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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 k....

Protocol

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

  1. Preparation of samples and mutagenic treatment
    1. Collect whole blood samples by venipuncture from healthy adult individuals in blood collection (e.g., Vacutainer) tubes containing lithium heparin as an anticoagulant.
    2. In order to guarantee proper blood sample preserva.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

We are grateful to the whole blood donors and all the health personnel who took the blood samples.

....

Materials

NameCompanyCatalog NumberComments
AlexaFluor 568 goat anti-mouse IgG (γ1)InvitrogenA2112453BP1 secondary antibody
BleoprimSanofibleomycin sulfate (mutagen)
Penicillin-streptomycin solution 100XEurocloneECB3001Dantibiotics for culture medium
PBS 10XTermofisher14200075Phosphate-buffered saline
FBSEurocloneEC20180LFetal Bovine Serum for immunofluorescence
Goat anti-rabbit IgG (H+L) DyLight 488 ConiugatedTermofisher#35552γH2AX secondary antibody
Mouse anti-53BP1 monoclonal antibodyMerckMAB 380253BP1 primary antibody
Labophot 2NikonFluorescence microscope
P-histone H2AX (Ser139) rabbit antibodyCell Signaling#2577γH2AX primary antibody
PhytohemoagglutininTermofisherR30852801component of culture medium
Prolong gold antifade reagent with DAPICell Signaling#8961Antifade solution with DAPI for counterstaining
RPMI 1640EurocloneECB9006LCulture medium
Triton-X100SigmaT9284Nonionic detergent for permeabilization

References

  1. Chatterjee, N., Walker, G. C. Mechanisms of DNA damage, repair and mutagenesis. Environmental and Molecular Mutagenesis. 58 (5), 235-263 (2017).
  2. Aleksandrov, R., Hristova, R., Stoynov, S., Gospodinov, A. The chromatin response to double....

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Dual ImmunofluorescenceH2AX53BP1Human Peripheral LymphocytesDNA DamageDouble strand BreaksBleomycinCell CultureFixationAntibody StainingFluorescence Microscopy

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