Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In investigating new methods for the treatment of pancreatic cancer, we have explored the possibility of using a combination of radiation and gene therapies. We demonstrate herein that the early growth response gene 1 promoter (Egr-1) is sufficient to confer selective expression of the luciferase gene (Luc) in a human pancreatic tumor cell line (AsPc-1) when exposed to ionizing radiation. The Egr-1 promoter directed the radioinducible expression of luciferase, and yielded higher levels of Luc activity than that in nonirradiated lines. The radioisotopes Tc-99m, I-131, and Ga-67-citrate were selected as Egr-1 activators for their potential to accumulate in tumors. We studied Ga-67-citrate, a radioisotope employed in tumor scintigraphy, for its suitability for selective gene induction. The plasmid vector pEgr-1-Luc was transfected into AsPc-1 cells and then exposed to radioisotopes. Luciferase activity increased by 100-300 times over control. We also inserted the herpes thymidine kinase gene (TK) downstream of Egr-1 and transfected this construct into AsPc-1 cells. Ga-67-citrate and ganciclovir were added to the cells and cell survival was assessed by MTT assay. The growth of AsPc-1 cells transfected with the pEgr-TK construct was suppressed 2 days after exposure of the cells to Ga-67-citrate. The results indicate that Ga-67-citrate may be useful in combining radiation and gene therapies.
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PMID:Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes. 914 8

Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.
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PMID:Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters. 1075 12

Estrogen receptor (ER)-positive breast cancers initially respond well to estrogen ablation treatment but finally acquire refractoriness, the phenomenon that is a major clinical problem. Because some breast cancers synthesize estradiol (E(2)) and E(2) synthesis is regulated by gonadotropins in normal ovaries, and because circulating gonadotropins are elevated in postmenopausal women and during estrogen ablation treatment, we hypothesized that gonadotropins might modulate estrogen synthesis/metabolism in breast cancer tissue as well. To test this possibility, MCF-7 cells were treated with dehydroepiandrosterone (DHEA) or human chorionic gonadotropin (hCG; approximately LH), each alone or in combination. Cell growth (3-day treatment) was assayed by the MTT method and estrogen synthesis (24-hour treatment) was measured using the ERE-luciferase reporter system. First, MCF-7 cell growth was stimulated by DHEA in a concentration-dependent manner with a maximal effect at 10(-4) M. Although hCG alone did not have a significant proliferative effect, hCG significantly and dose dependently stimulated MCF-7 cell growth in the presence of a submaximal concentration of DHEA (10(-7 )M). This stimulatory effect of DHEA and hCG was blocked by a pure antiestrogen ICI182,780 and an aromatase inhibitor, arimidex. Using MCF-7 cells transfected with the ERE-luciferase reporter system, hCG treatment was shown to increase ERE-mediated transcription. These results indicate that MCF-7 cells intrinsically converted DHEA into E(2) upon hCG stimulation, then grew their own cells DHEA- and hCG-dependently. We conclude that gonadotropins can act on breast cancer cells and accelerate conversion of DHEA into estrogens, thereby stimulating growth of estrogen-dependent tumor cells. This phenomenon, at least in part, could explain: (1) an increased tissue concentration of E(2) in postmenopausal breast cancer; (2) acquisition of hormone refractoriness during estrogen ablation treatment, and (3) the effectiveness of GnRH antagonist/superagonist in some postmenopausal breast cancer patients.
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PMID:Gonadotropins stimulate growth of MCF-7 human breast cancer cells by promoting intracellular conversion of adrenal androgens to estrogens. 1109 52

Folate-polyethylene glycol-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized by linking folic acid to both ends of a mono-functional PEG and then grafting to PEI. The graft ratio was determined using Beer's law by measuring the UV absorbance at 363 nm. The pH profile, diameter and shape of the carriers were determined. Transfection efficiencies were optimized in normal smooth muscle cells (SMC) and CT-26 colon adenocarcinoma cells using plasmid DNA encoding luciferase reporter gene. Free folic acid was shown to inhibit transfection with FPF-2.3 g-PEI at neutral charge ratio. Relative toxicity between PEI and the modified carrier was measured using MTT colorimetric assay. Therapeutic potential of pmIFN-gamma complexed with these polymeric carriers in terms of gene expression was determined at protein and mRNA levels using ELISA and RT-PCR. FPF-g-PEI was determined to have 2.3 folate-PEG-folate (FPF) linear polymers grafted to each PEI molecule. The average molecular weight was measured to be approximately 33,500 Mw and the pH profile was characteristic of endosomal disruption capacity. Atomic Force Microscopy (AFM) and Dynamic Laser Light Scattering (DLLS) indicated FPF-2.3 g-PEI and PEI (at 2 w/w ratio) efficiently condensed plasmid DNA resulting in oblique spheroid polyplexes with a mean diameter of approximately 150 nm. FPF-2.3 g-PEI was superior to PEI in terms of cytotoxicity and transfection efficiency in cancer cells. Smooth muscle cells showed no specificity for folate tethered complexes, where PEI/pLuc complexes yielded higher efficiencies.
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PMID:Folate-PEG-folate-graft-polyethylenimine-based gene delivery. 1169 7

