Persistence of γ-H2AX and 53BP1 foci in proliferating and non-proliferating human mammary epithelial cells after exposure to γ-rays or iron ions

Abstract

Purpose: To investigate γ-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionising radiation under different cell culture conditions.

Material and methods: HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure.

Results: Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced γ-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both γ-H2AX and 53BP1 foci decreased to control levels during the 24–48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after γ-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition.

Conclusions: The disappearance of radiation-induced γ-H2AX and 53BP1 foci in HMEC has different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent γ-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double-strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodelling.

Keywords:

• Foci
• MCF10A
• HZE
• iron
• 3D
• HMEC

Acknowledgements

We thank Dr Marcelo Vazquez, Dr Peter Guida, Dr Betsy Sutherland, and Dr Adam Rusek and their groups for support during the NSRL runs at Brookhaven National Laboratory, Dr Janice Pluth (LBNL) for her help with flow cytometry analysis, Dr Martha Stampfer and Dr James Garbe for providing the 184v HMEC cells and for their cell culture support, and Christopher Pham for his help with fitting the curves. The research was support by NASA Grant no. T6275W (awarded to Dr. Mary-Helen Barcellos-Hoff, NSCOR), and in part by the Low Dose Radiation Program, Office of Biological and Environmental Research of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

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