Title: Characterizing the DNA damage response by cell tracking algorithms and cell features classification using high-content time-lapse analysis

Abstract

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolvemore » with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 microm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.« less

Authors:

Georgescu, Walter ^[1]; Osseiran, Alma ^[1]; Rojec, Maria ^[1]; Liu, Yueyong ^[2]; Bombrun, Maxime ^[3]; Tang, Jonathan ^[1]; Costes, Sylvain V. ^[1]; Huen, Michael Shing-Yan ^[4]

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+ Show Author Affiliations

1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
2. Harvard Medical School, Boston, MA (United States)
3. CNRS, Aubiere (France)
4. Univ. of Hong Kong (Hong Kong)

Publication Date:

Wed Jun 24 00:00:00 EDT 2015

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1212447

Alternate Identifier(s):

OSTI ID: 1469157

Report Number(s):

LBNL-185582
Journal ID: ISSN 1932-6203

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

PLoS ONE

Additional Journal Information:

Journal Volume: 10; Journal Issue: 6; Journal ID: ISSN 1932-6203

Publisher:

Public Library of Science

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; ionizing radiation; cell death; DNA repair; DNA damage; epithelial cells; image processing; linear programming; nuclear bodies