RESUMO
In most eukaryotes, poly(ADP-ribose) polymerase (PARP) recognizes DNA strand interruptions generated in vivo. DNA binding by PARP triggers primarily its own modification by the sequential addition of ADP-ribose units to form polymers; this modification, in turn, causes the release of PARP from DNA ends. Studies on the effects of the disruption of the gene encoding PARP (Adprt1, formerly Adprp) in mice have demonstrated roles for PARP in recovery from DNA damage and in suppressing recombination processes involving DNA ends. Telomeres are the natural termini of chromosomes and are, therefore, potential targets of PARP. Here, by the use of two different techniques, we show that mice lacking PARP display telomere shortening compared with wild-type mice. Telomere shortening is seen in different genetic backgrounds and in different tissues, both from embryos and adult mice. In vitro telomerase activity, however, is not altered in Adprt1-/- mouse fibroblasts. Furthermore, cytogenetic analysis of mouse embryonic fibroblasts reveals that lack of PARP is associated with severe chromosomal instability, characterized by increased frequencies of chromosome fusions and aneuploidy. The absence of PARP does not affect the presence of single-strand overhangs, naturally present at the ends of telomeres. This study therefore reveals an unanticipated role for PARP in telomere length regulation and provides insights into its functions in maintaining genomic integrity.
Assuntos
Cromossomos/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/fisiologia , Telômero/fisiologia , Aneuploidia , Animais , Aberrações Cromossômicas , Cruzamentos Genéticos , Enzimas de Restrição do DNA/metabolismo , Fibroblastos , Genótipo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hibridização de Ácido Nucleico , Telômero/genéticaRESUMO
To study the effect of continued telomere shortening on chromosome stability, we have analyzed the telomere length of two individual chromosomes (chromosomes 2 and 11) in fibroblasts derived from wild-type mice and from mice lacking the mouse telomerase RNA (mTER) gene using quantitative fluorescence in situ hybridization. Telomere length at both chromosomes decreased with increasing generations of mTER-/- mice. At the 6th mouse generation, this telomere shortening resulted in significantly shorter chromosome 2 telomeres than the average telomere length of all chromosomes. Interestingly, the most frequent fusions found in mTER-/- cells were homologous fusions involving chromosome 2. Immortal cultures derived from the primary mTER-/- cells showed a dramatic accumulation of fusions and translocations, revealing that continued growth in the absence of telomerase is a potent inducer of chromosomal instability. Chromosomes 2 and 11 were frequently involved in these abnormalities suggesting that, in the absence of telomerase, chromosomal instability is determined in part by chromosome-specific telomere length. At various points during the growth of the immortal mTER-/- cells, telomere length was stabilized in a chromosome-specific man-ner. This telomere-maintenance in the absence of telomerase could provide the basis for the ability of mTER-/- cells to grow indefinitely and form tumors.
Assuntos
Telomerase/deficiência , Telômero/genética , Telômero/ultraestrutura , Animais , Transformação Celular Neoplásica , Células Cultivadas , Aberrações Cromossômicas , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Modelos Genéticos , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/genética , Telomerase/genéticaRESUMO
Reactive oxygen species (ROS) have been closely associated with both apoptotic and non-apoptotic/necrotic cell death. Our previous study has illustrated that c-Jun-N-terminal kinase 1 (JNK1) is the main executor in hydrogen peroxide (H(2)O(2))-induced nonapoptotic cell death. The main objective of this study is to further elucidate the molecular mechanisms downstream of JNK1 in H(2)O(2)-induced cell death. In this study, poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair protein, was readily activated by H(2)O(2) and inhibition of PARP-1 activation by either a pharmacological or genetic approach offered significant protection against H(2)O(2)-induced cell death. More importantly, H(2)O(2)-mediated PARP-1 activation is subject to regulation by JNK1. Suppression of JNK1 activation by a chemical inhibitor or genetic deletion markedly suppressed the late-phase PARP-1 activation induced by H(2)O(2), suggesting that JNK1 contributes to the sustained activation of PARP-1. Such findings were supported by the temporal pattern of nuclear translocation of activated JNK and a direct protein-protein interaction between JNK1 and PARP-1 in H(2)O(2)-treated cells. Finally, in vitro kinase assay suggests that PARP-1 may serve as the direct phosphorylation target for JNK1. Taken together, data from our study reveal a novel underlying mechanism in H(2)O(2)-induced nonapoptotic cell death: JNK1 promotes a sustained PARP-1 activation via nuclear translocation, protein-protein interaction and PARP-1 phosphorylation.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Trifosfato de Adenosina/deficiência , Animais , Antracenos/farmacologia , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
