Suzuki, K., Mori, I., Nakayama, Y., Miyakoda, M., Kodama, S. and Watanabe, M. Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening. Normal human diploid cells irradiated with X rays showed permanent cell cycle arrest and exhibited senescence-like phenotypes including the expression of senescence-associated beta-galactosidase (SA-beta-gal). X irradiation caused persistent phosphorylation of TP53 at Ser 15 and accumulation of the TP53 protein, followed by the induction of CDKN1A (also known as p21(Waf1/Cip1)) and CDKN2A (also known as p16), preceded the expression of SA-beta-gal. NCI-H1299 human lung carcinoma cells, in which no TP53 protein was expressed, were irradiated with X rays with or without the exogenous expression of TP53 gene. Although induction of TP53 protein alone could induce SA-beta-gal expression, the frequency of SA-beta-gal-positive cells was significantly increased when TP53-induced H1299 cells were exposed to X rays. The mean terminal restriction fragment length in normal human cells was approximately 12 kb and did not change in SA-beta-gal-positive cells. These results indicate that ionizing radiation induces senescence-like growth arrest that is dependent on TP53 function but independent of telomere shortening. Our findings suggest that cells harboring irreparable DNA damage are programmed to undergo premature senescence to maintain the integrity of the genome.

Radiation generates reactive oxygen species (ROS) that interact with cellular molecules, including DNA, lipids, and proteins. To know how ROS contribute to the induction of genetic instability, we examined the effect of the anti-ROS condition, using both ascorbic acid phosphate (APM) treatment or a low oxygen condition, on the induction of delayed reproductive cell death and delayed chromosome aberrations. The primary surviving colonies of mouse m5S-derived cl. 2011-14 cells irradiated with 6 Gy of X-rays were replated and allowed to form secondary colonies. The anti-ROS treatments were applied to either preirradiation culture or postirradiation cultures for primary or secondary colony formation. Both anti-ROS conditions relieved X-ray-induced acute cell killing to a similar extent. These anti-ROS conditions also relieved genetic instability when those conditions were applied during primary colony formation. However, no effect was observed when the conditions were applied during preirradiation culture and secondary colony formation. We also demonstrated that the amounts of ROS in X-ray-irradiated cells rapidly increase and then decrease at 6 hr postirradiation, and the levels of ROS then gradually decrease to a baseline within 2 weeks. The APM treatment kept the ROS production at a lower level than an untreated control. These results suggest that the cause of genetic instability might be fixed by ROS during a 2-week postirradiation period.

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The product of the ATM gene, which is mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it involves the activation of the p53 pathway after ionizing radiation. Here we show that the ATM protein is constitutively associated with double strand DNA and that the interaction increases when the DNA is exposed to ionizing radiation. The ATM protein also had affinity to restriction endonuclease PvuII-digested DNA, but not to UV-irradiated DNA nor X-irradiated single-stranded DNA. The immunoprecipitation experiment detected very weak association between ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or no effect on the interaction of the ATM protein with damaged DNA, indicating that an interaction with DNA-PK might not be required for the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are Chinese hamster ovary mutant cell lines defective in Ku80 function. These results indicate that the ATM protein is recruited to the site of DNA damage and it recognizes double strand breaks by itself or through an association with other DNA-binding protein other than DNA-PK and Ku80 proteins. Ataxia telangiectasia (AT)1 is a human autosomal recessive disorder characterized by a variety of clinical symptoms. At the cellular level, AT cells exhibit hypersensitivity to ionizing radiation, radio-resistant DNA synthesis, and a high frequency of chromosome aberrations (reviewed in Refs. 1-3). Recently, the gene defective in AT cells has been cloned and designated ATM (4). Sequence analysis revealed that the ATM protein shows homology to a family related to phosphatidylinositol 3-kinase, those include phosphatidylinositol 3-kinase p110 subunit, FRAP, RAFT1, and DNA-PK proteins in mammalian cells (2,(5)(6)(7). Because p53 accumulation is diminished or severely delayed in AT cells irradiated with ionizing radiation, ATM protein has been suggested to be involved in p53 activation (8 -11). p53 is a nuclear protein whose phosphorylation is required for its stability and activity (reviewed in Refs. 12-15). Our study and others (16,17) have reported that ionizing radiation causes p53 protein accumulation, and recently phosphorylation of p53 at serine 15 has been shown to control p53 accumulation through inhibition of its interaction with MDM2 (18 -20). Furthermore, recent in vitro experiments have indicated that ATM phosphorylates p53 protein at serine 15 (21, 22). Although there has been a report indicating the phosphorylation of serine 15 in AT cells (23), it is highly possible that the activity of p53 protein is mediated by ATM kinase activity.Very little is known about the mechanism by which ATM protein is activated by DNA damage. Previous studies have shown that ATM protein levels do not change during cell cycle progression or after exposure to ionizing radiation (24 -26), and therefore, it has been hypothesized that ATM protein may not be an inducible effector, but may act as a transducer or, more likely, a sensor for DNA dama...

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.

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Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1 Gy of X-rays. While the initial foci size was approximately 0.6 µm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6 µm or more in diameter at 24 h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1 and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1 Gy of X-rays and incubated for 24 h, the grown large phosphorylated ATM foci (≥1.6 µm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6 µm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM 4 by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint.These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.5

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