Protein activation dynamics in cells and tumor micro arrays assessed by time resolved Förster resonance energy transfer

Véronique Calleja, Pierre Leboucher, Banafshé Larijani

Research output: Chapter or section in a book/report/conference proceedingChapter or section

3 Citations (SciVal)

Abstract

Analytical time resolved Förster resonance energy transfer (FRET) can be exploited for assessing, in cells and tumor micro arrays, the activation status and dynamics of oncoproteins such as epidermal growth factor receptor (EGFR1) and their downstream effectors such as protein kinase B (PKB) and 3-phosphoinositide-dependent protein kinase 1 (PDK1). The outcome of our research involving the application of quantitative imaging for investigating molecular mechanisms of phosphoinositide-dependant enzymes, such as PKB and PDK1, has resulted in a refined model describing the dynamics and regulation of these two oncoproteins in live cells. Our translational research exploits a quantitative FRET method for establishing the activation status of predictive biomarkers in tumor micro arrays. We developed a two-site FRET assay monitored by automated frequency domain Fluorescence lifetime imaging microscopy (FLIM). As a proof of principle, we tested our methodology by assessing EGFR1 activation status in tumor micro arrays from head and neck patients. Our two-site FRET assay, by high-throughput frequency domain FLIM, has great potential to provide prognostic and perhaps predictive biomarkers.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherElsevier Academic Press Inc
Pages225-246
Number of pages22
DOIs
Publication statusPublished - 16 Feb 2012

Publication series

NameMethods in Enzymology
Volume506
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
We are grateful to Peter J. Parker for his critical insights and scientific discussions in these projects. We would like to thank Paul Barber from the Gray Institute for Radiation, Oncology & Biology and Medical Sciences Division (University of Oxford) for the analysis software (TRI2) of the two-photon FLIM. We thank Christopher Applebee for the setup of live imaging on the two-photon FLIM. We also thank Nirmal Jethwa and Christopher Applebee for commenting on the manuscript. This work was supported by Cancer Research UK core funding to London Research Institute and by EU grant-QLK3-CT-2000.

Keywords

  • FLIM
  • FRET
  • Keywords
  • PDK1
  • PKB/Akt
  • Tumor micro arrays

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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