RESUMO
Ultraviolet (UV) radiation influences development and genome stability in organisms; however, its impact on meiosis, a special cell division essential for the delivery of genetic information across generations in eukaryotes, has not yet been elucidated. In this study, by performing cytogenetic studies, we reported that UV radiation does not damage meiotic chromosome integrity but attenuates centromere-mediated chromosome stability and induces unreduced gametes in Arabidopsis thaliana. We showed that functional centromere-specific histone 3 (CENH3) is required for obligate crossover formation and plays a role in the protection of sister chromatid cohesion under UV stress. Moreover, we found that UV specifically alters the orientation and organization of spindles and phragmoplasts at meiosis II, resulting in meiotic restitution and unreduced gametes. We determined that UV-induced meiotic restitution does not rely on the UV Resistance Locus8-mediated UV perception and the Tapetal Development and Function1- and Aborted Microspores-dependent tapetum development, but possibly occurs via altered JASON function and downregulated Parallel Spindle1. This study provides evidence that UV radiation influences meiotic genome stability and gametophytic ploidy consistency in flowering plants.
Assuntos
Arabidopsis , Centrômero , Instabilidade Genômica , Meiose , Ploidias , Raios Ultravioleta , Meiose/efeitos da radiação , Meiose/genética , Centrômero/genética , Centrômero/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Células Germinativas Vegetais/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Histonas/metabolismo , Fuso Acromático/efeitos da radiaçãoRESUMO
The effects of ionizing radiation on centrosomes have been well documented and reviewed by Saladino et al. (2012) and are only briefly addressed here. These results showed that exposure of tumor cells to ionizing radiation causes centrosome overduplication and the formation of multipolar mitotic spindles, resulting in nuclear fragmentation and subsequent cell death (Sato et al. 2000). By using a variety of cell lines derived from different types of human solid tumors, it was shown that exposure to 10 Gy γ-radiation resulted in a substantial increase in cells containing an abnormally high number of aberrant centrosomes that formed multipolar spindles, resulting in imbalanced chromosome separation followed by mitotic cell death and formation of multi- or micronucleated cells.
Assuntos
Centrossomo , Fuso Acromático , Humanos , Centrossomo/metabolismo , Centrossomo/patologia , Centrossomo/efeitos da radiação , Fuso Acromático/metabolismo , Fuso Acromático/efeitos da radiação , Segregação de Cromossomos , Morte Celular , Linhagem CelularRESUMO
Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.
Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Dano ao DNA , Meduloblastoma/genética , Meduloblastoma/patologia , MicroRNAs/metabolismo , Fuso Acromático/metabolismo , Vincristina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/metabolismo , Camundongos Nus , MicroRNAs/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/efeitos da radiaçãoRESUMO
BACKGROUND/AIM: Owing to the frequent observation of centrosome amplification in human cancers, cancer cells have a unique mechanism to suppress detrimental multipolar division by clustering multiple centrosomes into two functional spindle poles, known as centrosome clustering. This study investigated whether inhibition of centrosome clustering enhances the radiation sensitivity of breast cancer cells. MATERIALS AND METHODS: In this study, inhibition of centrosome clustering was examined by using various centrosome-declustering agents and KIFC1 siRNA in three breast cancer cell lines and two normal fibroblast cell lines. The combination effect of radiation and centrosome declustering was evaluated by cell viability, clonogenic, immunofluorescence assay. RESULTS: This study showed that targeting centrosome clustering enhanced the efficacy of radiotherapy of breast cancer cells with less damage to normal cells. Ionizing radiation induced centrosome amplification in breast cancer cells, but not in normal fibroblast cells. Notably, we showed that centrosome declustering efficiently radiosensitized the centrosome-amplified breast cancer cells through induction of multipolar spindles but did not affect the viability of normal fibroblasts in response to irradiation. Furthermore, KIFC1 mediated the radiosensitivity of the centrosome-amplified breast cancer cells. CONCLUSION: Our data provided the first evidence that centrosome clustering is a tumor-selective target for the improvement of radiotherapy in breast cancer cells.
