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Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Mondesert O, Frongia C, Clayton O, Boizeau ML, Lobjois V, Ducommun B - PLoS ONE (2015)

Bottom Line: Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation.Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation.We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse; ITAV-USR3505, F-31106 Toulouse, France; CNRS; ITAV-USR3505, F-31106 Toulouse, France.

ABSTRACT
Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

No MeSH data available.


Related in: MedlinePlus

A Fluorescent assay to monitor DDR pathway activation.(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.
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pone.0134411.g001: A Fluorescent assay to monitor DDR pathway activation.(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.

Mentions: A reporter of the DNA Damage Response (DDR) pathway activation was constructed based on the schematic summary presented in Fig 1. The coding sequence of the mRFP fluorescent protein was cloned under the control of the human p21/CIP1 whole promoter sequence [10] with the aim of monitoring the p53-dependent transcriptional regulation of p21 expression. Thus upon activation of the DDR pathway, increase in p53 levels should result in a sustained expression of mRFP and enhanced fluorescence levels. This reporter system was designed to allow monitoring of the DDR pathway activation in 2D cultured cells and in 3D spheroid models using a high content imaging approach (Fig 1B).


Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Mondesert O, Frongia C, Clayton O, Boizeau ML, Lobjois V, Ducommun B - PLoS ONE (2015)

A Fluorescent assay to monitor DDR pathway activation.(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520595&req=5

pone.0134411.g001: A Fluorescent assay to monitor DDR pathway activation.(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.
Mentions: A reporter of the DNA Damage Response (DDR) pathway activation was constructed based on the schematic summary presented in Fig 1. The coding sequence of the mRFP fluorescent protein was cloned under the control of the human p21/CIP1 whole promoter sequence [10] with the aim of monitoring the p53-dependent transcriptional regulation of p21 expression. Thus upon activation of the DDR pathway, increase in p53 levels should result in a sustained expression of mRFP and enhanced fluorescence levels. This reporter system was designed to allow monitoring of the DDR pathway activation in 2D cultured cells and in 3D spheroid models using a high content imaging approach (Fig 1B).

Bottom Line: Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation.Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation.We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse; ITAV-USR3505, F-31106 Toulouse, France; CNRS; ITAV-USR3505, F-31106 Toulouse, France.

ABSTRACT
Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

No MeSH data available.


Related in: MedlinePlus