Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay

Li, Xinping; Shen, Fuwen; Zhang, Yuwu; Zhu, Jiang; Shi, Qunfang

doi:10.1016/j.ejpb.2007.01.004

Cited by 10 publications

(5 citation statements)

References 3 publications

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“…19–23 The generation of monoclonal cell lines requires the expansion of single cells over tens of generations to provide a sufficiently large number of cells to process using conventional tissue culture techniques. The evolution of monoclonal cell lines over these timescales has not been well characterized.…”

Section: Resultsmentioning

confidence: 99%

“…18 In an attempt to reduce cell-to-cell variability to permit more sensitive measurements pooled over multiple cells, monoclonal cell lines are often employed for fluorescent reporters. 19–23 Noise from polyclonal variability can also be reduced by averaging measurements over a larger number of cells but this leads to increased reagent consumption, reduced throughput and limited dynamic range. These dynamics and variability of fluorescent transcriptional reporters for β-catenin have not been well characterized in the literature, particularly in the context of the evolution of reporter performance in monoclonal cell lines.…”

Section: Introductionmentioning

confidence: 99%

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Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

et al. 2014

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Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (< 6) and long (> 25) generational timescales. To characterize phenotypic divergence these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1,119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provide insight into the pathway’s mechanisms of positive feedback and self-inhibition.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

et al. 2014

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“…These secondary pathogenic cascades have been shown to occur and have implications in the molecular mechanism of several LSDs [24]. Several cell-based assays have been developed for HTS including second messenger mobilization after GPCR activation [25], reporter gene assays [26] and phenotypic assays for cellular processes ( e.g., cell migration [27], cytokinesis [28]). However, none of the previously described HTS assays utilized patient-derived cells.…”

Section: Discussionmentioning

confidence: 99%

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

et al. 2011

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Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.

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“…Instead, many cell-based assays aim to identify modulators of a pathway of interest in the more physiological environment of a cell, complete with intact regulatory networks and feedback control mechanisms. Examples of cell-based assays include functional assays (e.g., second messenger mobilization after GPCR activation [19,20]), reporter gene assays [21,22], and phenotypic assays for cellular processes (e.g., cell migration [23], cytokinesis [24]). In such assays, entire pathways of interest can be interrogated providing the opportunity for multiple potential intervention points, as opposed to a single pre-defined step under the biochemical approach.…”

Section: Assay Types: Cell-based Versus Biochemical Screensmentioning

confidence: 99%

Cell-Based Assays for High-Throughput Screening

2010

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Cell-based assays represent approximately half of all high-throughput screens currently performed. Here, we review in brief the history and status of high-throughput screening (HTS), and summarize some of the challenges and benefits associated with the use of cell-based assays in HTS. Approaches for successful experimental design and execution of cell-based screens are introduced, including strategies for assay development, implementation of primary and secondary screens, and target identification. In doing so, we hope to provide a comprehensive review of the cell-based HTS process and an introduction to the methodologies and techniques used.

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Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay

Cited by 10 publications

References 3 publications

Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

Cell-Based Assays for High-Throughput Screening

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