Luciferase Expression In Cells Research Articles

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 μg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

NF-κB controls four genes encoding core enzymes of tricarboxylic acid cycle

NF-κB may promote tumor progression by altering cell metabolism. Hence, finding its target genes that are involved in cell metabolism is helpful for understanding its role in tumor growth. Here we discovered four metabolism-related target genes of this transcription factor. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) data that characterizing the global binding sites (BSs) of NF-κB RelA in the TNFα-stimulated HeLa cells, we found that four genes that encode core enzymes of the tricarboxylic acid (TCA) cycle, including IDH1, IDH3A, ACO2, and SUCLA2, were multiply bound by this transcription factor. The subsequent bioinformatic analysis revealed that the NF-κB BSs contained many canonical κB sequences and the NF-κB-like DNA-binding motifs. Detection of ChIPed DNA with polymerase chain reaction (ChIP-PCR) also indicated that the NF-κB BSs were bound by NF-κB in both TNFα-treated HeLa and HepG2 cells. The reporter construct showed that the NF-κB BSs could activate the luciferase expression in cells in a NF-κB-specific manner. The quantitative PCR and Western blot detections demonstrated that NF-κB could regulate the expressions of IDH1, IDH3A, and ACO2 genes at both mRNA and protein levels and that of SUCLA2 gene at mRNA level in the TNFα-treated HeLa and HepG2 cells. Based on these investigations we identified the four genes as new target genes of NF-κB. The finding provides new insights into the role of NF-κB in cellular energetic metabolism, which may be beneficial for understanding the metabolic physiology of tumor growth.

Novel Small-Molecule Human G-CSF Receptor Agonist Stimulates Neutrophil Counts In Monkeys and Displays Anti-Proliferative Effects On Tumor Cells Mediated By a Reduction Of Intracellular Iron

Previously we reported on the discovery of a series of novel, non-peptidyl, small molecules that selectively activate the human granulocyte colony stimulating factor receptor (hG-CSFR) in a manner distinct from G-CSF and similar to the mechanism of small-molecule human thrombopoietin receptor (hTPOR) agonists [Marschke KB, et al. ASH 53rd Annual Meeting Abstracts, Blood 2011 118(21):1453, Abstract 3391]. The lead compound, LG7455, activates STAT5 and STAT3 signal transduction and induces G-CSF-dependent hematopoietic cell growth, but not TPO-dependent or erythropoietin-dependent cell growth. LG7455 promotes the differentiation of human bone marrow cells into granulocytes, significantly increasing expression of the granulocyte-specific marker CD15 (FUT4). The G-CSF-like effect of LG7455 requires the expression of human or monkey G-CSFR, and a histidine in the transmembrane domain is needed for activity, similar to what has been found for small-molecule hTPOR agonists, such as eltrombopag. LG7455 increases the binding of GCSF in a manner consistent with allosteric receptor modulation. To test the ability of LG7455 to stimulate neutrophil counts in vivo, cynomolgus monkeys were dosed intravenously (IV) 3 times per day (TID) to increase LG7455 exposure. Increases in peripheral blood neutrophils were observed in male cynomolgus monkeys with LG7455 (2 mg/kg/dose, TID). Because of the potential for LG7455 to interact with metal ions, their role in the activity of LG7455 was investigated. The ability of LG7455 to activate luciferase expression in cells transiently transfected with an hG-CSFR expression vector and a STAT5-responsive luciferase reporter was dependent on the addition of zinc (II) ion (up to 30 µM) to the cell culture media. The addition of Zn(II) also significantly enhanced the efficacy (63% vs. 23% relative to rhG-CSF) and potency ( EC50 of 28 nM vs. 300 nM) by which LG7455 stimulated the growth of UT-7 cells made dependent on G-CSF by stable transfection of hGCSFR (UTP-hGCSFR). LG7455 did not activate luciferase expression or stimulate growth of cells incubated in the presence of additional iron (III), copper (II), magnesium (II) or calcium (II) ions. Similar to the antitumor effect of the well-described iron chelator salicylaldehyde isonicotinyl hydrazine (SIH), LG7455 inhibited the growth of a selection of human tumor cell lines, including the myeloid leukemia cell lines HL-60 and Kasumi-1, the lung carcinoma cell line A549, the embryonic kidney cell line HEK293, and the hepatocellular carcinoma cell line HepG2 (IC50's of 5.0, 12.1, 6.1, 1.4 and 1.0 µM, respectively). SIH did not stimulate but instead inhibited the growth of UTP-hGCSFR cells. The anti-proliferative effect of LG7455 on HL-60 cells was accompanied by an increase in apoptosis as determined by Annexin V/propidium iodide staining and FACS analysis. The addition of excess Fe(III) to the tumor cells was able to overcome the anti-proliferative effect of LG7455 in a concentration-dependent manner, indicating that a reduction in intracellular iron plays a role in the effect. The addition of Zn(II) and Cu(II) were less efficient at blocking the anti-proliferative effect of LG7455 than Fe(III). Addition of Mg(II) or Ca(II) had no effect. LG7455 increased the fluorescence of HL-60 cells loaded with calcein-AM, a fluorescent dye quenched when bound to iron, further supporting that a reduction of free intracellular iron occurs in cells treated with LG7455. Lastly, as has been shown with other iron chelators with anti-proliferative activity, LG7455 increased intracellular reactive oxygen species (ROS) formation in HL-60 cells loaded with the cell-permeant ROS indicator, CM-H2-DCFDA, and evaluated by flow cytometry. Further optimization of the LG7455 series may provide a novel, orally-available adjuvant anticancer therapy that is also a supportive care agent to treat neutropenia in patients receiving bone-marrow suppressive treatments. Disclosures:Marschke:Ligand Pharmaceuticals Incorporated: Employment. Vajda:Ligand Pharmaceuticals Incorporated: Employment. Zhi:Ligand Pharmaceuticals Incorporated: Employment.

