Delayed γH2AX foci disappearance in mammary epithelial cells from aged women reveals an age-associated DNA repair defect.

Teresa Anglada,Anna Espinal,Anna Genescà,Martha R Stampfer,Mark A Labarge,Marta Martín,Joan Repullés

doi:10.18632/aging.101849

Abstract

Aging is a degenerative process in which genome instability plays a crucial role. To gain insight into the link between organismal aging and DNA repair capacity, we analyzed DNA double-strand break (DSB) resolution efficiency in human mammary epithelial cells from 12 healthy donors of young and old ages. The frequency of DSBs was measured by quantifying the number of γH2AX foci before and after 1Gy of γ-rays and it was higher in cells from aged donors (ADs) at all times analyzed. At 24 hours after irradiation, ADs retained a significantly higher frequency of residual DSBs than young donors (YDs), which had already reached values close to basal levels. The kinetics of DSB induction and disappearance showed that cells from ADs and YDs repair DSBs with similar speed, although analysis of early times after irradiation indicate that a repair defect may lie within the firing of the DNA repair machinery in AD cells. Indeed, using a mathematical model we calculated a constant factor of delay affecting aged human epithelial cells repair kinetics. This defect manifests with the accumulation of DSBs that might eventually undergo illegitimate repair, thus posing a relevant threat to the maintenance of genome integrity in older individuals.

Highlights

The aging process is related to a loss of function and an increased probability of developing several diseases, such as cancer
human mammary epithelial cells (HMECs) were obtained from reduction mammoplasty tissue of 12 donors, which were classified according to age into young donors (YDs, ≤ 27, age in parentheses): YD48R(16), YD240L(19), YD168R(19), YD184(21), YD59L(23) and YD123(27) and aged donors (ADs, ≥ 60, age in parentheses): AD153L(60), AD112R(61), AD122L(66), AD29(68), AD429ER(72), AD353P(72)
In order to rule out radiationsensitivity differences between the two breast cell types, cells from young and aged donors were exposed to 1Gy of γ-rays and labelled with γH2AX and claudin-4 (Cl4), a cytoplasmic membrane protein mostly expressed by www.aging‐us.com luminal cells (Figure 1D)

Summary

Introduction

The aging process is related to a loss of function and an increased probability of developing several diseases, such as cancer. One of the earliest events in DSB signaling is the phosphorylation of the histone H2AX on serine 139, γH2AX [2]. Phosphorylation of H2AX spreads over megabases surrounding the break site, resulting in a platform that enables the recruitment of effector proteins at the damaged DNA [3]. The modification of H2AX can be www.aging‐us.com identified as discrete foci forming at DSB sites and scoring of γH2AX foci is a widely used tool to estimate the number of DSBs induced after exposure to damaging agents [4]. ΓH2AX foci disappearance over time is a good approach to assess kinetics of DSB repair because once DNA has been repaired, H2AX phosphorylation disappears and foci are no longer detectable [5]. The DSB repair kinetics follow a biphasic pattern: most of the DSBs are repaired by the fast component of repair within the first two hours after induction, while the remaining DSBs can be repaired by the slow component of repair, which acts with slower kinetics and might require several hours –or even days– to complete repair [6,7,8]

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