Down-regulation of a DNA damage barrier in aging human mammary epithelial cells

We aimed to investigate the capability of human mammary epithelial cells (HMEC) to respond to ionising radiation with the activation of the ATM-Chk2-p53 DNA damage response. HMEC at different passages were exposed to 0.1–6 Gy of ionising radiation, and cellular radiation responses were assessed by flow cytometry and by immunoblot analyses. Early passages of HMEC phosphorylated p53(Ser15), Nbn(Ser343) and Chk2(Thr68) in response to irradiation with 0.1 to 6 Gy in a dose-dependent manner. Inhibitor studies with KU–55933 revealed that these phosphorylation events were strictly dependent on the ataxia-telangiectasia mutated kinase, ATM. Irradiation at doses above 0.5 Gy altered the cell cycle profile of young HMEC towards a gradual loss of G1 and S phase cells and concomitant G2 accumulation. By contrast, aged HMEC showed a strongly reduced capability to phosphorylate p53 and Chk2 after irradiation, and aneuploidy became apparent in about 5% of the cells. Levels of ATM protein were normal in young and in aged HMEC, indicating that ATM activation rather than ATM expression is impaired in aging HMEC. Altogether, our data indicate that the radiation-inducible ATM-Chk2-p53 damage barrier is down-regulated in aging HMEC which might be a prerequisite for distinct cells to develop genomic instability and tumorigenic properties.

ATM - HMEC - ionising radiation - p53 - senescence