A reporter cell line for real-time imaging of autophagy and apoptosis.

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.

BACKGROUND: NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening. METHOD: We recombined the reporter gene vector with inserting the "neo" transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela. RESULT: In this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes. CONCLUSION: In conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

Saucerneol G, a new lignan, from Saururus chinensis inhibits matrix metalloproteinase-9 induction via a nuclear factor κB and mitogen activated protein kinases in lipopolysaccharide-stimulated RAW264.7 cells.

This study was conducted to demonstrate the inhibitory effect of saucerneol G (SG), a new lignan, isolated from the aerial part of Saururus chinensis (Saururaceae) on lipopolysaccharide (LPS)-stimulated matrix metalloproteinase-9 (MMP)-9 inductions in RAW 264.7 cells. Aimed at evaluating the mechanism of action by which SG inhibits the LPS-mediated induction of MMP-9, the effects of SG on nuclear factor-κB (NF-κB) DNA binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation, degradation and p65 nuclear translocation were assessed. SG profoundly suppressed the DNA binding activity and the reporter gene activity as well as translocation of NF-κB p65 subunit. Furthermore, SG also dose dependently inhibited LPS-stimulated activation of mitogen-activated protein kinases (MAPKs). These findings suggest that SG may inhibit LPS-stimulated MMP-9 induction by blocking NF-κB and MAPKs activation.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64 percent, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Evaluation of anti-Wnt/ß-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/ß-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator ß-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/ß-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X ß-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3ß inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant ß-catenin signaling cells and decreased ß-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/ß-catenin signaling inhibitor NCTD.

Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans.

Epigallocatechin gallate (EGCG) is a major green tea polyphenol with pronounced antioxidative activity. The effects of EGCG on lifespan and stress resistance in wild-type N2 and transgenic strains of Caenorhabditis elegans [ HSP-16.2/GFP, MEV-1(KN1), FEM-1(HC17)] were investigated. The expression of HSP-16.2 (induced by the pro-oxidant juglone) and the intracellular levels of H (2)O (2) were inhibited by EGCG treatment. Daily administration of 220 muM EGCG increased the mean lifespan by 10.14 % and 14.27 % in N2 and FEM-1(HC17) strains, respectively, and 55 muM EGCG increased the mean lifespan in MEV-1(KN1) by 16.11 %. The survival rate was also increased under lethal oxidative stress by 65.05 %. These findings suggest that the increased mean lifespan and stress resistance in C. ELEGANS apparently depend, among other factors, on the antioxidant properties of EGCG.

The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling.

Cell integrity in Saccharomyces cerevisiae is ensured by a rigid cell wall whose synthesis is controlled by a highly conserved MAP kinase signal transduction cascade. Stress at the cell surface is detected by a set of sensors and ultimately transmitted through this cascade to the transcription factor Rlm1, which governs expression of many genes encoding enzymes of cell wall biosynthesis. We here report on a number of versatile reporter constructs which link activation of a hybrid, Rlm1-lexA, by the MAP kinase Mpk1/Slt2 to the expression of the bacterial lacZ gene. This system was adapted to automated microwell screening and shown to be activated by a number of compounds inhibiting cell wall biosynthesis or interfering with plasma membrane function. In addition, we tested tea tree oil and two of its purified constituents (alpha-terpineol, terpinen-4-ol) for their effects on growth and on cell integrity signalling using such reporter strains. Tea tree oil was found to inhibit growth of wild-type and slg1/wsc1 mutant cells at a threshold of approximately 0.1% v/v, with the purified compounds acting already at half these concentrations. A mid2 deletion displayed hyper-resistance. Tea tree oil also induces the signalling pathway in a dose-dependent manner.

Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters.

Tumor necrosis factor-alpha (TNF-alpha) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF-alpha in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF-alpha upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF-alpha activates canonical pro-inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin-1beta, interleukin-6, interleukin-10 and suppressor of cytokine signalling-3, respectively. In addition, TNF-alpha induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF-alpha may act directly in the hypothalamus inducing a pro-inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.

Strengths and weaknesses of in vitro assays for estrogenic and androgenic activity.

The endocrine and reproductive effects of xenobiotics are believed to be due to (1) their mimicking the effects of endogenous hormones; (2) their antagonizing the effects of endogenous hormones; (3) their altering the pattern of synthesis and metabolism of natural hormones; and (4) their modifying hormone receptor levels. It has been suggested that endocrine disruptors may play a role in the decrease in human semen quantity and quality, an increase in the anomalies of male genital tract, and an increase in the testicular and breast cancer incidence during the last 50 years. Testing these hypotheses will require: (1) identifying estrogen and androgen agonists and antagonists among the chemicals present in the environment; (2) assessing the interactions among the endocrine disruptors to which humans are exposed; and (3) finding markers of estrogen (and androgen) exposure. The development of fast and sensitive bioassays is central to the achievement of these three goals.

Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.

In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.

Polynorbornene polycationic polymers as gene transfer agents. Influence of the counterion for in vitro transfection.

