Microscopic imaging of DNA repair foci in irradiated normal tissues

Purpose: It is now feasible to detect DNA double strand breaks (DSB) in tissues by measuring the induction and resolution of DNA repair foci, such as γ-H2AX, using immunofluorescent microscopy and digital image analysis. This review will highlight principal tools and approaches to tissue microscopy and analysis. It will also discuss the practical considerations of using microscopy in vitro and in vivo in measuring intranuclear foci following irradiation. Conclusions: Computer-based image analysis algorithms allow an objective and quantitative analysis of foci and protein-protein interactions using 3D confocal images. Finally, we review the literature in which DNA repair foci have been investigated as a biodosimeter or a biomarker of DNA repair in normal tissues.

[1]  T. Zimmermann,et al.  Live cell spinning disk microscopy. , 2005, Advances in biochemical engineering/biotechnology.

[2]  A. Giaccia,et al.  Hypoxic Conditions Atm Activation and Signaling Under , 2008 .

[3]  J. Kobayashi,et al.  Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain. , 2004, Journal of radiation research.

[4]  I. Turesson,et al.  Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients. , 2008, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[5]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[6]  C. Rübe,et al.  DNA double-strand break rejoining in complex normal tissues. , 2008, International journal of radiation oncology, biology, physics.

[7]  J. Bartek,et al.  Differences in DNA double strand breaks repair in male germ cell types: lessons learned from a differential expression of Mdc1 and 53BP1. , 2007, DNA repair.

[8]  P. Olive,et al.  Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays , 2003, International journal of radiation biology.

[9]  Jan Nyman,et al.  DNA double strand break quantification in skin biopsies. , 2004, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[10]  F. Boussin,et al.  Radiation-Induced H2AX Phosphorylation and Neural Precursor Apoptosis in the Developing Brain of Mice , 2006, Radiation research.

[11]  J. Harper,et al.  Induction and quantification of γ-H2AX foci following low and high LET-irradiation , 2006 .

[12]  Stephen P. Jackson,et al.  A role for Saccharomyces cerevisiae histone H2A in DNA repair , 2000, Nature.

[13]  Kai Rothkamm,et al.  Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle , 2003, Molecular and Cellular Biology.

[14]  E. Scanziani Immunohistochemical staining of fixed tissues. , 1998, Methods in molecular biology.

[15]  A. Gingras,et al.  PP4 is a γH2AX phosphatase required for recovery from the DNA damage checkpoint , 2008, EMBO reports.

[16]  M. Kastan DNA Damage Responses: Mechanisms and Roles in Human Disease , 2008, Molecular Cancer Research.

[17]  M. Löbrich,et al.  DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing. , 1994, International journal of radiation biology.

[18]  M. Koike,et al.  Histone H2AX phosphorylation independent of ATM after X-irradiation in mouse liver and kidney in situ. , 2008, Journal of radiation research.

[19]  J. Moulder Report on an Interagency Workshop on the Radiobiology of Nuclear Terrorism , 2002, Radiation research.

[20]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[21]  Thomas Helleday,et al.  DNA repair pathways as targets for cancer therapy , 2008, Nature Reviews Cancer.

[22]  H. Burkhardt,et al.  Spatial quantitative analysis of fluorescently labeled nuclear structures: Problems, methods, pitfalls , 2008, Chromosome Research.

[23]  N. Chao Accidental or intentional exposure to ionizing radiation: biodosimetry and treatment options. , 2007, Experimental hematology.

[24]  A. Jeyasekharan,et al.  HP1-β mobilization promotes chromatin changes that initiate the DNA damage response , 2008, Nature.

[25]  M. Koike,et al.  Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ. , 2007, Biochemical and biophysical research communications.

[26]  F. Cordelières,et al.  A guided tour into subcellular colocalization analysis in light microscopy , 2006, Journal of microscopy.

[27]  M. Koike,et al.  p53 phosphorylation in mouse skin and in vitro human skin model by high-dose-radiation exposure. , 2005, Journal of radiation research.

[28]  S. Friend,et al.  Cancer Biomarkers—An Invitation to the Table , 2006, Science.

[29]  J. Swedlow,et al.  A workingperson's guide to deconvolution in light microscopy. , 2001, BioTechniques.

[30]  A. Nakano Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic. , 2002, Cell structure and function.

[31]  M. Engelhard,et al.  gamma-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: Dependence on the dose-distribution, irradiated site and time from start of treatment , 2007, International journal of radiation biology.

[32]  Michel Nussenzweig,et al.  H2AX: the histone guardian of the genome. , 2004, DNA repair.

[33]  E. F. Stanley,et al.  A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization , 2004, The Journal of Neuroscience.

[34]  John C. Russ,et al.  The Image Processing Handbook , 2016, Microscopy and Microanalysis.

[35]  D. Firsanov,et al.  Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ. , 2006, Biochemical and biophysical research communications.

[36]  E. Rogakou,et al.  Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo , 1999, The Journal of cell biology.

[37]  A. Chott,et al.  Effect of formalin tissue fixation and processing on immunohistochemistry. , 2000, The American journal of surgical pathology.

[38]  O. Hammarsten,et al.  An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans , 2007, Nucleic acids research.

[39]  Junjie Chen,et al.  Tumor Suppressor P53 Binding Protein 1 (53bp1) Is Involved in DNA Damage–Signaling Pathways , 2001, The Journal of cell biology.

[40]  P. Levendag,et al.  Quantitative analysis of radiation-induced DNA break repair in a cultured oral mucosal model. , 2006, Tissue engineering.

[41]  D. Delia,et al.  The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity. , 2001, The Journal of biological chemistry.