Assuntos
Centrossomo/efeitos da radiação , Fibroblastos/efeitos da radiação , Fuso Acromático/efeitos da radiação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/efeitos da radiação , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Griseofulvina/farmacologia , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Células MCF-7 , Fenantrenos/farmacologia , Interferência de RNA , Radiossensibilizantes/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismoRESUMO
Taxane-based radiochemotherapy is a central treatment option for various cancer entities in locally advanced stages. The therapeutic synergism of this combined modality approach due to taxane-mediated radiosensitization of cancer cells is well-known. However, the underlying molecular mechanisms remain largely elusive, and mechanism-derived predictive markers of taxane-based radiochemotherapy are currently not available. Here, we show that clinically relevant doses of Paclitaxel, the prototype taxane, stimulate a tripolar mode of mitosis leading to chromosomal missegregation and aneuploidization rather than interfering with cell cycle progression. This distinct mitotic phenotype was interlinked with Paclitaxel-mediated radiosensitization via overexpression of mitotic Aurora kinase A (AURKA) and its cofactor TPX2 whose knockdown rescued the bipolar mode of cell division and largely attenuated the radiosensitizing effects of Paclitaxel. In the cancer genome atlas (TCGA) lung adenocarcinoma cohort, high expression levels of AURKA and TPX2 were associated with specifically improved overall survival upon taxane-based radiochemotherapy, but not in case of non-taxane-based radiochemotherapy, chemo- or radiotherapy only. Thus, our data provide insights into Paclitaxel-mediated radiosensitization on a mechanistic and molecular level and identify AURKA and TPX2 as the first potential mechanism-based, predictive markers of taxane-based radiochemotherapy.
Assuntos
Adenocarcinoma/terapia , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias Pulmonares/terapia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Radiossensibilizantes/farmacologia , Taxoides/farmacologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Aneuploidia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aurora Quinase A/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Estudos de Coortes , Conjuntos de Dados como Assunto , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Proteínas Associadas aos Microtúbulos/genética , Mitose/efeitos da radiação , Proteínas Nucleares/genética , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/uso terapêutico , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/efeitos da radiação , Análise de Sobrevida , Taxoides/uso terapêutico , Resultado do TratamentoRESUMO
Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology.
Assuntos
Campos Magnéticos , Mitose/efeitos da radiação , Fuso Acromático/efeitos da radiação , Linhagem Celular , Cromossomos Humanos/efeitos da radiação , HumanosRESUMO
Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule-kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.
Assuntos
Pontos de Checagem do Ciclo Celular , Dano ao DNA , Meiose , Oócitos/citologia , Fuso Acromático/metabolismo , Animais , Dano ao DNA/efeitos da radiação , Feminino , Meiose/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Oócitos/efeitos da radiação , Fuso Acromático/efeitos da radiação , Raios UltravioletaRESUMO
Leukemia is a leading cause of cancer death. Recently, the latexin (Lxn) gene was identified as a potential tumor suppressor in several types of solid tumors and lymphoma, and Lxn expression was found to be absent or downregulated in leukemic cells. Whether Lxn functions as a tumor suppressor in leukemia and what molecular and cellular mechanisms are involved are unknown. In this study, the myeloid leukemogenic FDC-P1 cell line was used as a model system and Lxn was ectopically expressed in these cells. Using the protein pull-down assay and mass spectrometry, ribosomal protein subunit 3 (Rps3) was identified as a novel Lxn binding protein. Ectopic expression of Lxn inhibited FDC-P1 growth in vitro. More surprisingly, Lxn enhanced gamma irradiation-induced DNA damages and induced cell-cycle arrest and massive necrosis, leading to depletion of FDC-P1 cells. Mechanistically, Lxn inhibited the nuclear translocation of Rps3 upon radiation, resulting in abnormal mitotic spindle formation and chromosome instability. Rps3 knockdown increased the radiation sensitivity of FDC-P1, confirming that the mechanism of action of Lxn is mediated by Rps3 pathway. Moreover, Lxn enhanced the cytotoxicity of chemotherapeutic agent, VP-16, on FDC-P1 cells. Our study suggests that Lxn itself not only suppresses leukemic cell growth but also potentiates the cytotoxic effect of radio- and chemotherapy on cancer cells. Lxn could be a novel molecular target that improves the efficacy of anti-cancer therapy.