Polycationic derivatives of polynorbornene with different non-cytotoxic counterions, have been prepared by organometallic polymerization of methyleneammonium norbornene and subsequent exchange of the counterion. In this paper the effect of the counterion on the polycationic polymer binding onto plasmid DNA was studied via different ethidium bromide assays, heparin displacement and protection against degradation by DNAse. According to the nature of the counterions and consequently the size of the polymer particles, their complexation with the DNA led to aggregates with variable binding affinity for the plasmid. The relative transfection efficiency of each polyplex was compared, on the basis of reporter gene expression, in cells in culture. The nature of the counterion was seen to affect gene delivery. The order of transfection efficiency of the counterions studied at equivalent charge ratios (NH3+/PO4-) is lactobionate, acetate, chloride. The results obtained with the polynorbornene methyleneammonium lactobionate and acetate are particularly encouraging.

Activity-dependent transcription regulation of PSD-95 by neuregulin-1 and Eos.

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. Here we report that, in the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD-Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of gamma-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD-Eos complex provides a molecular basis for activity-dependent synaptic plasticity.

An extract of Uncaria tomentosa inhibiting cell division and NF-kappa B activity without inducing cell death.

Previous reports have demonstrated that extracts of the plant Uncaria tomentosa inhibit tumor cell proliferation and inflammatory responses. We have confirmed that C-Med 100, a hot water extract of this plant, inhibits tumor cell proliferation albeit with variable efficiency. We extend these findings by showing that this extract also inhibits proliferation of normal mouse T and B lymphocytes and that the inhibition is not caused by toxicity or by induction of apoptosis. Further, the extract did not interfere with IL-2 production nor IL-2 receptor signaling. Since there was no discrete cell cycle block in C-Med 100-treated cells, we propose that retarded cell cycle progression caused the inhibition of proliferation. Collectively, these data suggested interference with a common pathway controlling cell growth and cell cycle progression. Indeed, we provide direct evidence that C-Med 100 inhibits nuclear factor kappa B (NF-kappa B) activity and propose that this at least partially causes the inhibition of proliferation.

Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis.

A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated. Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a K(m) for glucose in the micromolar range. In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose. Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte. AtSTP9 expression is not found in other floral organs or vegetative tissues. Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes. This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth. The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars.

Screening of chemopreventive tea polyphenols against PAH genotoxicity in breast cancer cells by a XRE-luciferase reporter construct.

Polycyclic aromatic hydrocarbons (PAHs) are established cancer initiators that can be found in our food and environment. Some dietary plant polyphenols are strong inhibitors to PAH-induced mutagenesis, whereas others may not be as effective. To identify the chemopreventive compounds from a huge volume of dietary components, the development of an efficient screening method is required. In this study, a xenobiotic response element (XRE)-luciferase reporter plasmid was constructed to screen for some potential chemopreventive agents in tea against PAH-induced DNA damage. Tea is one of the most consumed beverages worldwide, and its beneficial effects on health have been documented. Previous studies have claimed that tea polyphenols could be protective against various cancers, and the rich database can be a source for comparison. Among the green and black tea polyphenols, the XRE-luciferase reporter assays suggested that only epigallocatechin gallate (EGCG) was effective in reducing XRE-driven luciferase assay in MCF-7 cells at the concentrations tested. Further study indicated EGCG could reduce CYP1A1 and CYP1B1 mRNA abundances and decrease the DMBA-DNA lesions. The results of DNA covalent binding of all tea polyphenols tested were consistent with the XRE-reporter assays. This study illustrated that the XRE-reporter assay was a viable screening test for dietary chemopreventive agents against PAH-initiated breast mutagenesis. It has the advantages of shorter sample processing time and producing no radioactive waste over directly measuring the CYP1A1/1B1 expressions, DNA lesion, or gel mobility shift assay.

Activation of the aryl hydrocarbon receptor by some vegetable constituents determined using in vitro reporter gene assay.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many aromatic environmental pollutants. In this study, we investigated the activation of the AhR by some vegetable constituents using the AhR-based bioassay for dioxins, i.e., the chemical activated luciferase gene expression (CALUX) assay. Ninety-five vegetable constituents, including flavonoids, tannins, saponins, and terpenes, were tested in vitro. Among them, isoflavones such as daidzein, resveratrol having a stilbene structure, and some flavonoids such as naringenin, hesperetin, and baicalein showed AhR activation.

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies.

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.

Properties of voltage-gated Ca(2+) channels in rabbit ventricular myocytes expressing Ca(2+) channel alpha(1E) cDNA.

Using the whole-cell patch-clamp technique, we have studied the properties of alpha(1E) Ca(2+) channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current (I(Ca,L)). Expression of green fluorescent protein significantly decreased the I(Ca,L). By contrast, expression of alpha(1E) with beta(2b) and alpha(2)/delta significantly increased the total Ca(2+) current, and in these cells a Ca(2+) antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni(2+) and was little affected by changing the charge carrier from Ca(2+) to Ba(2+) or by beta-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of alpha(1E) did not generate T-like current in cardiac myocytes. On the other hand, expression of alpha(1E) decreased I(Ca,L) and slowed the I(Ca,L) inactivation. This inactivation slowing was attenuated by the beta(2b) coexpression, suggesting that the alpha(1E) may slow the inactivation of I(Ca,L) by scrambling with alpha(1C) for intrinsic auxiliary beta.