[42]  George Iliakis,et al.  γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin , 2008, Nucleic acids research.

[43]  J. Moulder Radiobiology of nuclear terrorism: report on an interagency workshop (Bethesda, MD, December 17-18, 2001). , 2002, International Journal of Radiation Oncology, Biology, Physics.

[44]  I. Belyaev,et al.  Kinetics and dose-response of residual 53BP1/γ-H2AX foci: Co-localization, relationship with DSB repair and clonogenic survival , 2007, International journal of radiation biology.

[45]  John R Yates,et al.  The hMre11/hRad50 Protein Complex and Nijmegen Breakage Syndrome: Linkage of Double-Strand Break Repair to the Cellular DNA Damage Response , 1998, Cell.

[46]  G. Moschini,et al.  DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study. , 2000, International journal of radiation biology.

[47]  R. Bristow,et al.  Biomarkers for DNA DSB inhibitors and radiotherapy clinical trials , 2008, Cancer and Metastasis Reviews.

[48]  T. Kashimoto,et al.  Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay. , 2007, Mutation research.

[49]  Michael Uder,et al.  In vivo formation and repair of DNA double-strand breaks after computed tomography examinations. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[50]  G J Brakenhoff,et al.  Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. , 1992, Journal of cell science.

[51]  Bo Stenerlöw,et al.  Focus Formation of DNA Repair Proteins in Normal and Repair-Deficient Cells Irradiated with High-LET Ions , 2004, Radiation research.

[52]  J. Dolling,et al.  Dose-Rate Effects for Apoptosis and Micronucleus Formation in Gamma-Irradiated Human Lymphocytes , 2000, Radiation research.

[53]  M. Tilby,et al.  γH2AX Foci Form Preferentially in Euchromatin after Ionising-Radiation , 2007, PloS one.

[54]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[55]  Richard P. Hill,et al.  The Basic Science of Oncology , 1989 .

[56]  Michael Lichten,et al.  Distribution and Dynamics of Chromatin Modification Induced by a Defined DNA Double-Strand Break , 2004, Current Biology.

[57]  P. Jeggo,et al.  Harmonising the response to DSBs: a new string in the ATM bow. , 2005, DNA repair.

[58]  F. Cucinotta,et al.  Induction and quantification of gamma-H2AX foci following low and high LET-irradiation. , 2006, International journal of radiation biology.

[59]  P. Olive,et al.  Phosphorylated histone H2AX in relation to cell survival in tumor cells and xenografts exposed to single and fractionated doses of X-rays. , 2006, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[60]  P. Jeggo,et al.  ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. , 2008, Molecular cell.

[61]  Y. Shiloh,et al.  Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway , 2006, Nature Cell Biology.

[62]  Jung-Ae Kim,et al.  Heterochromatin is refractory to γ-H2AX modification in yeast and mammals , 2007, The Journal of cell biology.

[63]  K. Luger,et al.  Histone chaperones and nucleosome assembly. , 2003, Current opinion in structural biology.

[64]  James B. Mitchell,et al.  Molecular and Cellular Biology of Moderate-Dose (1–10 Gy) Radiation and Potential Mechanisms of Radiation Protection: Report of a Workshop at Bethesda, Maryland, December 17–18, 20011 , 2003, Radiation research.

[65]  S. Lovett,et al.  The DNA Damage Response , 2011 .

[66]  Danny Vesprini,et al.  Homologous recombination and prostate cancer: a model for novel DNA repair targets and therapies. , 2007, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[67]  T. Halazonetis,et al.  P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks , 2000, The Journal of cell biology.

[68]  Winfried Denk,et al.  New developments in multiphoton microscopy , 2002, Current Opinion in Neurobiology.

[69]  M. Löbrich,et al.  DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing , 2008, Clinical Cancer Research.

[70]  M. Löbrich,et al.  Measurement of DNA double-strand breaks in mammalian cells by pulsed-field gel electrophoresis: a new approach using rarely cutting restriction enzymes. , 1994, Radiation research.

[71]  Alberto Diaspro,et al.  Two-photon fluorescence excitation and related techniques in biological microscopy , 2005, Quarterly Reviews of Biophysics.

[72]  E. Rogakou,et al.  Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint‐blind DNA damage , 2003, EMBO reports.

[73]  H T van der Voort,et al.  Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. , 1996, Journal of cell science.

[74]  Takamitsu A Kato,et al.  γ-H2AX Foci after Low-Dose-Rate Irradiation Reveal Atm Haploinsufficiency in Mice , 2006, Radiation research.

[75]  P. Olive,et al.  Phosphorylation of histone H2AX as a measure of radiosensitivity. , 2004, International journal of radiation oncology, biology, physics.

[76]  Jiri Bartek,et al.  Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks , 2006, The Journal of cell biology.

[77]  M. Mumby PP2A: Unveiling a Reluctant Tumor Suppressor , 2007, Cell.

[78]  Sylvain V Costes,et al.  Automatic and quantitative measurement of protein-protein colocalization in live cells. , 2004, Biophysical journal.

[79]  Richard P. Hill,et al.  Hypoxia and metabolism: Hypoxia, DNA repair and genetic instability , 2008, Nature Reviews Cancer.

[80]  P. Olive,et al.  DNA damage and repair in individual cells: applications of the comet assay in radiobiology. , 1999, International journal of radiation biology.

[81]  Colin G. Coates,et al.  Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution. , 2004, Journal of biomedical optics.

[82]  Y. Shiloh,et al.  Nuclear retention of ATM at sites of DNA double strand breaks. , 2001, The Journal of biological chemistry.

[83]  P. Shaw Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging , 2006 .