Development of a Positive-readout Mouse Model of siRNA Pharmacodynamics

Development of RNAi-based therapeutics has the potential to revolutionize treatment options for a range of human diseases. However, as with gene therapy, a major barrier to progress is the lack of methods to achieve and measure efficient delivery for systemic administration. We have developed a positive-readout pharmacodynamic transgenic reporter mouse model allowing noninvasive real-time assessment of siRNA activity. The model combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli. Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active. Five founder luciferase-expressing and three founder Lac-expressing lines were generated and characterized. Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line. Administration of isopropyl β-D-1-thiogalactopyranoside either in drinking water or given intraperitoneally increased luciferase expression in eight of the mice examined, which fell rapidly when withdrawn. Intraperitoneal administration of siRNA targeting lac in combination with Lipofectamine 2000 resulted in increased luciferase expression in the liver while control nontargeting siRNA had no effect. We believe a sensitive positive readout pharmacodynamics reporter model will be of use to the research community in RNAi-based vector development.

Discovery of Non-Peptidyl Small-Molecule Human GCSF Receptor Agonists for the Potential Treatment of Neutropenia,

Abstract 3391Granulocyte colony stimulating factor (GCSF) is the essential cytokine for the regulation of neutrophilic granulocytes. Binding of GCSF to its receptor (GCSFR) triggers receptor dimerization, leading to activation of JAK1 and JAK2, phosphorylation of GCSFR, STAT3, STAT5, and Ras/mitogen-activated protein kinase (MAPK), and results in proliferation and differentiation of granulocytic cells. Recombinant human GCSF (rhGCSF) is used successfully to alleviate chemotherapy-induced neutropenia, neutropenia associated with hematopoietic stem cell transplantation, and severe chronic neutropenia. A small molecule oral GCSFR agonist may offer a safer and more convenient alternative to the current injectable rhGCSF therapy. Whereas a previous effort to identify small-molecule mimetics of GCSF found SB-247464 that selectively activated the murine GCSFR, no small-molecule human GCSF mimetics have been developed. Recently, we have discovered a series of novel non-peptidyl small molecules that selectively activate human GCSFR (hGCSFR) function, and may provide a significant innovation in the treatment of neutropenia. In cells transiently transfected with an hGCSFR expression vector and a STAT3-responsive luciferase reporter, a lead compound, LG7455, activates luciferase expression with an efficacy of 50% relative to rhGCSF, and potency (EC50) of 100 nM. LG7455 also activates luciferase expression in cells transfected with hGCSFR and a STAT5-responsive luciferase reporter (65%, 40 nM EC50). The activity of LG7455 is dependent on the expression of hGCSFR, and LG7455 is not active in luciferase assays when human thrombopoietin receptor (hTPOR) or erythropoietin receptor (hEPOR) is expressed. In UT-7 cells made responsive to GCSF by stable transfection of hGCSFR (UTP-hGCSFR), LG7455 stimulated cell growth and increased the phosphorylation of STAT3 and STAT5. LG7455 did not increase growth of TPO- or EPO-responsive UT-7 cells. