RESUMO
How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.
Assuntos
Transformação Celular Neoplásica/genética , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/química , Cromatina/metabolismo , Ciclina D1/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Treonina/metabolismo , UbiquitinaçãoRESUMO
In experimental organisms such as fruit flies and mice, increased frequencies in germ cell mutations have been detected following exposure to ionizing radiation. In contrast, there has been no clear evidence for radiation-induced germ cell mutations in humans that lead to birth defects, chromosome aberrations, Mendelian disorders, etc. This situation exists partly because no sensitive and practical genetic marker is available for human studies and also because the number of people exposed to large doses of radiation and subsequently having offspring was small until childhood cancer survivors became an important study population. In addition, the genome of apparently normal individuals seems to contain large numbers of alterations, including dozens to hundreds of nonfunctional alleles. With the number of mutational events in protein-coding genes estimated as less than one per genome after 1 gray (Gy) exposure, it is unsurprising that genetic effects from radiation have not yet been detected conclusively in humans.
Assuntos
Genoma Humano/efeitos da radiação , Anormalidades Induzidas por Radiação/etiologia , Anormalidades Induzidas por Radiação/genética , Animais , Aberrações Cromossômicas , Cromossomos/efeitos da radiação , Dano ao DNA , Relação Dose-Resposta à Radiação , Drosophila melanogaster/efeitos da radiação , Feminino , Seguimentos , Mutação em Linhagem Germinativa/efeitos da radiação , Humanos , Masculino , Camundongos , Modelos Animais , Mutagênese , Neoplasias Induzidas por Radiação/epidemiologia , Neoplasias Induzidas por Radiação/genética , Armas Nucleares , Exposição Ocupacional , Lesões por Radiação/genética , Tolerância a Radiação , Liberação Nociva de Radioativos , Radioterapia/efeitos adversos , Razão de Masculinidade , SobreviventesRESUMO
This study was designed to learn if asymptomatic heterozygotes with mutations in a DNA repair gene are at an increased risk for cancer. To examine this, we focused on carriers of an XPA founder mutation because the frequency of xeroderma pigmentosum (XP) patients is much greater among Japanese than Caucasians, more than half of Japanese XP patients are affected at the XPA gene, and the majority of XP-A patients carry the same founder mutation in the XPA gene. Here we show that the frequency of XPA heterozygote was 14/1698 (0.8%) in cancer-free controls, and the corresponding frequency in patients with nonmelanocytic skin cancer that developed in sun-exposed areas was 11/440 (2.5%, OR = 3.08, p = 0.0097) for basal cell carcinoma, and 3/272 (1.1%, OR = 1.34, p = 0.72) for squamous cell carcinoma. These results suggest a moderately elevated risk for skin cancer among XPA heterozygotes.
Assuntos
Adenocarcinoma/genética , Povo Asiático/genética , Carcinoma de Células Escamosas/genética , Efeito Fundador , Heterozigoto , Mutação , Neoplasias Cutâneas/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Idoso , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-IdadeRESUMO
After an exposure to ionising radiation, cells can quickly repair damage to their genomes; however, a few unrepairable DNA double-strand breaks (DSBs) emerge in the nucleus in a prolonged culture and perpetuate as long as the culture continues. These DSBs may be retained forever in cells such as non-dividing ageing tissues, which are resistant to apoptosis. We show that such unrepairable DSBs, which had been advocated by the classical target theory as the 'radiation hit', could account for permanent growth arrest and premature senescence. The unrepairable DSBs build up with repeated irradiation, which accounts for an accumulated dose. Because these DSBs tend to be paired, we propose that the untethered and 'torn-off' molecular structures at the broken ends of the DNA result in an alteration of chromatin structure, which protects the ends of the DNA from genomic catastrophe. Such biochemical responses are important for cell survival but may cause gradual tissue malfunction, which could lead to the late effects of radiation exposure. Thus, understanding the biology of unrepairable damage will provide new insights into the long-term effects of radiation.