Quaternized modifications of chitosan present characteristics that might be useful in DNA condensing and efficient gene delivery. Trimethylated chitosan (TMO) was synthesized from oligomeric chitosan (<20 monomer units). TMOs spontaneously formed complexes (chitoplexes) with RSV-alpha3 luciferase plasmid DNA. These complexes were characterized by photon correlation spectroscopy and were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum and compared with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulphate) lipoplexes. Additionally, their effect on the viability of the respective cell cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results showed that quaternized chitosan oligomers were able to condense DNA and form complexes with a size ranging from 200 to 500 nm. Chitoplexes proved to transfect COS-1 cells, however, to a lesser extent than DOTAP-DNA lipoplexes. The quaternized oligomer derivatives appeared to be superior to oligomeric chitosan. The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP DNA complexes was decreased. Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to approximately 50% in both cell lines. Both DOTAP-DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells; however quaternized chitosan oligomers proved to be superior to DOTAP. Effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells. We conclude that trimethylated chitosan-DNA complexes present suitable characteristics and the potential to be used as gene delivery vectors.
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PMID:Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines. 1176 33

Folate-poly(ethylene glycol)-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized over a range of grafting ratios of folate-poly(ethylene glycol)-folate (FPF) to polyethylenimine (PEI). The conjugation was determined using the absorbance at 363 nm for each polymer. FPF-g-PEIs were determined to have 2.3, 5.2, 9.3 and 20 FPF linear polymers grafted to each PEI. The average molecular weight was calculated to be approximately 34,848, 47,266, 64,823 and 110,640 Da, respectively. The pH profiles of FPF-g-PEIs suggest that the polymers have endosomal disruption capacity, and the gel electrophoretic band retardation showed efficient condensation of DNA. The transfection efficiency, determined using plasmid encoding luciferase, was dependent on the cell type and was different for CT-26 colon adenocarcinoma, KB oral epidermoid, and normal smooth muscle cells (SMC). The relative toxicity of polymer/plasmid complexes was determined using the MTT colorimetric assay. At neutral charge ratio, FPF-g-PEI/pLuc complexes were less toxic to cells and showed higher transfection in cancer cells compared to PEI/pLuc complexes. Smooth muscle cells showed no specificity for FPF-g-PEI/pLuc complexes, whereas PEI/pLuc complexes showed a higher transfection efficiency. The transfection efficiency increased when neutral polymer/DNA complex concentrations increased, but decreased when positively charged polymer/DNA complex concentrations increased. There was little increase in toxicity when FPF-5.2g-PEI/pLuc complex concentrations increased.
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PMID:Optimization of factors influencing the transfection efficiency of folate-PEG-folate-graft-polyethylenimine. 1185 36

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.
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PMID:Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome. 1217 22

kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2-3 days after removal of doxycycline, cells underwent G(2)/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G(2)/M arrest and apoptotic cell death through its kinase activity.
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PMID:Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor. 1262 1

Oligomycin at 0.01 microM produces very rapid decrease of [3H]estradiol (E2)-binding capacity in MCF-7 cells maintained in culture in glucose- and serum-free medium. Loss of binding capacity was associated with elimination of the estrogen receptor (ER) as well as a decrease of basal expression of ERE-luciferase reporter gene. These effects were not due to major cell death as shown by MTT assay. Hence, the inhibition of ATP synthesis produced by oligomycin seems to influence ER turnover, resulting in very rapid loss of receptor. Withdrawal of oligomycin and maintenance of glucose in the medium led to only a partial reappearance of ER and failed to restore optimal ERE-dependent transcription. Oligomycin significantly down-regulated progesterone receptor (PR) level and partially abrogated E2-induced PR up-regulation, indicating that this drug also affects other nuclear receptors. Treatment of cytosol from MCF-7 cells with acid and alkaline phosphatases decreased [3H]E2-binding capacity, indicating the requirement of ER phosphorylation for optimal hormone binding. On the other hand, oligomycin-induced ER loss was partly compensated by E2 and partial anti-estrogens (AEs; 4-OH-TAM or RU 39 411); i.e. oligomycin failed to improve the E2-induced ER down-regulation and very weakly suppressed partial AE-induced receptor up-regulation. The known ability of these ligands to stabilize ER in the cell nucleus before regulating ER level may explain this phenomenon since such antagonism was not recorded with pure AE RU 58 668, which is known to impede nuclear translocation of the receptor. Interestingly, ligands able to down-regulate ER (i.e. E2 or RU 58 668) increased ER phosphorylation while 4-OH-TAM which up-regulate the receptor had little effect in this regard. Oligomycin failed to strongly affect such phosphorylation enhancements while it produced a weak decrease of basal phosphorylation level. Hence, phosphorylations/dephosphorylations of specific sites on ER and/or co-regulators seem to govern its turnover.
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PMID:Decrease of estrogen receptor expression and associated ERE-dependent transcription in MCF-7 breast cancer cells after oligomycin treatment. 1262 89

A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent pK(a) value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(alpha-myristoyloxy)ethyl]imidazole (alpha-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.
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PMID:Cytosolic delivery of macromolecules: I. Synthesis and characterization of pH-sensitive acyloxyalkylimidazoles. 1265 55


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