Sudden cardiac death (SCD) can be caused by a number of reasons. Previous works have identified the genetic causes, such as alterations in the DNA sequence, for many of these diseases. We hypothesize that some patients may show genomic imbalances and changes in the gene copy number leading to genetic instability. To clarify this, we analysed DNA samples from SCD victims using comparative genomic hybridization (CGH), a molecular cytogenetic technique that permits the genome-wide screening of chromosomal imbalances, and telomere length measurement. DNA derived from peripheral blood and heart tissue of 14 SCD cases and six apparently healthy control individuals were subjected to CGH analysis. Telomere length measurements were done by the Southern blotting method. Eight out of 14 SCD cases exhibited changes in DNA/gene copy number. CGH analysis showed variation in the gene copy number of some of the genes associated with potassium (KCNAB1, KCNH2, and KCNA4) and calcium (RyR2, ATP2A2) ions which are involved in maintaining the ionic balance of the heart. Alterations in TERC and TERT genes were also detected in SCD victims. In nine SCD victims shorter telomeres were detected. This might have resulted from excessive cellular proliferation and/or oxidative stress in these individuals. Copy number changes observed and telomere shortening detected in SCD cases would possibly explain at least some of the causes of SCD at early ages in humans. Identification of biomarkers of SCD is of great importance and thus the present study will facilitate the identification of some of the biomarkers.
Assuntos
Desequilíbrio Alélico , Morte Súbita Cardíaca/patologia , Parada Cardíaca/genética , Canais Iônicos/genética , Telômero/genética , Adolescente , Adulto , Criança , Cromossomos Humanos/genética , Hibridização Genômica Comparativa , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Deleção de Sequência , Telômero/ultraestrutura , Adulto JovemRESUMO
DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.
Assuntos
Antígenos Nucleares , Cromossomos , DNA Helicases , Reparo do DNA , DNA/genética , Proteínas de Saccharomyces cerevisiae , Telômero , Animais , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hibridização in Situ Fluorescente , Autoantígeno Ku , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.
Assuntos
RNA , Telomerase/fisiologia , Telômero/fisiologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Marcação de Genes , Camundongos , Telomerase/genética , Telomerase/metabolismoRESUMO
Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.
Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Telômero/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Aberrações Cromossômicas , Cromossomos/metabolismo , Dano ao DNA , Primers do DNA/genética , Feminino , Genes p53 , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Knockout , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Poli(ADP-Ribose) Polimerases/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.
Assuntos
Biomarcadores Tumorais , Fragilidade Cromossômica/genética , Exonucleases , Deleção de Genes , Proteínas de Neoplasias , Proteínas/metabolismo , Telômero/genética , Telômero/efeitos da radiação , Proteínas 14-3-3 , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Bandeamento Cromossômico , Quebra Cromossômica/genética , Relação Dose-Resposta à Radiação , Exorribonucleases , Fase G1 , Fase G2 , Raios gama , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Índice Mitótico , Matriz Nuclear/metabolismo , Proteínas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequências Repetitivas de Ácido Nucleico/efeitos da radiação , Cromossomos em Anel , Telômero/metabolismo , Translocação Genética/genética , Translocação Genética/efeitos da radiação , Células Tumorais CultivadasRESUMO
Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.
Assuntos
Telomerase/fisiologia , Telômero/fisiologia , Animais , Sequência de Bases , Sobrevivência Celular , Clonagem Molecular , DNA , DNA Complementar , Camundongos , Dados de Sequência Molecular , Células-Tronco , Telomerase/genéticaRESUMO
TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.
