Comparison of methods to quantify histone H2AX phosphorylation and its usefulness for prediction of radiosensitivity.
OBJECTIVE
The number of radio-induced double-strand breaks is correlated with the number of histone gamma-H2AX (γ-H2AX) foci. For this reason, foci quantification is a useful tool to measure radiation-induced DNA damage and the number of foci has been suggested as a predictive biomarker of radiosensitivity. The aim of the present study was to evaluate the reproducibility of different microscopic methodologies and flow cytometry analysis to score γ-H2AX induction, and its suitability to distinguish a radiosensitive (RS) cell line from a radioresistant (RR) one.


METHODS
γ-H2AX analyses were performed by semi-automated and automated microscopic methods and by flow cytometry before and after irradiation in two human lymphoblastoid cell lines and in lymphocytes from three healthy donors.


RESULTS
Reproducible results were obtained by all the methodologies tested, although not all showed the same sensitivity. The RS cell line always showed higher foci counts and higher levels of immunofluorescence intensity after irradiation than the RR cell line.


CONCLUSIONS
Our results suggest that microscopic methodologies with z-stage capacity give the most accurate results after 1 Gy irradiation. However, for high doses of ionizing radiation, flow cytometry gives reliable results. Further studies will be necessary to determine the usefulness of γ-H2AX analysis to predict adverse side reactions in radiotherapy patients.
a Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular, Fisiologia i Immunologia , Universitat Autònoma de Barcelona , Bellaterra, Catalonia , Spain ;
Mireia
M
Borràs
b Unitat d'Antropologia Biològica, Departament de Biologia Animal, Biologia Vegetal i Ecologia , Universitat Autònoma de Barcelona , Bellaterra, Catalonia , Spain ;
Gemma
G
Armengol
c Servei de Microscòpia , Universitat Autònoma de Barcelona , Bellaterra, Catalonia , Spain.
Martí
M
De Cabo
b Unitat d'Antropologia Biològica, Departament de Biologia Animal, Biologia Vegetal i Ecologia , Universitat Autònoma de Barcelona , Bellaterra, Catalonia , Spain ;
Joan-Francesc
JF
Barquinero
a Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular, Fisiologia i Immunologia , Universitat Autònoma de Barcelona , Bellaterra, Catalonia , Spain ;
Leonardo
L
Barrios
D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008214:Lymphocytes / Q000378:metabolism / Q000528:radiation effects; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010766:Phosphorylation / Q000528:radiation effects; D011836:Radiation Tolerance
C468783:H2AX protein, human; D006657:Histones
Histone γ-H2AX; flow cytometry; foci; radiosensitivity