Page 124 - 2014 Printable Abstract Book
P. 124
(PS1-38) Superresolution microscopy visualizes nanostructure of carbon ion-induced DSB repair foci.
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Ramon Lopez Perez, MS ; Gerrit Best, PhD ; Nils Nicolay, MD PhD ; Christoph Cremer, PhD ; Peter Huber,
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MD PhD, German Cancer Research Center, Heidelberg, Germany ; KIP/Ophtalmology, Heidelberg,
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Germany ; and Institute of Molecular Biology/KIP, Mainz/Heidelberg, Germany
Experimental and clinical data using carbon-ion radiotherapy show that the biological effectiveness of
heavy-ion irradiation is enhanced in comparison to conventional radiotherapy using for example photon-
radiation. It is assumed that one reason for this endpoint dependent observation are clustered DNA
lesions, and in particular, clusters of DNA double-strand breaks (DSBs). Such multiple lesion sites can arise
because heavy-ions deposit their energy in form of densely spaced ionizations along their trajectory
through the nucleus. So far, the clustering of DSBs could not be directly observed using
immunofluorescent staining of γH2AX foci or other DSB repair proteins. It is known that heavy-ions induce
larger γH2AX foci than γ-radiation does, however the inner structure of the foci has remained largely
obscure due to the limited resolution of conventional microscopes, including confocal microscopes. Using
a combination of two superresolution microscopy techniques, we were able to show in human
glioblastoma cells that carbon ion-induced γH2AX foci are composed of several small subfoci of round
shape with a typical diameter of ~60 nm. The physical peculiarity of superresolution microscopy is its
ability to circumvent the classical resolution limit given by Abbe’s law to enable an optical resolution in
the order of 10-100 nm. The subfoci may represent the elementary repair machinery for individual DSBs
or may indicate the presence of multiple repair complexes; both interpretations would however support
the hypothesis of clustered DSBs as one reason for the high relative biological effectiveness of heavy-ion
radiation. Taken together, the new optical technology opens completely new insights in the repair
processes upon radiation.
(PS1-39) Irradiation techniques for facilitating the study of DNA damage induction and repair following
high or low-LET irradiation. Mark A. Hill, PhD; James Thompson, PhD; Pamela Reynolds, PhD; and David
L. Stevens,CRUK/MRC Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford,
United Kingdom
The induction of DNA damage and its subsequent repair is of critical importance with regards to
the fate of individual cells, not only with respect to cell killing which is important for radiotherapy, but
also genetic alteration potentially relevant for carcinogenesis. Many studies currently use fluorescently
labelled antibodies or proteins to follow the induction and subsequent repair of radiation induced DNA
damage and associated recruitment of repair proteins. Irradiation techniques have been developed to
help facilitate these studies for both low-LET x-rays and high-LET alpha-particle irradiation. A soft x-ray
irradiation facility has been designed to irradiate cells with 1 µm wide stripes of x-rays at 10 micrometre
intervals. The production of damage in a geometric fashion helps facilitate the identi-fication of rad-
iation induced foci where the signal is low compared to background levels. This has the advantages over
laser induced stripe-irradiation systems in that all the damage produced are common to all low-LET
radiation. Irradiation of cells with high-LET alpha-particles produces correlated damage along the path
of the particle, these are difficult to resolve following conventional irradiation perpendicular to a cell
mono layer and typically produce a single foci per track. Irradiation techniques have been developed to
irradiate a cell monolayer with alpha - particles at a shallow angle so enabling the visualization of the
induction and repair of damage sites along the individual alpha-particle tracks. The development,
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2
1
3
Ramon Lopez Perez, MS ; Gerrit Best, PhD ; Nils Nicolay, MD PhD ; Christoph Cremer, PhD ; Peter Huber,
1
1
MD PhD, German Cancer Research Center, Heidelberg, Germany ; KIP/Ophtalmology, Heidelberg,
3
2
Germany ; and Institute of Molecular Biology/KIP, Mainz/Heidelberg, Germany
Experimental and clinical data using carbon-ion radiotherapy show that the biological effectiveness of
heavy-ion irradiation is enhanced in comparison to conventional radiotherapy using for example photon-
radiation. It is assumed that one reason for this endpoint dependent observation are clustered DNA
lesions, and in particular, clusters of DNA double-strand breaks (DSBs). Such multiple lesion sites can arise
because heavy-ions deposit their energy in form of densely spaced ionizations along their trajectory
through the nucleus. So far, the clustering of DSBs could not be directly observed using
immunofluorescent staining of γH2AX foci or other DSB repair proteins. It is known that heavy-ions induce
larger γH2AX foci than γ-radiation does, however the inner structure of the foci has remained largely
obscure due to the limited resolution of conventional microscopes, including confocal microscopes. Using
a combination of two superresolution microscopy techniques, we were able to show in human
glioblastoma cells that carbon ion-induced γH2AX foci are composed of several small subfoci of round
shape with a typical diameter of ~60 nm. The physical peculiarity of superresolution microscopy is its
ability to circumvent the classical resolution limit given by Abbe’s law to enable an optical resolution in
the order of 10-100 nm. The subfoci may represent the elementary repair machinery for individual DSBs
or may indicate the presence of multiple repair complexes; both interpretations would however support
the hypothesis of clustered DSBs as one reason for the high relative biological effectiveness of heavy-ion
radiation. Taken together, the new optical technology opens completely new insights in the repair
processes upon radiation.
(PS1-39) Irradiation techniques for facilitating the study of DNA damage induction and repair following
high or low-LET irradiation. Mark A. Hill, PhD; James Thompson, PhD; Pamela Reynolds, PhD; and David
L. Stevens,CRUK/MRC Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford,
United Kingdom
The induction of DNA damage and its subsequent repair is of critical importance with regards to
the fate of individual cells, not only with respect to cell killing which is important for radiotherapy, but
also genetic alteration potentially relevant for carcinogenesis. Many studies currently use fluorescently
labelled antibodies or proteins to follow the induction and subsequent repair of radiation induced DNA
damage and associated recruitment of repair proteins. Irradiation techniques have been developed to
help facilitate these studies for both low-LET x-rays and high-LET alpha-particle irradiation. A soft x-ray
irradiation facility has been designed to irradiate cells with 1 µm wide stripes of x-rays at 10 micrometre
intervals. The production of damage in a geometric fashion helps facilitate the identi-fication of rad-
iation induced foci where the signal is low compared to background levels. This has the advantages over
laser induced stripe-irradiation systems in that all the damage produced are common to all low-LET
radiation. Irradiation of cells with high-LET alpha-particles produces correlated damage along the path
of the particle, these are difficult to resolve following conventional irradiation perpendicular to a cell
mono layer and typically produce a single foci per track. Irradiation techniques have been developed to
irradiate a cell monolayer with alpha - particles at a shallow angle so enabling the visualization of the
induction and repair of damage sites along the individual alpha-particle tracks. The development,
122 | P a g e
