Enhanced induction of abnormal telomere FISH signals in response to oxidative DNA damage.

Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we investigated X-ray-induced abnormalities in telomere signals detected by fluorescence in situ hybridization (telomere FISH) in mouse embryo fibroblast cells. The abnormalities were categorized as either extra telomere signals (ETSs) or loss of telomere signals (LTSs).

High susceptibility of mouse newborns to delayed appearance of DNA double-strand breaks in neural stem/progenitor cells exposed to ionizing radiation.

Fetal brains are known to be extremely sensitive to ionizing radiation, which can induce structural and functional defects in the developing brain. However, there is less data on the effects of radiation on newborn brains. To determine the radiation sensitivity in newborn brains, we determined the number of DNA double-strand breaks (DSBs) appearing at later stage post-irradiation in neural stem/progenitor cells (NSPCs) of mouse newborns <3 days old, and compared it with the numbers of DSBs of fetal, 1-week-neonate, 2-week-neonate, and adult mice.

Repair kinetics of DNA double-strand breaks and incidence of apoptosis in mouse neural stem/progenitor cells and their differentiated neurons exposed to ionizing radiation.

Neuronal loss leads to neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Because of their long lifespans, neurons are assumed to possess highly efficient DNA repair ability and to be able to protect themselves from deleterious DNA damage such as DNA double-strand breaks (DSBs) produced by intrinsic and extrinsic sources. However, it remains largely unknown whether the DSB repair ability of neurons is more efficient compared with that of other cells.

A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein-Protein Interactions by Epitope and Arginine Grafting.

The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix.

Induction of genetic instability by transfer of a UV-A-irradiated chromosome.

Exposure of cells to ultraviolet (UV)-A radiation induces oxidative damage in DNA, such as 8-oxo-7,8-dihydroguanine (8-oxoG), single-strand breaks, a-basic sites, and DNA-protein cross-links, via reactions with reactive oxygen species (ROS). In this study we examine whether the damage other than double-strand breaks (non-DSB damage), which is UV-A-induced oxidative damage, plays a role in the induction of chromosomal instability. We exposed human chromosome 21 to UV-A and transferred the chromosome into non-irradiated mouse recipient cells by microcell-mediated chromosome transfer.

Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen).

In vitro thermal effects on embryonic cells of endangered hawksbill turtle Eretmochelys imbricata.

The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro.

Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss.

Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells.

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells.

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials And Methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy).

Persistent amplification of DNA damage signal involved in replicative senescence of normal human diploid fibroblasts.

Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts.

Epigenetic gene silencing is a novel mechanism involved in delayed manifestation of radiation-induced genomic instability in mammalian cells.

We examined mechanisms involved in delayed mutagenesis in CHO-LacZeo cells harboring the fusion gene between the bacterial LacZ and the Zeocin-resistance genes. After X irradiation, Zeocin-resistant primary colonies were isolated, and the primary clones were subjected to the secondary colony formation in the absence of Zeocin. We found that the surviving primary clones showed a significantly higher delayed mutation frequency compared with those derived from nonirradiated CHO-LacZeo cells.

Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation.

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined.

Long-term persistence of X-ray-induced genomic instability in quiescent normal human diploid cells.

Ionizing radiation can induce genomic instability in the progeny of irradiated cells, as was demonstrated in various experimental systems. Most in vitro studies have utilized replicating cells, but it is not clear whether radiation-induced genomic instability persists in quiescent cells. Here we show the induction of X-ray-induced genomic instability in normal human diploid cells irradiated and maintained in a quiescent state for up to 24 months while cells were subcultured approximately once every 2-3 months.

Introduction of a normal human chromosome 8 corrects abnormal phenotypes of Werner syndrome cells immortalized by expressing an hTERT gene.

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging and caused by mutations of the WRN gene mapped at 8p12. To examine functional complementation of WS phenotypes, we introduced a normal human chromosome 8 into a strain of WS fibroblasts (WS3RGB) immortalized by expressing a human telomerase reverse transcriptase subunit (hTERT) gene. Here, we demonstrate that the abnormal WS phenotypes including cellular sensitivities to 4-nitroquinoline-1-oxide (4NQO) and hydroxy urea (HU), and chromosomal radiosensitivity at G(2) phase are corrected by expression of the WRN gene mediated by introducing a chromosome 8.

Different involvement of radical species in irradiated and bystander cells.

Purpose: To examine whether nitric oxide (NO) and other radical species are involved in radiation-induced bystander effects in normal human fibroblasts.

Materials And Methods: Bystander effects were modeled by co-culture of non-irradiated cells with X-irradiated cells, and induction levels of micronuclei in co-cultured non-irradiated cells were examined. Three types of radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), dimethylsulfoxide (DMSO) and ascorbic acid phosphoric ester magnesium salt (APM), were used to discover which types of radicals are involved in bystander responses.

p53 status-dependent sensitization of human tumour cells to hyperthermia by plant flavonol.

Purpose: Quercetin (QCT), an important flavonol, is known to sensitize tumour cells to hyperthermia by suppressing heat shock protein 72 (Hsp72) induction, and is also reported to inhibit p53 accumulation. This study was conducted to examine the effects of QCT on the heat sensitivities of human tumour cell lines with different p53 statuses.

Material And Methods: Cell lines derived from human cancers and p53-inducible cells were used.

Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling.

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.

Effective suppression of bystander effects by DMSO treatment of irradiated CHO cells.

Evidence is accumulating that irradiated cells produce some signals which interact with non-exposed cells in the same population via a bystander effect. Here, we examined whether DMSO is effective in suppressing radiation induced bystander effects in CHO and repair deficient xrs5 cells. When 1 Gy-irradiated CHO cells were treated with 0.

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells.

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced.

Early response of neural stem/progenitor cells after X-ray irradiation in vitro.

We investigated the effect of oxidative stress on cell cycle regulation of neural stem/progenitor cells in neurosphere culture. We exposed murine neural stem/progenitor cells to 2 Gy of X-ray irradiation at 48 h after first passage. We found that G2 and G1-arrested cells increased at 3 and 12 h after X-ray irradiation, respectively by using laser scanning cytometer.

Increased chromosome instability and accumulation of DNA double-strand breaks in Werner syndrome cells.

Werner syndrome (WS) is a premature aging syndrome caused by mutations of the WRN gene. Here, we demonstrate that a strain of WS fibroblast cells shows abnormal karyotypes characterized by several complex translocations and 50-fold more frequency of abnormal metaphases including dicentric chromosomes without fragments than normal cells when examined at a similar culture stage. Further, telomere fluorescence in situ hybridization indicates that the abnormal signals, extra telomere signal and loss of telomere signal, emerge two- to three-fold more frequently in WS cells than in normal cells.

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells.

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells.

[Low-dose radiation effects and intracellular signaling pathways].

Accumulated evidence has shown that exposure to low-dose radiation, especially doses less than 0.1 Gy, induces observable effects on mammalian cells. However, the underlying molecular mechanisms have not yet been clarified.

Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair.

DNA repair within the cell nucleus is a dynamic process involving a close interaction between repair proteins and chromatin structure. Recent studies have indicated a quantitative relationship between DNA double-strand break induction and histone H2AX phosphorylation. The dynamics of this process within individual cell nuclei is unknown.

Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation.

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.