, gene transfer studies were performed in a placental cell line. 5'-Deletional constructs demonstrated that cis-acting elements important for rGnRH promoter activity in placenta were contained within a region of the proximal promoter between -73 and -16 bp upstream of the transcription initiation site. In addition, an inhibitory region was found from -903 to -424. Deletion of sequences to -424 which removed the inhibitory region, produced a promoter fragment which exhibited a 50% inhibition of GnRH promoter activity in the presence of estrogen (E) and cotransfected estrogen receptor. Negative regulation by E was retained in constructs deleted to -73 bp. We conclude that sequences important for placental cell expression and estrogen regulation of rGnRH lie within the region from -73 to -16. In addition, potential repressor sequences active in placental cells are present between -903 to 424.

Structure of the rat gonadotropin releasing hormone (rGnRH) gene promoter and functional analysis in hypothalamic cells.

The gonadotropin releasing hormone (GnRH) gene encodes a protein which plays a critical role in mammalian reproductive physiology. Its expression is predominantly restricted to the hypothalamus although it has also been described in the placenta. To begin to determine the promoter elements important for tissue specific expression and to examine the mechanisms of developmental and hormonal regulation of the rat GnRH (rGnRH) gene, we cloned the rGnRH gene from a rat liver genomic DNA library. The nucleotide sequence of greater than 3 kb of 5'-flanking region was determined. The transcriptional initiation site in rat hypothalamic tissue and a mouse hypothalamic cell line were mapped by primer extension analysis and found to be different. In addition, transient transfection studies demonstrated that multiple regions of the distal promoter are important for tissue specific and basal promoter activity in hypothalamic cells. Furthermore, in these cells a potent activation region resides between -3026 and -1031 bp and suppressor region between -1031 and -903 bp upstream of the transcriptional start site. We conclude that different portions of the 5'-flanking region, which are activating and suppressing in nature, are critical for hypothalamic expression of the rGnRH gene.