RESUMO
The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1ß and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1ß serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1ß, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1ß remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1ß interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1ß protein; thus, HP1ß-S88ph could be considered as an important marker of DNA damage.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Serina/metabolismo , Homólogo 5 da Proteína Cromobox , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Imagem Óptica , Fosforilação , Células Tumorais CultivadasRESUMO
Replication stress (RS) is a major driver of genomic instability and tumorigenesis. Here, we investigated whether RS induced by the nucleotide analog fludarabine and specific kinase inhibitors [e.g. targeting checkpoint kinase 1 (Chk1) or ataxia telangiectasia and Rad3-related (ATR)] led to apoptosis or senescence in four cancer cell lines differing in TP53 mutation status and expression of lamin A/C (LA/C). RS resulted in uneven chromatin condensation in all cell types, as evidenced by the presence of metaphasic chromosomes with unrepaired DNA damage, as well as detection of less condensed chromatin in the same nucleus, frequent ultrafine anaphase bridges, and micronuclei. We observed that responses to these chromatin changes may be distinct in individual cell types, suggesting that expression of lamin A/C and lamin B1 (LB1) may play an important role in the transition of damaged cells to senescence. MCF7 mammary carcinoma cells harboring wild-type p53 (WT-p53) and LA/C responded to RS by transition to senescence with a significant reduction of lamin B receptor and LB1 proteins. In contrast, a lymphoid cancer cell line WSU-NHL (WT-p53) lacking LA/C and expressing low levels of LB1 died after several hours, while lines MEC-1 and SU-DHL-4, both with mutated p53, and SU-DHL-4 with mutations in LA/C, died at different rates by apoptosis. Our results show that, in addition to being influenced by p53 mutation status, the response to RS (apoptosis or senescence) may also be influenced by lamin A/C and LB1 status.
Assuntos
Apoptose/fisiologia , Senescência Celular/fisiologia , Replicação do DNA/fisiologia , Linhagem Celular Tumoral , Humanos , Lamina Tipo A/metabolismo , Células MCF-7 , Mutação , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.
Assuntos
Cromatina/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento/efeitos adversos , Fase S/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Fibroblastos , Humanos , Células MCF-7 , Pele/citologiaRESUMO
The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.
Assuntos
Apoptose/fisiologia , Núcleo Celular/fisiologia , Modelos Teóricos , Tamanho das Organelas/fisiologia , Análise de Célula Única/métodos , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/metabolismo , Cromatina/ultraestrutura , Empacotamento do DNA , Células Hep G2 , Humanos , Imageamento Tridimensional , Cinética , Microscopia Confocal , Imagem com Lapso de Tempo/métodosRESUMO
Anchoring of heterochromatin to the nuclear envelope appears to be an important process ensuring the spatial organization of the chromatin structure and genome function in eukaryotic nuclei. Proteins of the inner nuclear membrane (INM) mediating these interactions are able to recognize lamina-associated heterochromatin domains (termed LAD) and simultaneously bind either lamin A/C or lamin B1. One of these proteins is the lamin B receptor (LBR) that binds lamin B1 and tethers heterochromatin to the INM in embryonic and undifferentiated cells. It is replaced by lamin A/C with specific lamin A/C binding proteins at the beginning of cell differentiation and in differentiated cells. Our functional experiments in cancer cell lines show that heterochromatin in cancer cells is tethered to the INM by LBR, which is downregulated together with lamin B1 at the onset of cell transition to senescence. The downregulation of these proteins in senescent cells leads to the detachment of centromeric repetitive sequences from INM, their relocation to the nucleoplasm, and distension. In cells, the expression of LBR and LB1 is highly coordinated as evidenced by the reduction of both proteins in LBR shRNA lines. The loss of the constitutive heterochromatin structure containing LADs results in changes in chromatin architecture and genome function and can be the reason for the permanent loss of cell proliferation in senescence.
RESUMO
Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (â¼135 keV µm-1) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.
