Long time persistence of residual 53BP1/γ-H2AX foci in human lymphocytes in relationship to apoptosis, chromatin condensation and biological dosimetry

Abstract

Purpose: Novel assay for radiosensitivity is based on measurements of residual DNA repair foci produced by several proteins including phosphorylated H2AX (γ-H2AX), recombinase Rad51 (Rad51) and tumour suppressor p53 binding protein 1 (53BP1), which co-localise with radiation-induced DNA double-strand breaks (DSB). Here, we studied dose-response for residual 53BP1, Rad51, and γ-H2AX foci in relationship to apoptosis and chromatin condensation in human G₀-lymphocytes.

Materials and methods: Residual foci, apoptosis and condensation of chromatin were studied following irradiation with γ-rays at doses of 0.5–10 Gy.

Results: No clear dose response for residual Rad51 was seen. Residual 53BP1/γ-H2AX foci remained in human lymphocytes up to four weeks after irradiation. No foci formed during radiation-induced apoptosis. We provide evidence that irreversible apoptotic condensation of chromatin is responsible for arrest of residual foci and preventing de novo focus formation. Similar linear dose dependences up to 2 Gy were observed for the 53BP1/γ-H2AX foci at all studied time points. At higher doses, saturation and decline were caused by preferential elimination of apoptotic lymphocytes with residual foci. While primary 53BP1 and γ-H2AX foci almost completely co-localised, co-localisation of residual foci did not exceed 17%, indicating that 53BP1 and γ-H2AX proteins may remain for different times at the locations of DSB repair.

Conclusions: Prolonged persistence of residual 53BP1/γ-H2AX foci may be used for biological dosimetry within the dose range up to 2 Gy. While foci are not formed during radiation-induced apoptosis in human lymphocytes, elimination of apoptotic cells with residual foci may affect the dose response.

Keywords:

• apoptosis
• DNA repair foci
• chromatin condensation
• human lymphocytes
• radiation
• biodosimetry

Glossary:

• acridine orange (AO)
• DNA double-strand break (DSB)
• fetal bovine serum (FBS)
• homologous recombination (HR)
• non-homologous end-joining (NHEJ)
• phosphate buffered saline (PBS)
• phosphorylated histone 2A family member X (γ-H2AX)
• propidium iodide (PI)
• Rad 51 recombinase essential for homologous recombination and double-strand break repair (Rad51)
• room temperature (RT)
• tumor suppressor p53 binding protein 1 (53BP1)

Acknowledgements

The Swedish National Board for Laboratory Animals, the Swedish Authority for Radiation Protection, the National Scholarship Program of the Slovak Republic, the Research & Development Operational Program TRANSMED, ITMS: 26240120008 funded by the ERDF, and the VEGA Grant Agency (2/0167/08) of the Slovak Republic supported these studies.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.