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), LG7455, increased the percentage of cells positive for the granulocyte-specific marker CD15 (FUT4). The effect of LG7455 in BM-HCs was additive to the effect of rhGCSF. LG7455 is active in luciferase assays with expressed cynomolgus monkey GCSFR, but not mouse, guinea pig or rabbit GCSFR. Similar to what has been demonstrated for small-molecule human TPOR agonists such as eltrombopag, the activity of LG7455 is dependent on a specific residue in the hGCSFR transmembrane domain. When histidine 627 (His-627) in hGCSFR is changed to asparagine present at a similar location in the mouse GCSFR (Asp-602), unlike rhGCSF, LG7455 is no longer active. LG7455 is active, however, on mouse GCSFR with Asp-602 replaced by His. In radioligand-binding experiments using UTP-hGCSFR cells, LG7455 did not displace [125I]rhGCSF, however binding of [125I]rhGCSF was augmented in a concentration dependent manner consistent with allosteric receptor modulation. These data demonstrate that LG7455 is a novel small-molecule selective hGCSFR agonist that activates the receptor in a manner distinct from GCSF and similar to the mechanism of small-molecule hTPOR agonists. Further optimization of the LG7455 chemical series should provide orally-available molecules to treat neutropenia with improved safety and convenience compared to current injectable rhGCSF. Disclosures:Marschke:Ligand Pharmaceuticals: Employment. Rungta:Ligand Pharmaceuticals: Employment. Slavin:Ligand Pharmaceuticals: Employment. Sanders:Ligand Pharmaceuticals: Employment. Roach:Ligand Pharmaceuticals: Employment. Pickens:Ligand Pharmaceuticals: Employment. Shen:Ligand Pharmaceuticals: Employment. van Oeveren:Ligand Pharmaceuticals: Employment. Hong:Ligand Pharmaceuticals: Employment. Sun:Ligand Pharmaceuticals: Employment. Bissonnette:Ligand Pharmaceuticals: Employment. Syka:Ligand Pharmaceuticals: Employment. Zhi:Ligand Pharmaceuticals: Employment.

Ang II and EGF synergistically induce COX‐2 expression via CREB in intestinal epithelial cells

Cyclooxygenase (COX)-2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX-2 is the rate-limiting step in the production of PGs, mechanisms that regulate COX-2 expression control PG production in the cell. Using the non-tumorigenic, rat intestinal epithelial cell, IEC-18, we demonstrate that co-activation of endogenously expressed AT(1) receptor and EGFR resulted in synergistic expression of COX-2 mRNA and protein involving transcriptional and post-transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38(MAPK) and CREB. Co-stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38(MAPK), and CREB. The p38(MAPK) selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II-dependent COX-2 expression and CREB phosphorylation. EGF-dependent COX-2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX-2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II- and EGF-dependent induction of the COX-2 promoter. Ang II induced luciferase expression in cells transfected with the CRE-luc reporter vector and cells co-transfected with Gal4-luc reporter vector and a Gal4-CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX-2 promoter region containing a CRE cis-acting element. These results indicate that co-stimulation with Ang II and EGF synergistically induced COX-2 expression in these intestinal epithelial cells through p38(MAPK) mediated signaling cascades that converge onto CREB.