Assuntos
Proteínas de Transporte/fisiologia , Telômero/fisiologia , Animais , Proteínas de Transporte/metabolismo , Catálise , Embrião de Mamíferos/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Modelos Genéticos , Mutagênese Sítio-Dirigida , Testes de Precipitina , RNA/metabolismo , Proteínas de Ligação a RNA , Recombinação Genética , Baço/citologia , Células-Tronco/metabolismo , Telomerase , Telômero/ultraestrutura , Timo/citologiaRESUMO
Multiple genetic alterations are common in cancers including those of the breast. The mechanisms leading to these alterations such as point mutations, gene amplifications, deletions and replication error are often associated with frequent and consistent loss of heterozygosity (LOH) or microsatellite instability (MSI). Several cytological and molecular studies have shown high frequency loss of genetic information on the long arm of chromosome 11 (i.e., 11q) in various primary breast cancers. In the present study allelic alterations in a refined position on the long arm of chromosome 11 were studied to identify the spectrum of induced damage at different stages of malignant transformation of MCF-10F cell lines after exposure to high-LET radiation using alpha-particles and exposure to estradiol by using PCR-single strand conformation polymorphism (SSCP) and fluorescence in situ hybridization (FISH) analysis. Microsatellite markers were selected from chromosome 11 (11q23-q24 loci) and it was found that frequency of allelic imbalance occurs at different stages of tumor progression with a range of 15-45% depending on the marker studied. These results strongly suggested the presence of several tumor suppressor genes in this critical region of chromosome 11 (11q23-q24). It also represents the first indication of allele loss at these loci in human breast epithelial cells induced by radiation and estrogen treatment suggesting a potential interventional target in breast carcinogenesis.
Assuntos
Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 11/genética , Estrogênios/farmacologia , Perda de Heterozigosidade , Partículas alfa , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Repetições de Microssatélites/genéticaRESUMO
Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.
Assuntos
DNA Helicases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Neoplasias/genética , Proteínas Nucleares/genética , Animais , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga/genética , Humanos , Camundongos , Camundongos Knockout , Neoplasias/patologiaRESUMO
Telomerase is a ribonucleoprotein whose activity has been detected in germline cells and in neoplastic and immortal cells. Telomerase compensates the telomere loss arising by the end replication problem by synthesizing telomeric repeats at the 3' end of the eukaryotic chromosomes. Telomerase is reactivated during cancer progression in human and mice. In order to determine whether the telomerase activity can be upregulated in vitro in response to DNA damaging agents, we examined the telomerase activity in five Chinese hamster cell lines following exposure to 5 J/m2 or 40 J/m2 UV-C radiation. All the cell lines tested showed an increase in telomerase activity in the PCR-based telomeric repeat amplification protocol (TRAP) in a dose dependent manner. This increase in telomerase activity correlated well with the number of cells being in the S and G2/M phase after UV exposure. However, in unirradiated control cells, similar levels of telomerase activity were observed in different phases of the cell cycle. Furthermore, telomeric signals were clustered in one or more parts of the disintegrating nuclear particles of the apoptotic cell as detected by fluorescence in situ hybridization (FISH). This is the first study to demonstrate the induction of telomerase activity following exposure to DNA-damaging agents like UV radiation in Chinese hamster cells in vitro.
Assuntos
Telomerase/metabolismo , Animais , Células CHO , Ciclo Celular , Cricetinae , Dano ao DNA/efeitos da radiação , Indução Enzimática/efeitos da radiação , Hibridização in Situ Fluorescente , Telômero/metabolismo , Raios UltravioletaRESUMO
Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.
Assuntos
Ataxia Telangiectasia/patologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , RNA , Telomerase/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Testes de Carcinogenicidade , Ciclo Celular/efeitos da radiação , Linhagem Celular Transformada , Senescência Celular , Cromossomos Humanos/genética , Cromossomos Humanos/efeitos da radiação , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Masculino , Camundongos , Camundongos Nus , Tolerância a Radiação , Radiação Ionizante , Valores de Referência , Retroviridae/genética , Telomerase/genética , Telômero/genéticaRESUMO
BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81(-/-) and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.
Assuntos
Transformação Celular Neoplásica/genética , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Linfoma/genética , RecQ Helicases/genética , Animais , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/efeitos da radiação , Aberrações Cromossômicas/induzido quimicamente , Aberrações Cromossômicas/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Raios gama/efeitos adversos , Linfoma/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitomicina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologiaRESUMO
Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/- cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53-/- cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/-PARP-/- mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.