Assuntos
Antígenos/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Raios gama , Leucemia/metabolismo , Leucemia/patologia , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos da radiação , Animais , Carcinogênese , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Instabilidade Cromossômica/efeitos da radiação , Dano ao DNA , Técnicas de Silenciamento de Genes , Camundongos , Células NIH 3T3 , Ligação Proteica/efeitos da radiação , Transporte Proteico/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Fuso Acromático/efeitos da radiação , Estresse Fisiológico/efeitos da radiaçãoRESUMO
We provide a detailed method to generate arrays of mitotic spindles in vitro. Spindles are formed in extract prepared from unfertilized Xenopus laevis eggs, which contain all the molecular ingredients of mitotic spindles. The method is based on using deep UV photochemistry to attach chromatin-coated beads on a glass surface according to a pattern of interest. The immobilized beads act as artificial chromosomes, and induce the formation of mitotic spindles in their immediate vicinity. To perform the experiment, a chamber is assembled over the chromatin pattern, Xenopus egg extract is flowed in and after incubation the spindles are imaged with a confocal microscope.
Assuntos
Cromatina/metabolismo , Fotoquímica/métodos , Fuso Acromático/metabolismo , Fuso Acromático/efeitos da radiação , Raios Ultravioleta , Animais , Extratos Celulares , Feminino , Óvulo/metabolismo , Óvulo/efeitos da radiação , Xenopus laevisRESUMO
This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained.
Assuntos
Actinas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Fuso Acromático/metabolismo , Animais , Aves , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Lasers , Microscopia Confocal , Microtúbulos/efeitos da radiação , Proteínas Nucleares/metabolismo , Fuso Acromático/efeitos da radiaçãoRESUMO
Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.
Assuntos
Pontos de Checagem do Ciclo Celular , Centrossomo/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Centríolos/efeitos dos fármacos , Centríolos/metabolismo , Centríolos/efeitos da radiação , Centrossomo/efeitos dos fármacos , Centrossomo/efeitos da radiação , Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Células HeLa , Humanos , Hidroxiureia/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Radiação Ionizante , Separase , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/efeitos da radiação , Quinase 1 Polo-LikeRESUMO
The hypogeomagnetic field (HGMF; magnetic fields <200 nT) is one of the fundamental environmental factors of space. However, the effect of HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two-cell stage, but no later than the four-cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α-tubulin showed reorientation of the spindle of four-cell stage blastomeres. These results indicate that a brief (2-h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos.
Assuntos
Embrião não Mamífero/efeitos da radiação , Campos Magnéticos , Xenopus laevis/embriologia , Animais , Feminino , Morfogênese/efeitos da radiação , Fuso Acromático/efeitos da radiaçãoRESUMO
The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10mW/cm(2)), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41°C, while apoptosis was induced only when the medium temperature achieved 40°C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line.
