Luciferase reporter gene

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

Legler J, van den Brink C, Brouwer A, et al. 1998. Assessment of (anti-)estrogenic compounds using a stably transfected luciferase reporter gene assay in the human T47-D breast cancer cell line. Organohalogen Compounds 37 265. 

Recent studies have demonstrated that lithium (and to a lesser extent VPA) produces, at therapeutically relevant concentrations, complex alterations in basal and/or stimulated DNA-binding of 12-o-tetradecanoyl-phorbol 13-acetate (TPA) response element (TRE) to AP-1 transcription factors. These alterations are produced not only in human SH-SY5Y cells in vitro, but also in rodent brain following chronic, in vivo administration [5, 7, 15-21]. Corresponding to an increase in basal AP-1 DNA-binding activity, lithium and VPA have been shown to increase the expression of a luciferase reporter gene driven by an SV40 promoter that contains TREs in a time- and concentration-dependent fashion. Mutations in the TRE... 

Fig. 19. The Cx43 promoter region. Partial analysis of the human Cx43 gene extending from -360 to the site of fusion where De Leon et al. [1994] inserted the luciferase reporter gene at position +143. The transcription start site is numbered -1 [redrawn from De Leon et al., 1994]. The TATA box and the putative AP-1-binding sequence are underlined.

The estrogenic and antiestrogenic activities of several PBDE congeners and three hydroxylated PBDEs were tested in vitro using human breast cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression (Mccrts et al. 2001). The hydroxylated PBDEs,... 

Luciferase reporter gene (pGL3 -control vector) activity measured in transfected fibroblast 183... 

LEGT.ER J, VAN DEN BRINK C E, BROUWER A, MURK A J, VAN DER SAAG P T, vethaak A D and van der BURG B, Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line , Toxicol Sci 1999 48 55-66. 

George, S.E., P.J. Bungay, and F.H. Naylor. 1997. Evaluation of a CRE-directed luciferase reporter gene assay as an alternative to measuring cAMP accumulation. J. Biomol. Screen. 2, 235-240. 

Figure 2.9. Luciferase reporter gene assay/promoter deletion analysis. Cells are transfected with the CYP1A1 promoter reporter (luciferase) constructs in which the promoter region is increasing deleted. The cells are then treated with benzo[a]pyrene (an inducer of CYP1A1), and cells are harvested 24-48hr later and luciferase activity is measured. Promoter deletion analysis revealed enhancer element between -1140 and -1029. Contained within this region is xenobiotic response element (XRE) to which the aryl hydrocarbon receptor (AHR) binds.

Wfe have recently carried out experiments with cell-based and animal models of inflammatory diseases. The experiments have revealed that human polymorphonuclear cells once activated with phorbol esters to cause an inflammatory response produce copious amounts of chlorinated polyphenol species. Rats treated with lipopolysaccharide (LPS) in an in vivo model of inflammatory disease heavily nitrate polyphenols in tissues (e.g., lung and liver) where inflammatory cell invasion occurs. These modified polyphenols are better antioxidants than their parent compound. Using a luciferase reporter gene assay in COS cells, both chloro- and nitrogenistein were shown to have 1-2 orders lower estrogen receptor activation than genistein itself. In summary, metabolism of polyphenols is rampant, but not always inactivating. 

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