Macrostemonoside A Promotes Visfatin Expression in 3T3-L1 Cells

The newly identified fat cell-secreted factor, visfatin, is insulin-mimetic and play a positive role in attenuating insulin resistance and diabetes. Natural steroidal saponins including tigogenin saponins have been reported to provide anti-diabetic activity and modulate glucose metabolism, but the mechanism remains unknown. In this study, we examined the effect of macrostemonoside A, a tigogenin steroidal saponin isolated from the bulbs of Allium macrostemon Bung on the expression of visfatin in differentiated 3T3-L1 adipocytes. It was found that macrostemonoside A markedly enhanced the synthesis and secretion of visfatin protein in 3T3-L1 adipocytes, and increased visfatin mRNA in a dose and time dependent manner as well. Moreover, visfatin promoter-driven luciferase expression in cells was elevated by macrostemonoside A, which was blocked by SB-203580, a specific inhibitor of p38 MAPK pathway. Lastly, we found that macrostemonoside A did not affect the expression of PPARgamma and its DNA-binding ability to visfatin promoter. These results indicate that macrostemonoside A could potently stimulate visfatin expression in 3T3-L1 adipocytes, which occurs at the transcriptional level and is mediated at least partially via p38 MAPK signaling pathway. Its regulation on visfatin in adipocytes may constitute an important element in its improvement of insulin resistance and diabetes.

Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake.

Two synthetic hammerhead ribozymes, one unmodified and the other with 2"-modifications and four phosphorothioate groups, targeting a single GUA site in the luciferase mRNA, were compared for their inhibition of gene expression in cell cultureand their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was treated with these ribozymes by liposome-mediated transfection to determine their activity. Luciferase expression in cells was inhibited to approximately 50% with little difference between the unmodified and the 2"-modified ribozyme. A similar degree of inhibition was observed with two catalytically inactive ribozymes, indicating that inhibition was mainly due to an antisense effect. A ribozyme carrying a cholesterol moiety, applied to the cells without carrier, showed no inhibition. Northern blotting indicated a similar amount of cellular uptake of all ribozymes. The unmodified ribozyme was essentially evenly distributed between cytoplasm and nucleus, whereas a higher proportion of the phosphorothioate-containing ribozyme was observed in the nucleus. Fluorescence microscopy, including confocal microscopy using 5"-fluorescein-labelled ribozymes, showed that the unmodified and 2"-modified ribozymes were present in the cytoplasm and in the nucleus to a similar extent, whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expression to a comparable degree, suggesting that the ribozyme in the nucleus did not contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their inability to interfere with gene expression.

Role of Egr‐1 in Cholinergic Stimulation of Phenylethanolamine N‐Methyltransferase Promoter

The effects of the cholinergic agonist carbachol on phenylethanolamine N-methyltransferase promoter activity and Egr-1 mRNA expression in PC12-derived RS1 cells were examined to investigate the potential involvement of Egr-1 in the neural regulation of phenylethanolamine N-methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N-methyltransferase promoter-luciferase reporter gene construct and also elevated Egr-1 mRNA levels in untransfected cells. Maximum induction of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr-1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was mediated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr-1 binding element in the phenylethanolamine N-methyl-transferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N-methyltransferase promotor through stimulation of Egr-1 expression, and are consistent with the potential involvement of Egr-1 in the cholinergic activation of the phenylethanolamine N-methyltransferase gene.

Cationic Lipid Enhances In Vitro Receptor-Mediated Transfection

The efficiency of cell-specific transfection by receptor-mediated uptake is improved by the use of cationic lipids. Asialoglycoprotein (AP) was conjugated to poly-L-lysine (PL) and complexed with the plasmid pCMVL that contains a luciferase reporter gene. The asialoglycoprotein-poly-L-lysine:pCMVL (AP-PL:pCMVL) complexes then were mixed with the cationic lipid dioctadecylamidoglycylspermine (DOGS). This complex was taken up by the hepatocyte-like cell line, Hep G2, via the asialoglycoprotein receptor. The expression of luciferase in cells transfected with the DOGS/AP-PL: pCMVL complexes were significantly increased compared with AP-PL:pCMVL complexes without DOGS. The ratio of AP-PL to DOGS is an important determinant for both transfection efficiency and for maintaining receptor specificity. Therefore, cationic lipids significantly increased the efficiency of asialoglycoprotein receptor mediated transfection in the hepatoblastoma cell line, Hep G2. The use of cationic lipids with receptor-mediated gene delivery systems could potentially increase transfection efficiency yet maintain cell-target specificity.

Monitoring foreign gene expression by a human adenovirus-based vector using the firefly luciferase gene as a reporter

We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.