Assuntos
Cromossomos/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA/fisiologia , Mamíferos/genética , Telômero/fisiologia , Envelhecimento/genética , Animais , Antígenos Nucleares/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/genética , Instabilidade Cromossômica/genética , Cromossomos/ultraestrutura , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/fisiologia , Autoantígeno Ku , Camundongos , Camundongos SCID , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Recombinação Genética , Células-Tronco/citologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de TumorRESUMO
Most mutagens and genotoxic carcinogens are efficient inducers of chromosomal alterations in exposed cells. Two important classes of aberrations, namely structural and numerical, are recognized and both types of aberrations are associated with congenital abnormalities and neoplasia in humans. These alterations can be easily detected and quantified in human peripheral blood lymphocytes. Conventional staining techniques can be used to detect these aberrations; this technique was used to estimate absorbed dose in the case of a radiation accident in Goiania, Brazil. A recently introduced fluorescent in situ hybridization technique (FISH) using DNA probes has increased the sensitivity and ease of detecting chromosome aberrations, especially stable chromosome aberrations. This technique allows, to some extent, the estimation of absorbed radiation dose from past exposures. Numerical aberrations can be directly estimated in metaphases by counting the number of FISH-painted chromosomes. Micronuclei are formed by lagging chromosome fragments or whole chromosomes during the anaphase stage of cell division. The nature of micronuclei as to whether they possess a centromere can be determined either by CREST staining (calcinosis, Raynoud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) or FISH with centromere-specific DNA probes. In several carcinogen-exposed populations, such as heavy smokers or people exposed to arsenic, aneuploidy appears to be more common than structural aberrations. In victims of radiation accidents, aneuploidy (hyperploidy) has been found to be common in addition to structural aberrations.
Assuntos
Testes de Carcinogenicidade , Citogenética/métodos , Testes de Mutagenicidade , Animais , Aberrações Cromossômicas , Exposição Ambiental , Humanos , Hibridização in Situ Fluorescente , Testes para Micronúcleos , Radiação IonizanteRESUMO
Pregnant Swiss albino mice were exposed to diagnostic ultrasound (3.5 MHz, approximately 65 mW) for 10 min on Day 3.5 (preimplantation period), 6.5 (early organogenesis period), or 11.5 (late organogenesis period) of gestation. Sham-exposed controls were maintained for comparison. Exposed as well as control fetuses were dissected out on the 18th day of gestation, and changes in total mortality, body weight, body length, head length, brain weight, sex ratio, and microphthalmia were recorded. Exposure on Day 3.5 of gestation resulted in a small increase in the resorption rate and a significant reduction in fetal body weight. A low fetal weight and an increase in the number of growth-retarded fetuses were produced by exposure on Day 6.5 postcoitus. A statistically nonsignificant increase in the incidence of microphthalmia was induced in fetuses exposed on Day 6.5 or Day 11.5 of gestation. These results indicate that ultrasound may have some adverse effects on the mouse embryos depending on the developmental stage at which the exposure occurred.
Assuntos
Desenvolvimento Embrionário e Fetal , Ultrassonografia Pré-Natal/efeitos adversos , Animais , Feminino , Morte Fetal/etiologia , Retardo do Crescimento Fetal/etiologia , Camundongos , Camundongos Endogâmicos , Microftalmia/etiologia , GravidezRESUMO
Pregnant Swiss albino mice were exposed to diagnostic ultrasound (3.5 MHz, 65 mW, ISPTP = 1 W/cm2,ISATA = 240 W/cm2) for 10, 20 or 30 min on day 14.5 (fetal period) of gestation. Sham-exposed controls were studied for comparison. Any changes in physiological reflexes (such as pinna detachment, opening of the eyes and development of fur), postnatal mortality and changes in adult behavior (open-field test, dark/bright arena test, hole board test and conditioned-avoidance test) were recorded. No change was observed in the physiological reflexes. The postnatal survival was also not affected significantly by the exposure. However, there were significant alterations in behavior in all three exposed groups as revealed by the decreased locomotor and exploratory activity and the increase in the number of trials needed for learning. These results indicate that ultrasound exposure during the early fetal period can impair brain function in the adult mouse.
Assuntos
Efeitos Tardios da Exposição Pré-Natal , Ultrassonografia Pré-Natal/efeitos adversos , Animais , Comportamento Animal , Encéfalo/fisiopatologia , Feminino , Camundongos , GravidezRESUMO
The abdominal region of pregnant Swiss mice was exposed to 0.05 to 0.50 Gy of gamma radiation on day 11.5 postcoitus. The animals were sacrificed on day 18 of gestation and the fetuses were examined for mortality, growth retardation, changes in head size and brain weight, and incidence of microphthalmia. No marked increase in fetal mortality or growth retardation was observed below 0.25 Gy; the increase in these parameters was significant only at 0.50 Gy. A significant reduction in head size and brain weight and a significant increase in the incidence of microphthalmia were observed at doses above 0.15 Gy. Detectable levels of microcephaly and microphthalmia were evident even at 0.10 Gy. A linear dose response was seen for these effects in the dose range of 0.05 to 0.15 Gy. It is concluded that the late period of organogenesis in the mouse, especially between days 10 and 12 postcoitus, is a particularly sensitive phase in the development of the skull, brain and eye.