Assuntos
Sobrevivência Celular/efeitos da radiação , Micro-Ondas/efeitos adversos , Fuso Acromático/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Divisão Celular/efeitos da radiação , Linhagem Celular , Cricetinae , Cricetulus , Temperatura Alta , Mitose , Índice MitóticoRESUMO
Combination therapy, in which two or more agents are applied, is more effective than single therapies for combating cancer. For this reason, combinations of chemotherapy with radiation are being explored in clinical trials, albeit with an empirical approach. We developed a screen to identify, from the onset, molecules that act in vivo in conjunction with radiation, using Drosophila as a model. Screens through two small molecule libraries from the NCI Developmental Therapeutics Program yielded microtubule poisons; this class of agents is known to enhance the effect of radiation in mammalian cancer models. Here we report an analysis of one microtubule depolymerizing agent, maytansinol isobutyrate (NSC292222; maytansinol), in Drosophila and in human cancer cells. We find that the effect of maytansinol is p53 dependent in Drosophila cells and human cancer cells, that maytansinol enhances the effect of radiation in both systems, and that the combinatorial effect of drug and radiation is additive. We also uncover a differential sensitivity to maytansinol between Drosophila cells and Drosophila larvae, which illustrates the value of studying cell behavior in the context of a whole organism. On the basis of these results, we propose that Drosophila might be a useful model for unbiased screens through new molecule libraries to find cancer drugs for combination therapy.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/efeitos da radiação , Maitansina/análogos & derivados , Maitansina/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Animais , Morte Celular/efeitos dos fármacos , Terapia Combinada , Células HCT116 , Humanos , Larva/efeitos dos fármacos , Larva/efeitos da radiação , Maitansina/farmacologia , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Mutação/genética , Radiação Ionizante , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/efeitos da radiação , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismoRESUMO
Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta1-dependent phenotype, and integrin beta1 might be a potentially effective therapeutic target in combination with radiotherapy.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/radioterapia , Integrina beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Fuso Acromático/efeitos da radiaçãoRESUMO
Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.
Assuntos
Estruturas Cromossômicas/ultraestrutura , Cromossomos de Mamíferos/ultraestrutura , Mitose , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Cafeína/toxicidade , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Núcleo Celular/ultraestrutura , Centrômero/efeitos dos fármacos , Centrômero/efeitos da radiação , Centrômero/ultraestrutura , Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Cromatina/ultraestrutura , Aberrações Cromossômicas , Estruturas Cromossômicas/efeitos dos fármacos , Estruturas Cromossômicas/efeitos da radiação , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos da radiação , Cricetinae , Cricetulus , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Fixadores/farmacologia , Proteínas Luminescentes/genética , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Fenótipo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/efeitos da radiação , Fuso Acromático/ultraestrutura , Transfecção , Raios Ultravioleta , Proteína Vermelha FluorescenteRESUMO
Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G(2)-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR-induced G(2)-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53(-/-) and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53(-/-) HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53(-/-) cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21(-/-) HCT116 cells displayed a similar pattern of response as p53(-/-) cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.
Assuntos
Ciclo Celular/efeitos dos fármacos , Proteínas Quinases/metabolismo , Radiossensibilizantes/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Citometria de Fluxo , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Células HCT116 , Humanos , Imuno-Histoquímica , Proteínas Mad2 , Camundongos , Índice Mitótico , Poliploidia , Quinolinas/farmacologia , Quinuclidinas/farmacologia , Radiação Ionizante , Proteínas Repressoras/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/efeitos da radiação , Ensaio Tumoral de Célula-TroncoRESUMO
The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.
Assuntos
Telefone Celular , Aberrações Cromossômicas/efeitos da radiação , Células Híbridas/citologia , Células Híbridas/efeitos da radiação , Fuso Acromático/efeitos da radiação , Fuso Acromático/ultraestrutura , Animais , Linhagem Celular , Cricetinae , Relação Dose-Resposta à Radiação , Humanos , Micro-Ondas , Doses de RadiaçãoRESUMO
Cells exposed to ionizing radiation die via different mechanisms, including apoptosis and mitotic catastrophe. To determine the frequency of mitotic catastrophe in tumor cells after irradiation, we used time-lapse imaging to track centrin-1 and histone H2B in U2OS osteosarcoma cells. We observed a dose-dependent increase in the frequency of mitotic catastrophe after irradiation, although a consistent 30% of cell death occurred through mitotic failure at doses from 2-10 Gy. One potential cause of mitotic catastrophe is centrosome amplification, which is induced by irradiation, and which can result in the formation of multipolar mitotic spindles. Up to 60% of mitotic catastrophes occurred in cells with >2 centrosomes after irradiation. We observed multipolar mitoses in p53(+) and p53(-) tumor cells after irradiation and found that the spindle assembly checkpoint is active in multipolar mitotic cells. However, we did not detect active caspase-3 in multipolar mitoses. These data demonstrate that a significant proportion of cell death induced by ionizing irradiation is through an apoptosis-independent mechanism involving centrosome amplification and mitotic catastrophe.