Assuntos
Linhagem da Célula/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Radiação Ionizante , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Diploide , Relação Dose-Resposta à Radiação , Ativação Enzimática/efeitos da radiação , Fibroblastos/metabolismo , Humanos , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação/efeitos da radiaçãoRESUMO
Although mammalian fetuses have been suggested to be sensitive to radiation, an increased frequency of translocations was not observed in blood lymphocytes from atomic bomb (A-bomb) survivors who were exposed to the bomb in utero and examined as adults. Since experiments using hematopoietic cells of mice and rats confirmed this finding, it was hypothesized that either irradiated fetal hematopoietic stem cells (f-HSCs) cannot generate exchange-type chromosomal aberrations or cells bearing induced aberrations are eliminated before the animals reach adulthood. In the present study, pregnant mice (12.5-15.5 days post coitum [dpc]) were irradiated with 2 Gy of X-rays and long-term HSCs (LT-HSCs) were isolated 24 h later. Multicolor fluorescence in situ hybridization (mFISH) analysis of LT-HSC clones proliferated in vitro showed that nine out of 43 (21%) clones from fetuses and 21 out of 41 (51%) clones from mothers bore translocations. These results indicate that cells with translocations can arise in mouse f-HSCs but exist at a lower frequency than in the mothers 24 h after X-ray exposure. Thus, it seems likely that translocation-bearing f-HSCs are generated but subsequently disappear, so that the frequency of lymphocyte translocations may decrease and reach the control level by the time the animals reach adulthood.
Assuntos
Aberrações Cromossômicas , Translocação Genética , Gravidez , Feminino , Ratos , Animais , Hibridização in Situ Fluorescente , Células-Tronco Hematopoéticas , Feto/efeitos da radiação , MamíferosRESUMO
The frequency of stable chromosome aberrations (sCA) in lymphocytes is a recognized radiation biological dosimeter. Its analysis can provide insights into factors that affect individual susceptibility as well as into the adequacy of radiation dose estimates used in studies of atomic bomb survivors. We analyzed the relationship between atomic bomb radiation exposure using the most recent DS02R1 dose estimates and the frequency of sCA as determined by FISH in 1,868 atomic bomb survivors. We investigated factors that may affect the background sCA rate and the shape and magnitude of the dose response. As in previous analyses of sCA in atomic bomb survivors that were based on Giemsa staining methods and used older DS86 dose estimates, the relationship between radiation dose and sCA rate was significant (P < 0.0001) with a linear-quadratic relationship at lower doses that did not persist at higher doses. As before, age at the time of the bombing and type of radiation shielding were significant dose-effect modifiers (P < 0.0001), but in contrast the difference in dose response by city was not so pronounced (P = 0.026) with a city effect not evident at doses below 1.25Gy. Background sCA rate increased with age at the time of examination (P < 0.0001), but neither sex, city, nor smoking was significantly associated with background rate. Based on FISH methods and recent dosimetry, the relationship between radiation dose and sCA frequency is largely consistent with previous findings, although the lesser importance of city as an effect modifier may reflect better dosimetry as well as more reproducible scoring of sCA. The persisting difference in sCA dose response by shielding category points to remaining problems with the accuracy or precision of radiation dose estimates in some A-bomb survivors.
Assuntos
Guerra Nuclear , Exposição à Radiação , Humanos , Sobreviventes de Bombas Atômicas , Radiometria/métodos , Exposição à Radiação/efeitos adversos , Aberrações Cromossômicas , Sobreviventes , Japão , Relação Dose-Resposta à RadiaçãoRESUMO
We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene (TetO-EGFP) and tetracycline repressor (TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8-3.4×10(-6)/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.
