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Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.

ABSTRACT: Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.

SUBMITTER: Fradet-Turcotte A

PROVIDER: S-EPMC3154085 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.

Fradet-Turcotte Amélie A Morin Geneviève G Lehoux Michaël M Bullock Peter A PA Archambault Jacques J

Virology 20100115 1

Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gen ...[more]

PMID: 20079917

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Project description:PURPOSE:Metabolism, and especially glucose uptake, is a key quantitative cell trait that is closely linked to cancer initiation and progression. Therefore, developing high-throughput assays for measuring glucose uptake in cancer cells would be enviable for simultaneous comparisons of multiple cell lines and microenvironmental conditions. This study was designed with two specific aims in mind: the first was to develop and validate a high-throughput screening method for quantitative assessment of glucose uptake in "normal" and tumor cells using the fluorescent 2-deoxyglucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and the second was to develop an image-based, quantitative, single-cell assay for measuring glucose uptake using the same probe to dissect the full spectrum of metabolic variability within populations of tumor cells in vitro in higher resolution. PROCEDURE:The kinetics of population-based glucose uptake was evaluated for MCF10A mammary epithelial and CA1d breast cancer cell lines, using 2-NBDG and a fluorometric microplate reader. Glucose uptake for the same cell lines was also examined at the single-cell level using high-content automated microscopy coupled with semi-automated cell-cytometric image analysis approaches. Statistical treatments were also implemented to analyze intra-population variability. RESULTS:Our results demonstrate that the high-throughput fluorometric assay using 2-NBDG is a reliable method to assess population-level kinetics of glucose uptake in cell lines in vitro. Similarly, single-cell image-based assays and analyses of 2-NBDG fluorescence proved an effective and accurate means for assessing glucose uptake, which revealed that breast tumor cell lines display intra-population variability that is modulated by growth conditions. CONCLUSIONS:These studies indicate that 2-NBDG can be used to aid in the high-throughput analysis of the influence of chemotherapeutics on glucose uptake in cancer cells.

Project description:CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

Dataset Information

Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.

Publications

Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.

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