Kinetics and dose-response of residual 53BP1/γ-H2AX foci: Co-localization, relationship with DSB repair and clonogenic survival

Abstract

Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (γ-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/γ-H2AX foci formation in relation to their co-localization, DSB repair and cell survival.

Materials and methods: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci.

Results: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of γ-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/γ-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair.

Conclusions: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.

Keywords:

• DSB co-localizing foci
• human cells
• radiation
• repair

Abbreviations
DSB	=	DNA double-strand break
IOD	=	integrated optical density
γ-H2AX	=	phosphorylated histone 2A family member X
PFGE	=	pulsed-field gel electrophoresis
53BP1	=	tumor suppressor p53 binding protein 1

Abbreviations
DSB	=	DNA double-strand break
IOD	=	integrated optical density
γ-H2AX	=	phosphorylated histone 2A family member X
PFGE	=	pulsed-field gel electrophoresis
53BP1	=	tumor suppressor p53 binding protein 1