Assuntos
Proteínas de Fluorescência Verde/genética , Testes de Mutagenicidade/métodos , Mutação/efeitos da radiação , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Raios X , Linhagem Celular Tumoral , Células Cultivadas , Fibrossarcoma , Deleção de Genes , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/genética , Sensibilidade e Especificidade , Tetraciclina/metabolismoRESUMO
PURPOSE: Cancer risks for Nagasaki survivors once appeared to be lower than for Hiroshima survivors. The possibility that this was due to overestimation of the doses for the Nagasaki survivors was tested by measuring biological doses of Nagasaki survivors and comparing them with DS02R1 individual doses as previously done for Hiroshima survivors. MATERIALS AND METHODS: The electron spin resonance (ESR) method and cytogenetic method were used to estimate radiation doses for 24 Nagasaki survivors, and the results were compared to calculated DS02R1 doses. RESULTS: Six factory workers and 10 other survivors showed ESR or cytogenetically estimated doses that were in reasonably good agreement with their DS02R1 doses, while one factory worker was found to have an ESR dose estimate of nearly one half of the DS02R1 dose to the eye lens (a proxy organ for teeth). A few outliers were also observed. CONCLUSIONS: Although apparently lower cancer risks were observed in the past for Nagasaki survivors when compared to Hiroshima survivors, the present results do not indicate the existence of a trend that DS02R1 doses are overestimated when compared with biologically estimated tooth or cytogenetic doses. This observation is in line with the recent disappearance of the city difference in cancer risks.
Assuntos
Análise Citogenética , Esmalte Dentário/metabolismo , Esmalte Dentário/efeitos da radiação , Armas Nucleares , Radiometria/métodos , Sobreviventes , Relação Dose-Resposta à Radiação , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Exposição Ocupacional/análiseRESUMO
Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.
Assuntos
Translocação Genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Ásia , Criança , Pré-Escolar , Coloração Cromossômica , Etnicidade , Europa (Continente) , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , América do Norte , Fatores de Risco , Fatores Sexuais , FumarRESUMO
Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.
Assuntos
Citometria de Fluxo/métodos , Linfócitos T/efeitos da radiação , Adulto , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Reparo do DNA , Relação Dose-Resposta à Radiação , Feminino , Fase G1/efeitos da radiação , Humanos , Antígenos Comuns de Leucócito/análise , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/efeitos da radiação , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Tolerância a Radiação , Receptores de IgG/análise , Fase de Repouso do Ciclo Celular/efeitos da radiação , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Tempo , Raios XRESUMO
Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Escherichia coli/enzimologia , Metilação de DNA , Desoxirribonucleases de Sítio Específico do Tipo I/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Plasmídeos , Especificidade por Substrato , Transformação GenéticaRESUMO
Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic 60Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the 60Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated 60Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.
Assuntos
Análise Citogenética , Raios gama/efeitos adversos , Células-Tronco Hematopoéticas/efeitos da radiação , Armas Nucleares , Fótons/efeitos adversos , Doses de Radiação , Sobreviventes , Criança , Radioisótopos de Cobalto/efeitos adversos , Esmalte Dentário/metabolismo , Esmalte Dentário/efeitos da radiação , Células-Tronco Hematopoéticas/metabolismo , Humanos , RadiometriaRESUMO
Individuals who are homozygotes for mutations in DNA repair genes are at high risk for cancer. It is not well documented, however, if the heterozygous carriers of the mutation are also predisposed to cancer. To address the issue, xeroderma pigmentosum (XP) in Japan is an interesting candidate because of three major reasons: XP is an autosomal recessive disorder with an enormously elevated risk of skin cancer, the frequency of XP patients is higher in Japan than in other parts of the world, and more than half of Japanese XP patients are homozygous for the same founder mutation in the XPA gene. We screened archival blood samples from Japanese individuals who resided in Hiroshima or Nagasaki. A simple PCR-RFLP method was developed that is highly specific for detection of XPA heterozygotes carrying the founder mutation. We identified nine XPA heterozygotes among 1,020 individuals screened for a prevalence of 0.88%. This rate, if representative, implies that there are about 1 million carriers of the XPA founder mutation in the Japanese population. Thus, investigation of their cancer risk may be warranted.
