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Imaging proteolytic activity in live cells and animal models.

ABSTRACT: In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

SUBMITTER: Galban S

PROVIDER: S-EPMC3679058 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Imaging proteolytic activity in live cells and animal models.

Galbán Stefanie S Jeon Yong Hyun YH Bowman Brittany M BM Stevenson James J Sebolt Katrina A KA Sharkey Lisa M LM Lafferty Michael M Hoff Benjamin A BA Butler Braeden L BL Wigdal Susan S SS Binkowski Brock F BF Otto Paul P Zimmerman Kris K Vidugiris Gediminas G Encell Lance P LP Fan Frank F Wood Keith V KV Galbán Craig J CJ Ross Brian D BD Rehemtulla Alnawaz A

PloS one 20130611 6

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulat ...[more]

PMID: 23776643

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Project description:BackgroundNoninvasive live-animal longitudinal monitoring of xenograft tumor growth and metastasis by bioluminescent imaging (BLI) has been widely reported in cancer biology and preclinical therapy literature, mainly in athymic nude mice. Our own experience at calibrating BLI readout with tumor weight/volume in human prostate cancer xenograft models in haired, SCID-NSG mice through intraprostatic (orthotopic) and subcutaneous (SC) inoculations revealed either nonexistent or poor correlation (coefficient of determination, R 2 = ~0.01-0.3). The present work examined several technical and biological factors to improve BLI utility.MethodsAfter ruling out promoter-luciferase (luc) specificity and luc gene loss in the cell inoculum with LNCaP-AR-luc cells expressing an androgen receptor (AR) and tagged with AR-responsive probasin promoter-luc gene, we evaluated different routes of d-luciferin administration, imaging time during the day, charge-coupled device camera image acquisition settings, and hair removal methods to improve the imaging protocol. For most imaging sessions, BLI was carried out within the same day of tumor volume measurement. After necropsy, histological and immunohistochemical (IHC) analyses were performed on the tumors to evaluate necrosis and expression of luciferase and AR, respectively.ResultsInjection of d-luciferin by SC route, robust image-capture setting (30 000 counts and autoexposure), imaging in the morning and thorough hair removal resulted in a substantial improvement of R2 to ~0.6. Histological analyses confirmed the lack of BLI signal in necrotic tumor masses consistent with luciferase-mediated light emission only in oxygenated adenosine triphosphate-producing viable cells. IHC staining detected heterogeneous expression of luciferase tracking generally with AR expression in nonnecrotic tumor tissues.ConclusionsOur body of work highlighted a framework to validate imaging protocols to ensure the acquisition of interpretable BLI data as an indicator of xenograft tumor burden. The vast tissue heterogeneity in prostate tumor xenografts and variable luciferase expression constrained this technology from achieving a high correlation.

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Imaging proteolytic activity in live cells and animal models.

Publications

Imaging proteolytic activity in live cells and animal models.

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