Community Research and Development Information Service - CORDIS

Final Report Summary - PEROXIREDOXIN-CLOCK (A new intracellular metabolic timer mechanism to induce cell death selectively in cancer cells)

In this project we were aiming to identify new targetable links between cellular clocks and cell proliferation at the level of redox regulation. First we have developed a novel redox proteomics method allowing screening for direct targets of oxidative stress in non-cancerous fibroblast and in u2os cancer cell lines. Our data show that cell cycle related regulatory proteins are overrepresented to any other regulatory proteins in U2OS cells with enrichment of oxidized cysteine residues. In non-cancerous cells, proteins related to cell metabolism and signalling were found more prone to hyperoxidation if compared with other proteins. Thus, our proteomics screening has justified the potential and the relevance of the project.
For testing our hypothesis we have set up a live cell microscopy analysis platform. We have generated novel primary mammalian (mouse) cell lines stably expressing both cell cycle phase fluorescent nuclear reporters (FUCCI) and redox fluorescent cytoplasmic reporters (HyPER2, RoGFP2, RoGFP2-Orp1, RoGFP2-PRX1/2/3) using 3rd generation Lentiviral system. To our knowledge we are the first to successfully develop such cell lines which allow live analysis of redox and cell cycle events in the same mammalian cell. The redox status of proliferating cells have not been real time monitored in live cells successfully before. Thus our results are of great importance for both cell cycle and cancer researchers. Importantly, the live cell analysis platform developed by the Fellow provided crucial observations on the relationship of replicative activity and the circadian rhythm of cellular responses to virus infection. This was a collaborative research the Fellow got involved because of the platform he developed. Subsequently, results of this collaboration got published in a high ranked journal (PNAS).
An important innovative output of this project was the establishment of a new workflow for quantitative image analysis. The Fellow has acquired computational skills as of his training program and was able to develop javascript and R language based new scripts to execute algorithms for image clearing, segmentation, tracking and measurement, combined with data analysis and data visualisation. Importantly, the Fellow has been able to execute these programs as in one single pipeline launched from the command prompt, essential to handle large, time-lapse multidimensional images in an automated and unbiased way. The workflow allows unbiased nuclear and cytoplasmic measurements of over 100 cells per field over time across all fluorescent channels, and plots all measurements with single-cell resolution, allowing any time-lapse imaging to be implemented in a high throughput way. This is a significant innovation not previously available. Therefore, as an output of this project this can be considered as a cutting edge advancement in the field of microscopic image analysis in general, to be used in the host laboratory for all current and future projects with live cell imaging, and will likely result in new collaborations within UK and in Hungary.
Using this analysis pipeline, we have assayed fibroblasts of wild type and clock gene-deficient mice (cry1-/- cry2-/- double knockout mice; bmal1 -/- mice). Combining our approach with luminescent microscopy we have been able to analyse not only cell cycle and redox changes but also clock gene-based oscillations in live cells (with per2::luc posttranslational reporters). These are important control experiments for deciphering the role of the clock gene-independent oscillator in the context of this research project.
We have introduced cell cycle phase-dependent separation of these cells with FACS sorting and subsequent artificial perturbations of the peroxide levels in time-lapse multi-channel monitoring during cell cycle progression which allowed further characterisations of the interplay between redox and cell cycle regulation. We have successfully introduced these fluorescent reporters (cell cycle and redox) to U2OS cancer cells carrying per2::luc transcriptional reporters.
Results of our experiments are being prepared for publication in a well ranked journal. The peer review process will take some time beyond the Fellowship period. For the follow up, the Fellow has taught all methodologies for other postdocs and PhD students in the host laboratory which allows completion of additional experiments to be requested by reviewers of the journal.
Summary of the progress of the researcher training activities/transfer of knowledge activities:
The Fellow had no past experience with any of the procedures and developments successfully performed by him in this project. Beyond the training program at Cambridge University, the Fellow has made use of the knowledge acquired at an EMBO course (Leadership training for young leaders) in May 2015.


Renata Schaeffer, (European Policy Manager)
Tel.: +44 1223 333543
Fax: +44 1223 332988
Record Number: 196661 / Last updated on: 2017-04-05
Follow us on: RSS Facebook Twitter YouTube Managed by the EU Publications Office Top