Assuntos
Quebras de DNA de Cadeia Dupla , Histonas/química , Transferência Linear de Energia , Microscopia Confocal , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Apoptose , Células Cultivadas , Reparo do DNA , Fibroblastos/efeitos da radiação , Imunofluorescência , Humanos , Fosforilação , Radiação IonizanteRESUMO
53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.
Assuntos
Dano ao DNA , Mutação , Proteínas Nucleares/deficiência , Transativadores/deficiência , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Regulação para Baixo , Humanos , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cellular transition to senescence is associated with extensive chromatin reorganization and changes in gene expression. Recent studies appear to imply an association of lamin B1 (LB1) reduction with chromatin rearrangement in human fibroblasts promoted to senescence, while the mechanisms and structural features of these relationships have not yet been clarified. In this work, we examined the functions of LB1 and the lamin B receptor (LBR) in human cancer cells. We found that both LB1 and LBR tend to deplete during cancer cell transfer to senescence by γ-irradiation. A functional study employing silencing of LBR by small hairpin ribonucleic acid (shRNA) constructs revealed reduced LB1 levels suggesting that the regulation of both proteins is interrelated. The reduced expression of LBR resulted in the relocation of centromeric heterochromatin (CSH) from the inner nuclear membrane (INM) to the nucleoplasm and is associated with its unfolding. This indicates that LBR tethers heterochromatin to INM in cycling cancer cells and that LB1 is an integral part of this tethering. Down-regulation of LBR and LB1 at the onset of senescence are thus necessary for the release of heterochromatin binding to lamina, resulting in changes in chromatin architecture and gene expression. However, the senescence phenotype was not manifested in cell lines with reduced LBR and LB1 expression suggesting that other factors, such as deoxyribonucleic acid (DNA) damage, are needed to trigger senescence. We conclude that the primary response of cells to various stresses leading to senescence consists of the down-regulation of LBR and LB1 to attain reversal of the chromatin architecture.
Assuntos
Regulação Neoplásica da Expressão Gênica , Heterocromatina/metabolismo , Lamina Tipo B/genética , Osteoblastos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Linhagem Celular Tumoral , Senescência Celular/efeitos da radiação , Centrômero/metabolismo , Centrômero/efeitos da radiação , Centrômero/ultraestrutura , Raios gama , Heterocromatina/efeitos da radiação , Heterocromatina/ultraestrutura , Humanos , Lamina Tipo B/metabolismo , Células MCF-7 , Membrana Nuclear/metabolismo , Membrana Nuclear/efeitos da radiação , Membrana Nuclear/ultraestrutura , Osteoblastos/patologia , Osteoblastos/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Receptor de Lamina BRESUMO
DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.
Assuntos
Nucléolo Celular/genética , Quebras de DNA de Cadeia Dupla , Acetilação , Animais , Arginina/metabolismo , Linhagem Celular , Nucléolo Celular/ultraestrutura , Reparo do DNA , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Genes de RNAr , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metilação , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
We studied epigenetics, distribution pattern, kinetics, and diffusion of proteins recruited to spontaneous and γ-radiation-induced DNA lesions. We showed that PML deficiency leads to an increased number of DNA lesions, which was accompanied by changes in histone signature. In PML wt cells, we observed two mobile fractions of 53BP1 protein with distinct diffusion in spontaneous lesions. These protein fractions were not detected in PML-deficient cells, characterized by slow-diffusion of 53BP1. Single particle tracking analysis revealed limited local motion of 53BP1 foci in PML double null cells and local motion 53BP1 foci was even more reduced after γ-irradiation. However, radiation did not change co-localization between 53BP1 nuclear bodies and interchromatin granule-associated zones (IGAZs), nuclear speckles, or chromocenters. This newly observed interaction pattern imply that 53BP1 protein could be a part of not only DNA repair, but also process mediated via components accumulated in IGAZs, nuclear speckles, or paraspeckles. Together, PML deficiency affected local motion of 53BP1 nuclear bodies and changed composition and a number of irradiation-induced foci. J. Cell. Biochem. 117: 2583-2596, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Raios gama/efeitos adversos , Corpos de Inclusão Intranuclear/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Imunofluorescência , Humanos , Corpos de Inclusão Intranuclear/patologia , Corpos de Inclusão Intranuclear/efeitos da radiação , Leucemia Promielocítica Aguda/patologia , Leucemia Promielocítica Aguda/radioterapia , Microscopia Confocal , Células Tumorais CultivadasRESUMO
