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This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.
Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely γH2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of γH2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of γH2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of γH2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.
DNA damage is continuously induced by agents that can be endogenous, such as ROS generated by cellular oxidative metabolism, or exogenous, both chemicals and physical1. Among the most harmful lesions, double-strand breaks (DSBs) play a fundamental role in contributing to genomic instability, since they cause chromosome aberrations that in turn can initiate the carcinogenesis process. Thus, cells are provided with complex and efficient mechanisms of DSBs repairing2.
When a DSB occurs, the cell triggers the DNA damage response (DDR) where, together with the MRE11/RAD50/NBS1 complex, ATM or ATR kinases are recruited to activate other proteins that slow down or stop the cell cycle3. An essential target of these kinases is histone H2AX, which is phosphorylated on Ser-139 within a few megabases from the DSBs (namely γH2AX), thereby allowing the recruitment of several repair factors such as, among others, BRCA1 and p53 binding protein 1 (53BP1)3. Later, one pathway among homologous recombination (HR), non-homologous end joining (NHEJ), or single-strand annealing (SSA) is triggered to repair the DSBs4,5. Therefore, 53BP1 is involved in dictating the choice between HR or NHEJ, mainly promoting the activation of NHEJ rather than HR6. Moreover, both the phosphorylated form of H2AX histone and 53BP1 can form foci at the sites of DSBs. As these foci persist until the integrity of the double strand is restored, assessing the appearance/disappearance of γH2AX or 53BP1 foci within a time interval is considered a useful method to evaluate the occurrence and repair of DSBs in a cell system6,7. However, according to the molecular processes above described, since γH2AX and 53BP1 foci are expected to co-localize near the DSBs during DDR8,9, it can be useful to detect concurrently the presence of these markers in a dual immunofluorescence.
Thus, the aim of this manuscript was to evaluate the suitability of the simultaneous quantification of γH2AX and 53BP1 foci to assess the genomic damage induced in human peripheral lymphocytes by the radiomimetic agent bleomycin. Using the same methodology, we also attempted to delineate repair kinetics of bleomycin-induced DSBs according to a previously set up experimental procedure10.
The study was approved by the ethical committee of Pisa University, and informed and signed consent was obtained from each donor.
1. Formation of γH2AX and 53BP1 foci
2. Analysis through a fluorescence microscope
NOTE: "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths12. AlexaFluor568 and DyLight 488 absorb light of approximately 568 and 488 nm and emit light of 603 and 520 nm, respectively. Thus, they are visible as red or green fluorescence using a TRITC or a FITC filter, respectively.
Data obtained by the fluorescence microscope analysis of peripheral lymphocytes allow us to evaluate three main aspects: the effectiveness of bleomycin treatment in increasing the number of γH2AX and 53BP1 foci (and thus of DSBs) due to its mutagenic effect, at what extent both foci co-localized at the site of DSBs, and the time-course of γH2AX and 53BP1 foci to delineate the repair kinetics of bleomycin-induced DSBs. As expected, a very higher frequency of both γH2AX and 53BP1 foci was observed between un...
Immunofluorescence analysis of γH2AX and 53BP1 foci is a suitable method for assessing genomic damage in interphase nuclei of a cell system. This procedure has several critical points that can affect the outcome of the experiments, mainly, the agents used in fixation and permeabilization, the type of antibodies and their dilution factors, and the concentration of the mutagen.
The maintenance of protein integrity is fundamental since the immunofluorescence method expects to identify antige...
The authors have nothing to disclose.
We are grateful to the whole blood donors and all the health personnel who took the blood samples.
Name | Company | Catalog Number | Comments |
AlexaFluor 568 goat anti-mouse IgG (γ1) | Invitrogen | A21124 | 53BP1 secondary antibody |
Bleoprim | Sanofi | bleomycin sulfate (mutagen) | |
Penicillin-streptomycin solution 100X | Euroclone | ECB3001D | antibiotics for culture medium |
PBS 10X | Termofisher | 14200075 | Phosphate-buffered saline |
FBS | Euroclone | EC20180L | Fetal Bovine Serum for immunofluorescence |
Goat anti-rabbit IgG (H+L) DyLight 488 Coniugated | Termofisher | #35552 | γH2AX secondary antibody |
Mouse anti-53BP1 monoclonal antibody | Merck | MAB 3802 | 53BP1 primary antibody |
Labophot 2 | Nikon | Fluorescence microscope | |
P-histone H2AX (Ser139) rabbit antibody | Cell Signaling | #2577 | γH2AX primary antibody |
Phytohemoagglutinin | Termofisher | R30852801 | component of culture medium |
Prolong gold antifade reagent with DAPI | Cell Signaling | #8961 | Antifade solution with DAPI for counterstaining |
RPMI 1640 | Euroclone | ECB9006L | Culture medium |
Triton-X100 | Sigma | T9284 | Nonionic detergent for permeabilization |
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