Following the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a great concern. The induction of chromosomal aberrations in ...splenic lymphocytes of small Japanese field mice (Apodemus argenteus) and house mice (Mus musculus) inhabiting Fukushima Prefecture was investigated. In mice inhabiting the slightly contaminated area, the average frequency of dicentric chromosomes was similar to that seen in mice inhabiting a noncontaminated control area. In contrast, mice inhabiting the moderately and heavily contaminated areas showed a significant increase in the average frequencies of dicentric chromosomes. Total absorbed dose rate was estimated to be approximately 1 mGy d–1 and 3 mGy d–1 in the moderately and heavily contaminated areas, respectively. Chromosomal aberrations tended to roughly increase with dose rate. Although theoretically, the frequency of chromosomal aberrations was considered proportional to the absorbed dose, chromosomal aberrations in old mice (estimated median age 300 days) did not increase with radiation dose at the same rate as that observed in young mice (estimated median age 105 days).
The nuclear foci of phosphorylated histone H2AX (γH2AX) are frequently used as a marker for DNA double-strand breaks (DSBs) following ionizing radiation (IR). However, recent studies reported that ...γH2AX foci do not necessarily correlate with DSBs under other conditions. We showed that γH2AX foci induced by oxidative stress in hydrogen peroxide (H2O2)-treated cells displayed several different features from those induced by IR. The magnitude of γH2AX induction was heterogeneous among H2O2-treated cells. Some cells expressed small discrete γH2AX foci, whereas others expressed a gross γH2AX signal that was distributed throughout the nucleus. Oxidative stress-induced γH2AX was eliminated in DSB repair-deficient mutant cells as efficiently as in wild-type cells and was not necessarily accompanied by phosphorylated ataxia telangiectasia mutated (ATM) or 53BP1 foci. Analyses using specific inhibitors showed that ATM- and Rad3-related (ATR), rather than ATM, was the prominent kinase mediating the oxidative stress response. These results suggest that a major fraction of γH2AX induced by oxidative stress is not associated with DSBs. Single-stranded DNA arisen from stalled replication forks can cause the ATR-mediated induction of γH2AX. However, oxidative stress appeared to induce γH2AX in both S- and non-S-phase cells. These results suggest that there may be another pathway leading to the ATR-mediated induction of γH2AX in non-S-phase cells without DSBs.
Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. ...Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Non-homologous end-joining (NHEJ) is the predominant pathway for the repair of DNA double-strand breaks (DSBs) in human cells. XRCC4 is indispensable to NHEJ and functions together with DNA ligase IV ...in the rejoining of broken DNA ends. Artemis is a nuclease required for trimming of some, but not all, types of broken DNA ends prior to rejoining by the DNA ligase IV/XRCC4 complex. To better understand the roles of these factors, we generated XRCC4- and Artemis-deficient cells from the human colon adenocarcinoma cell line HCT116 by gene targeting and examined their cellular responses to several DNA-damaging agents including X-rays. As anticipated, kinetic analyses of γ-H2AX foci and chromosomal aberrations after ionizing radiation (IR) demonstrated a serious incompetence of DSB repair in the XRCC4-deficient cells, and relatively moderate impairment in the Artemis-deficient cells. The XRCC4-deficient cells were highly sensitive to etoposide and 5-fluoro-2'-deoxyuridine as well as IR, and moderately sensitive to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, aphidicolin and hydroxyurea, compared to the parental HCT116 cells. The Artemis-deficient cells were not as sensitive as the XRCC4-deficient cells, except to cisplatin and mitomycin C. By contrast, the Artemis-deficient cells were significantly more resistant to hydroxyurea than the parental cells. These observations suggest that Artemis also functions in some DNA damage response pathways other than NHEJ in human cells.
Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G
2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. To ...identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human
SMU1 gene. Comparison of sequences of the
Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39°C. Furthermore, cdc2 kinase was not activated during G
2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.
Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell-cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. We ...previously identified an amino acid substitution in Smu1 that underlies the temperature-sensitive phenotypes of tsTM18 cells. In the present study, we confirmed that Smu1 is associated with the temperature-sensitive defect of tsTM18 by RNA interference. We also found an early temperature effect in DNA synthesis. Because genetic studies of nematodes revealed that smu-1 is involved in splicing of the unc52/perlecan pre-mRNA, we analysed the perlecan transcript in tsTM18 cells by reverse transcription-polymerase chain reaction (RT-PCR). The perlecan PCR product amplified from RNA of tsTM18 cells cultured at 39°C appeared to be a mixture of variants. Sequence analysis identified at least six variants that result from alternative splicing and intron retention. Comparison of the results of perlecan RT-PCR analysis with those of analysis of four other genes suggested that the splicing defect in the perlecan gene is unique and that it is conserved through evolution.
Changes in the thymic microenvironment lead to radiation-induced thymic lymphomagenesis, but the phenomena are not fully understood. Here we show that radiation-induced chromosomal instability and ...bystander effects occur in thymocytes and are involved in lymphomagenesis in C57BL/6 mice that have been irradiated four times with 1.8-Gy -rays. Reactive oxygen species (ROS) were generated in descendants of irradiated thymocytes during recovery from radiation-induced thymic atrophy. Concomitantly, descendants of irradiated thymocytes manifested DNA lesions as revealed by -H2AX foci, chromosomal instability, aneuploidy with trisomy 15 and bystander effects on chromosomal aberration induction in co-cultured ROS-sensitive mutant cells, suggesting that the delayed generation of ROS is a primary cause of these phenomena. Abolishing the bystander effect of post-irradiation thymocytes by superoxide dismutase and catalase supports ROS involvement. Chromosomal instability in thymocytes resulted in the generation of abnormal cell clones bearing trisomy 15 and aberrant karyotypes in the thymus. The emergence of thymic lymphomas from the thymocyte population containing abnormal cell clones indicated that clones with trisomy 15 and altered karyotypes were prelymphoma cells with the potential to develop into thymic lymphomas. The oncogene Notch1 was rearranged after the prelymphoma cells were established. Thus, delayed nontargeted radiation effects drive thymic lymphomagenesis through the induction of characteristic changes in intrathymic immature T cells and the generation of prelymphoma cells.
Variants of the error-prone gene, mucAB, were found in Escherichia coli plasmids from 3 out of 63 healthy Japanese donors. As compared with mucAB of pKM101, one contained unaltered mucA only, while ...two others contained full-sized, mutated mucAB with mutation-enhancing activity in E. coli. Possible interaction between bacterial plasmid genes and host humans was discussed.
PDF
