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ABSTRACT: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays.
Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays.
Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function.
The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.
International journal of radiation oncology, biology, physics 02/2012; 83(2):e241-50. · 4.59 Impact Factor
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ABSTRACT: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.
Biochemical and Biophysical Research Communications 11/2011; 416(1-2):111-8. · 2.48 Impact Factor
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10/2011; , ISBN: 978-953-307-606-5
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ABSTRACT: The cohesin loading factor NIPBL is required for cohesin to associate with chromosomes and plays a role in DNA double-strand break (DSB) repair. Although the NIPBL homolog Scc2 is recruited to an enzymatically generated DSB and promotes cohesin-dependent DSB repair in yeast, the mechanism of the recruitment remains poorly understood. Here we show that the human NIPBL is recruited to the sites of DNA damage generated by micro-irradiation as well as to the sites of DSBs induced by homing endonuclease, I-PpoI. The recruitment of NIPBL was impaired by RNAi-mediated knockdown of MDC1 or RNF168, both of which also accumulate at DSBs. We also show that the recruitment of NIPBL to the sites of DNA damage is mediated by its C-terminal region containing HEAT repeats and Heterochromatin protein 1 (HP1) interacting motif. Furthermore, NIPBL accumulation at damaged sites was also compromised by HP1γ depletion. Taken together, our study reveals that human NIPBL is a novel protein recruited to DSB sites, and the recruitment is controlled by MDC1, RNF168 and HP1γ.
Biochemical and Biophysical Research Communications 08/2011; 411(4):762-7. · 2.48 Impact Factor
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ABSTRACT: We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2'-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 μM BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.
Nature Protocol 02/2011; 6(2):134-9. · 8.36 Impact Factor
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ABSTRACT: Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm(2)). Cells were grown for up to 3 days, and phosphorylated ATM at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process.
Journal of Radiation Research 01/2011; 52(6):766-74. · 1.68 Impact Factor
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ABSTRACT: An ataxia-telangiectasia mutated (ATM)-dependent DNA damage signal is amplified through the interaction of various factors, which are recruited to the chromatin regions with DNA double-strand breaks. Spatial and temporal regulation of such factors is analysed by fluorescence microscopy in combination with laser micro-irradiation. Here we describe a novel and simple technique for micro-irradiation that does not require a laser source. Cells were labelled with BrdU for 48-72 h, covered with porous polycarbonate membranes, and exposed to UVC. All BrdU-labelled cells showed localized foci of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 upon irradiation, showing that these foci were induced irrespective of the cell-cycle phase. They were also detectable in nucleotide excision repair-defective XPA cells labelled with BrdU, indicating that the foci did not reflect an excision repair-related process. Furthermore, an ATM-specific inhibitor significantly attenuated the foci formation, and disappearance of the foci was significantly abrogated in non-homologous end-joining-defective cells. Thus, it can be concluded that micro-irradiation generated DNA double-strand breaks in BrdU-sensitized cells. The present technique should accelerate research in the fields of DNA damage response, DNA repair and DNA recombination, as it provides more chances to perform micro-irradiation experiments without any specific equipment.
Nucleic Acids Research 04/2010; 38(12):e129. · 8.03 Impact Factor
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ABSTRACT: DNA double strand breaks induced by DNA damaging agents, such ionizing radiation, are repaired by multiple DNA repair pathways including non-homologous end-joining (NHEJ) repair and homologous recombination (HR) repair. ATM-dependent DNA damage checkpoint regulates a part of DNA repair pathways, however, the exact role of ATM activity remains to be elucidated. In order to define the molecular structure of DNA double strand breaks requiring ATM activity we examined repair of DNA double strand breaks induced by different restriction endonucleases in normal human diploid cells treated with or without ATM-specific inhibitor.
Synchronized G1 cells were treated with various restriction endonucleases. DNA double strand breaks were detected by the foci of phosphorylated ATM at serine 1981 and 53BP1. DNA damage was detectable 2 hours after the treatment, and the number of foci decreased thereafter. Repair of the 3'-protruding ends created by Pst I and Sph I was efficient irrespective of ATM function, whereas the repair of a part of the blunt ends caused by Pvu II and Rsa I, and 5'-protruding ends created by Eco RI and Bam HI, respectively, were compromised by ATM inhibition.
Our results indicate that ATM-dependent pathway plays a pivotal role in the repair of a subset of DNA double strand breaks with specific end structures.
Genome integrity. 01/2010; 1(1):4.
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ABSTRACT: Developmentally, every tissue accommodates different types of cells, for example, epitheliocytes and stromal cells of mesenchymal
origin in parenchymal organs. To gain insights into the particulars of radiation response, it is essential to evaluate possible
cross talk between different cellular components of a tissue. This work addressed the reciprocal influence of normal human
epithelial/ mesenchymal cells interactions on the extent of radiation-induced DNA damage in comparison with epithelial cancer/normal
mesenchymal cells. Individual or mixed epithelial/mesenchymal cell cultures, including primary human thyrocytes (PT), normal
diploid fibroblasts, normal mammary epithelial or endothelial cells, and several human thyroid and breast cancer cell lines,
or cell cultures after conditioned medium transfer, were tested for the number of γ-H2AX foci as a measure of double-strand
DNA breaks following exposure to gamma rays. In the mixed PT/ fibroblast cultures, the number of γ-H2AX foci was significantly
lower in both types of cells as compared to individual cultures. Reciprocal conditioned medium transfer to individual counterpart
cells before irradiation also resulted in the reduction in the number of γ-H2AX foci in both PT and fibroblasts. The reciprocal
DNAprotective effect was likewise observed in the endothelial cell/fibroblast but not in the epithelial cell/endotheliocyte
systems. In contrast to medium conditioned on PT cells, conditioned medium collected from cancer cell lines did not establish
a DNA-protected state in normal fibroblasts and vice versa. The results imply the existence of a soluble factor-mediated network
of reciprocal interactions between normal epithelial and some types of mesenchymal cells that act in a paracrine manner to
protect DNA from genotoxic stress.
12/2008: pages 294-301;
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ABSTRACT: Ionizing radiation causes deleterious effects in cells that have directly absorbed its energy as well as in those which are
not exposed to radiation directly. The latter are often referred to as radiation-induced nontargeted effects. Radiationinduced
genomic instability is one of those effects, and it is manifested as the expression of various delayed effects, such as delayed
cell death, delayed chromosomal instability, and delayed mutagenesis. Because this instability accumulates genetic changes
in the genome, it has been hypothesized to be a driving force to accelerate multistep carcinogenesis. Exposure to ionizing
radiation causes doublestrand breaks in DNA, which result in deletion of the genome through illegitimate rejoining of the
broken ends. Recently, we found that large deletions could be transmitted in the progeny of cells surviving ionizing radiation.
As large deletions disrupt higher-order chromatin structure and chromatin codes, they possibly comprise potentially unstable
chromatin regions, whose disintegration causes delayed manifestation of radiation-induced genomic instability. Our present
study defi nes the molecular nature of DNA damage memory, which is associated with nontargeted effects.
12/2008: pages 123-126;
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ABSTRACT: 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 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h 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 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) 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 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.
DNA Repair 03/2008; 7(3):405-17. · 4.14 Impact Factor
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ABSTRACT: We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.
Radiation Research 06/2006; 165(5):499-504. · 2.68 Impact Factor
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