skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells

Abstract

Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique representsmore » an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.« less

Authors:
 [1];  [1];  [2];  [1];  [2];  [3];  [4];  [1];  [5];  [1];  [2]
  1. Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States)
  2. (United States)
  3. Department of Medical Physics, The Ottawa Hospital Cancer Centre, Ottawa, Ontario (Canada)
  4. Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Centre, Dallas, Texas (United States)
  5. Crystal Growth Division, Landauer, Inc, Stillwater, Oklahoma (United States)
Publication Date:
OSTI Identifier:
22648800
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Radiation Oncology, Biology and Physics; Journal Volume: 96; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; CHARGED PARTICLES; DAMAGE; DNA REPAIR; DOSE RATES; FLUORESCENCE; FLUORINE COMPOUNDS; LIVER; PARTICLE TRACKS; PROTON BEAMS; RADIOTHERAPY; STRAND BREAKS

Citation Formats

McFadden, Conor H., Hallacy, Timothy M., Department of Physics and Astronomy, Rice University, Houston, Texas, Flint, David B., Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas, Granville, Dal A., Asaithamby, Aroumougame, Sahoo, Narayan, Akselrod, Mark S., Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org, and Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas. Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells. United States: N. p., 2016. Web. doi:10.1016/J.IJROBP.2016.04.007.
McFadden, Conor H., Hallacy, Timothy M., Department of Physics and Astronomy, Rice University, Houston, Texas, Flint, David B., Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas, Granville, Dal A., Asaithamby, Aroumougame, Sahoo, Narayan, Akselrod, Mark S., Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org, & Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas. Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells. United States. doi:10.1016/J.IJROBP.2016.04.007.
McFadden, Conor H., Hallacy, Timothy M., Department of Physics and Astronomy, Rice University, Houston, Texas, Flint, David B., Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas, Granville, Dal A., Asaithamby, Aroumougame, Sahoo, Narayan, Akselrod, Mark S., Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org, and Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas. 2016. "Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells". United States. doi:10.1016/J.IJROBP.2016.04.007.
@article{osti_22648800,
title = {Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells},
author = {McFadden, Conor H. and Hallacy, Timothy M. and Department of Physics and Astronomy, Rice University, Houston, Texas and Flint, David B. and Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas and Granville, Dal A. and Asaithamby, Aroumougame and Sahoo, Narayan and Akselrod, Mark S. and Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org and Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas},
abstractNote = {Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.},
doi = {10.1016/J.IJROBP.2016.04.007},
journal = {International Journal of Radiation Oncology, Biology and Physics},
number = 1,
volume = 96,
place = {United States},
year = 2016,
month = 9
}
  • Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peakmore » intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.« less
  • 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 ablemore » 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 resolve 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 µm 2 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. Furthermore, 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
  • Two successive transient electromagnetic surveys were carried out over an underground gas storage site in France. The idea was to monitor changes in the gas bubble from the differences in the data. If successful, the new methodology could help to reduce the number of monitoring wells and finally reduce costs. Preliminary 3D modeling indicated that the resistivity changes caused by movements of the gas/water contact should be detectable in the electric field transients provided that the signal-to-noise ratio is at least 100:1. The surveys were performed with the TEAMEX multichannel acquisition system, adapted from a seismics system. The highly redundantmore » data were analyzed by calculating the relative differences in the electric field transients. The differences were common-midpoint, sorted and spatially stacked. Another approach was the calculation of electric field time derivatives in a log-log domain, to eliminate static shift effects which are present in the data. Even though the data quality is excellent from a classical point of view, neither of the two approaches reveals changes in the data which might be caused by changes in the gas reservoir. In future applications to monitoring, transmitters and receivers should be installed permanently, and the transmitter input waveform should be monitored continuously, to avoid some of the problems encountered here. Moreover, the signal-to-noise ratio will have to be further increased by at least one order of magnitude.« less
  • Time-lapse crosswell seismic data acquired with a cemented receiver cable have been processed into P- and S-wave tomograms which image heavy oil sand lithofacies and changes as a result of steam injection. Twenty-seven crosswell surveys were acquired between two wells over a 3.5 month period before, during, and after a 34-day, 30 MBBL (4,770 m{sup 3}) steam injection cycle. Interpretation was based on correlations with reservoir data and models, observation well data, and engineering documentation of the production history and steam cycle. Interdisciplinary interpretation indicates that tomograms not only complement other borehole-derived reservoir characterization and temperature monitoring data but canmore » be used to quantitatively characterize interwell reservoir properties and monitor changes as a result of the thermal recovery process. Monitoring results over 3.5 months confirms that stratification has controlled the flow of steam, in contrast to gravity override. This suggests that tomographic images of reservoir flow-units and gas-bearing high temperature zones should be useful for positioning wells and optimizing injection intervals, steam volumes, and producing well completions.« less