Assuntos
Efeito Fundador , Heterozigoto , Mutação/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Adolescente , Adulto , Povo Asiático/genética , Estudos de Coortes , Frequência do Gene , Predisposição Genética para Doença/genética , Humanos , Japão , Reação em Cadeia da Polimerase/métodos , Xeroderma Pigmentoso/genéticaRESUMO
It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.
Assuntos
Duplicação Gênica , Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hipoxantina Fosforribosiltransferase/genética , Mutação , Animais , Éxons , Técnicas de Introdução de Genes , Genes , Intestino Delgado/citologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Mutação/efeitos da radiação , Pâncreas/citologia , Baço/citologiaRESUMO
INTRODUCTION: Progerin, the protein responsible for the Hutchinson-Gilford Progeria Syndrome (HGPS), is a partially deleted form of nuclear lamin A, and its expression has been suggested as a cause for dysfunctional nuclear membrane and premature senescence. To examine the role of nuclear envelop architecture in regulating cellular aging and DNA repair, we used ionizing radiation to increase the number of DNA double strand breaks (DSBs) in normal and HGPS cells, and analyzed possible relationship between unrepaired DSBs and cellular aging. RESULTS: We found that HGPS cells are normal in repairing a major fraction of radiation-induced double strand breaks (M-DSBs)but abnormal to show increased amount of residual unrepaired DSBs (R-DSBs). Such unrepaired DSBs were 2.6 times (CI 95 %: 2.2-3.2) higher than that in normal cells one week after the irradiation, and 1.6 times (CI 95 %: 1.3-1.9) higher even one month after the irradiation. These damages tend to increase as the nuclear envelope become abnormal, a characteristic of both HGPS and normal human cells which undergo replicative senescence. The artificial, enforced over-expression of progerin further impaired the repair of M-DSBs, implying lamin A-associated nuclear membrane has an important role for DNA DSB repair. Introduction of telomerase gene function in HGPS cells reversed such aging phenotypes along with upregulation of lamin B1 and downregulation of progerin, which is a hallmark of young cells. CONCLUSION: We suggest that lamin A- or progerin-associated nuclear envelope is involved in cellular aging associated with DNA damage repair.
RESUMO
The stem of kidney bean plant (Phaseolus vulgaris L., cv. Kentucky 101), a typical dextrorse climbing plant, was subjected to compulsorily sinistrorse-winding. The compulsory sinistrorse-winding induced changes in physiological activities. The number of pods with immature seeds (used as vegetable) was doubled and the fresh weight of the pods also significantly increased by sinistrorse-winding. Compulsory sinistrorse-winding increased chlorophyll content, photosynthetic rate, respiration, nodule formation, N(2)-fixation, glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming); E.C. 6.3.1.2] activity and protein content. Thus, it seems to affect the basic physiological processes that promote physiological activities though the action mechanism is unknown.
Assuntos
Phaseolus/fisiologia , Biomassa , Dióxido de Carbono/metabolismo , Clorofila/biossíntese , Glutamato-Amônia Ligase/metabolismo , Fixação de Nitrogênio , Nitrogenase/metabolismo , Phaseolus/crescimento & desenvolvimento , Phaseolus/microbiologia , Fotossíntese , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/biossíntese , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Caules de Planta/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimentoRESUMO
In both humans and mice, fetal exposure to radiation fails to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level response to radiation exposure is counterintuitive in view of the generally accepted belief that a fetus is sensitive to radiation. To determine if this is a general phenomenon, both mammary epithelial cells and spleen cells were studied in rats. Fetuses of 17.5 days postcoitus were irradiated with 2 Gy of gamma rays, and mammary tissues were removed 6-45 weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH method. The results showed that translocation frequencies were not only elevated in rats irradiated as fetuses, but were also almost as high as those in rats that were irradiated as adults (12 weeks old, pregnant mothers or young virgins) and examined 6-45 weeks later. There was no evidence of higher sensitivity in fetal cells with respect to the induction of translocations. In contrast, translocation frequencies in spleen cells were not elevated in adult rats irradiated as fetuses but were increased after irradiation of adults as previously seen in mouse spleen cells and human T lymphocytes. In the case of irradiation of adult rats, the induced translocation frequencies were similar between spleen cells and mammary epithelial cells. If we take translocation frequency as a surrogate marker of potential carcinogenic effect of radiation, the current results suggest that fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but this does not contribute to the increased risk of cancer since it has been reported that irradiation of fetal rats of the SD strain does not increase the risk of mammary cancers. Possible reasons for this discrepancy are discussed.
