BRCA1 and BRCA2 heterozygosity in embryonic stem cells reduces radiation-induced Rad51 focus formation but is not associated with radiosensitivity

Abstract

Purpose: The breast cancer susceptibility genes BRCA1 (breast cancer 1) and BRCA2 (breast cancer 2) encode proteins involved in double-strand break (DSB) repair, whose functions include facilitating homologous recombination through interactions with Rad51, the human homologue of bacterial RecA. Homozygous deficiency inhibits Rad51 focus formation and enhances radiosensitivity, but the effects of heterozygosity have not been investigated in detail. The purpose of this work was to examine the effect of heterozygosity on Rad51 activation and clonogenicity following X-irradiation (XR).

Materials and methods: We used quantitative assessment of immunofluorescent foci to assess Rad51 activation in wild type mouse embryonic fibroblasts (MEF) and in paired mutant and wild type BRCA1 and BRCA2 embryonic stem cells (ES cells). We measured radiosensitivity in the same cell lines using clonogenic survival assays.

Results: ES cells exhibit higher numbers of cells with Rad51 foci post radiation than MEF, likely due to differences in cell cycle distribution. Compared to wild type cells, BRCA1 and BRCA2 heterozygous ES cells demonstrate lower numbers of Rad51 foci per nucleus 4 and 24 hours post radiation. This was not associated with significantly enhanced radiosensitivity.

Conclusions: BRCA1/2 heterozygosity in ES cells is associated with a subtle reduction in Rad51 foci formation that is not associated with increased XR induced cytotoxicity.

Keywords:

• BRCA1
• BRCA2
• Rad51
• radiosensitivity
• stem cells

Acknowledgements

The authors wish to thank Nuala McCabe of The Institute of Cancer Research, London. This work has been supported by UCLH/UCL Comprehensive Biomedical Research Centre.

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