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Notes: Here the authors describe development of an HTS cell viability assay protocol for use with cultured cyropreserved human primary hepatocytes. Cryopreserved hepatocytes for culturing were prepared as suspensions and dispensed at 2,000 or 4,000 cells/5µl/well in collagen I-coated 1,536-well plates. Cells were allowed to attach and then 23nl of each test compound was added in a dilution series from 2.8nM to 92µM, and cells incubated for 24 or 40 hours. Five microliters of CellTiter-Glo^® Reagent was added and cells were incubated 30 minutes before reading the luminescent output. IC₅₀ values for 12 compounds were determined; a summary of the protocol is provided in Table 1 of the paper. Cultured cryopreserved hepatocytes were assayed for function using the P450-Glo^® CYP3A4 assay with the Luciferin-IPA substrate. (4182)

Notes: The authors tested if CYP1B1 removed cellular oxygenation products that induce oxidative stress and promote the release of antiangiogenic factors. The P450-Glo™ CYP1B1 Assay was used to determine CYP1B1 activity. The presence of glutathione was assessed using either 10⁴ retinal endothelial cells or 50µl of mouse retinal extracts dispensed into each well of a 96-well plate with the GSH-Glo™ Glutathione Assay. (4010)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

Notes: A CYP3A4 response element consisting of proximal and distal enhancer sequences with the CYP3A4 promoter was cloned into a pGL3 vector and used in transfection studies with HepG2 cells. The effects of either or both enhancer motifs on luciferase expression were studied in relation to various xenochemical treatments. The researchers also used the P450-Glo™ CYP3A4 Assay to analyze increases in CYP3A4 activities in a stably transfected cell line (DPX-2) and on primary hepatocytes. For these assays, the researchers incubated cells in 96-well or 24-well plates with or without CYP3A4 inhibitors and the P450-Glo™ CYP3A4 substrate. Following a 3 hour incubation, the P450-Glo™ Detection Reagent was added and the luminescence was recorded. In these studies cells were also pretreated with various chemicals including 10µM rifampicin, 1000µM phenobarbital, 100µM dexamethasone, 50µg/ml kava, 50µM methoxychlor, 50µM troglitazone, 100µM omeprazole, 100µM 2-acetylaminofluorene, and 25 µM chrysin. (3221)

Notes: The authors of this paper present proof-of-concept experiments showing that drug metabolism enzyme activity can be measured in whole animals (in vivo) in real time. Using a mouse that expresses a luciferase transgene at constitutively high levels in the liver, the authors evaluated CYP3A4 and CYP3A7 activity using a CYP3A P450 substrate (proluciferin substrate) that is converted into a luciferase substrate by CYP34 activity. The luciferase substrate produced by the P450 activity is then used by luciferase in a reaction that produces light. An increase in luminescence correlates with an increase in enzyme activity in this assay. The authors conclude that optical imaging of reporter mice will provide a new method for looking at drug actions in whole animals, with the caveat that the solubility of the proluciferin substrate is optimized and toxicity is minimized. (3996)

Notes: Microsomal extracts from Jurkat cells were assayed for cytochrome P450 1B1 (CYP1B1) using the P450-Glo™ CYP1B1 Assay. CYP1B1 activity increased when Jurkat cell cultures were supplemented with 10nM biotin. (3132)