Assuntos
Feto/metabolismo , Feto/efeitos da radiação , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/efeitos da radiação , Translocação Genética/efeitos da radiação , Adulto , Animais , Feminino , Feto/citologia , Feto/imunologia , Humanos , Linfócitos/efeitos da radiação , Masculino , Camundongos , Gravidez , Ratos , Especificidade da Espécie , Baço/imunologiaRESUMO
Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.
Assuntos
Reprogramação Celular/genética , Senescência Celular/genética , Instabilidade Cromossômica/genética , Telômero/genética , Síndrome de Werner/genética , Adulto , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neoplasias/genética , Fenótipo , Telomerase/metabolismo , Síndrome de Werner/metabolismoRESUMO
The electron paramagnetic resonance (EPR, or electron spin resonance) method was used to measure CO2â»· radicals recorded in tooth enamel by exposure to atomic-bomb gamma rays. The EPR-estimated doses (i.e. 6°Co gamma-ray equivalent dose) were generally in good correlation with cytogenetic data of the same survivors, whereas plots of EPR-estimated dose or cytogenetically estimated dose against DS02 doses turned out to scatter more widely. Because those survivors whose EPR doses were higher (or lower) than DS02 doses tended to show also higher (or lower) responses for cytogenetic responses, the apparent variation appears primarily due to problems in individual DS02 doses rather than the measurement errors associated with the EPR or cytogenetic technique. A part of the enamel samples were also used for evaluation of neutron doses by measuring 4¹Ca/4°Ca ratios using the accelerator mass spectrometry technique. The results for the measured ratios were on average ~85 % of the calculated ratios by DS02 (but within the 95 % confidence bounds of the simulated results), which lends support to DS02-derived neutron doses to the survivors.
Assuntos
Esmalte Dentário/efeitos da radiação , Espectroscopia de Ressonância de Spin Eletrônica , Raios gama , Espectrometria de Massas , Nêutrons , Armas Nucleares , Radiometria , Sobreviventes , Aberrações Cromossômicas , Humanos , Aceleradores de PartículasRESUMO
We previously reported that mouse fetuses or neonates exposed to 2 Gy of X rays showed an unexpectedly low incidence of chromosome damage in lymphocytes, bone marrow, and spleen cells when the mice were subsequently examined at 20 weeks of age. However, cells bearing translocations were occasionally observed that, on the basis of 2-color whole chromosome painting appeared to be clonal descendants. Unfortunately, this approach typically did not permit unequivocal confirmation of their clonality. To overcome this problem, multi-color FISH (mFISH) was employed, which assigns all 21 individual chromosome types of the mouse a unique color. After mFISH analyses of the same cell samples studied previously, it was confirmed that spleen cells of 20-week-old mice irradiated either as 15.5-day fetuses or as 3- to 4-day-old neonates showed translocation frequencies close to zero. Translocations previously suspected as being clonal in nature were confirmed as such by mFISH, which also revealed the presence of an additional clone not previously detected or suspected. Since no evidence of clonality was observed in the irradiated mother, we concluded that in both fetuses and neonates, there exists a small fraction of stem cells that are distinct from the bulk of the stem cell compartment in terms of their ability to acquire and transmit radiation-induced chromosome damage through clonal expansion.