Document Detail


Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.
MedLine Citation:
PMID:  21103341     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs) are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.
METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.
CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics.
Authors:
Michael S Weiss; Beatriz Peñalver Bernabé; Abigail D Bellis; Linda J Broadbelt; Jacqueline S Jeruss; Lonnie D Shea
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2010-11-17
Journal Detail:
Title:  PloS one     Volume:  5     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2010  
Date Detail:
Created Date:  2010-11-24     Completed Date:  2011-04-27     Revised Date:  2014-09-08    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e14026     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Activins / pharmacology
Cell Culture Techniques
Cell Line, Tumor
Collagen
Drug Combinations
Estradiol / pharmacology
Estrogens / pharmacology
Green Fluorescent Proteins / genetics,  metabolism*
Humans
Kinetics
Laminin
Luciferases / genetics,  metabolism*
Luminescent Measurements / instrumentation,  methods*
Proteoglycans
Recombinant Fusion Proteins / genetics,  metabolism
Signal Transduction / drug effects
Transcription Factors / genetics,  metabolism*
Transfection
Grant Support
ID/Acronym/Agency:
K22 CA138776/CA/NCI NIH HHS; R21CA125285/CA/NCI NIH HHS; T32GM008449/GM/NIGMS NIH HHS; UL1DE019587/DE/NIDCR NIH HHS
Chemical
Reg. No./Substance:
0/Drug Combinations; 0/Estrogens; 0/Laminin; 0/Proteoglycans; 0/Recombinant Fusion Proteins; 0/Transcription Factors; 0/activin A; 104625-48-1/Activins; 119978-18-6/matrigel; 147336-22-9/Green Fluorescent Proteins; 4TI98Z838E/Estradiol; 9007-34-5/Collagen; EC 1.13.12.-/Luciferases
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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