BACKGROUND: Tumor targeting of radiotherapy represents a great challenge. The addition of multimodal nanoparticles, such as 3 nm gadolinium-based nanoparticles (GdBNs), has been proposed as a promising strategy to amplify the effects of radiation in tumors and improve diagnostics using the same agents. This singular property named theranostic is a unique advantage of GdBNs. It has been established that the amplification of radiation effects by GdBNs appears due to fast electronic processes. However, the influence of these nanoparticles on cells is not yet understood. In particular, it remains dubious how nanoparticles activated by ionizing radiation interact with cells and their constituents. A crucial question remains open of whether damage to the nucleus is necessary for the radiosensitization exerted by GdBNs (and other nanoparticles). METHODS: We studied the effect of GdBNs on the induction and repair of DNA double-strand breaks (DSBs) in the nuclear DNA of U87 tumor cells irradiated with γ-rays. For this purpose, we used currently the most sensitive method of DSBs detection based on high-resolution confocal fluorescence microscopy coupled with immunodetection of two independent DSBs markers. RESULTS: We show that, in the conditions where GdBNs amplify radiation effects, they remain localized in the cytoplasm, i.e. do not penetrate into the nucleus. In addition, the presence of GdBNs in the cytoplasm neither increases induction of DSBs by γ-rays in the nuclear DNA nor affects their consequent repair. CONCLUSIONS: Our results suggest that the radiosensitization mediated by GdBNs is a cytoplasmic event that is independent of the nuclear DNA breakage, a phenomenon commonly accepted as the explanation of biological radiation effects. Considering our earlier recognized colocalization of GdBNs with the lysosomes and endosomes, we revolutionary hypothesize here about these organelles as potential targets for (some) nanoparticles. If confirmed, this finding of cytoplasmically determined radiosensitization opens new perspectives of using nano-radioenhancers to improve radiotherapy without escalating the risk of pathologies related to genetic damage.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Gadolínio/toxicidade , Glioblastoma/metabolismo , Nanopartículas Metálicas/toxicidade , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , HumanosRESUMO
Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.
Assuntos
Amifostina/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Amifostina/farmacocinética , Ensaio Cometa , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Raios gama , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células MCF-7/efeitos dos fármacos , Células MCF-7/efeitos da radiação , Mercaptoetilaminas/farmacocinética , Microscopia de Fluorescência/métodos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
BACKGROUND INFORMATION: The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. RESULTS: We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. CONCLUSION: We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. SIGNIFICANCE: Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent.
Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA/genética , DNA/genética , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilação/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Raios UltravioletaRESUMO
The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.
Assuntos
Ciclo Celular/efeitos da radiação , Fase G2/efeitos da radiação , Raios gama/efeitos adversos , Animais , Apoptose/efeitos da radiação , Linhagem Celular , Linhagem Celular Tumoral , Nucléolo Celular/efeitos da radiação , Biologia Computacional , Dano ao DNA/efeitos da radiação , Camundongos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Transcrição Gênica , Raios UltravioletaRESUMO
Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Nuclear architecture and nuclear processes, including DNA damage responses, are precisely organized in space and time. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone, but requires sophisticated structural probing and imaging. Based on the results obtained from studying the relationship between higher-order chromatin structure, DNA double-strand break induction and repair, and the formation of chromosomal translocations, we show the development of Omics solutions especially for radiation research (radiomics) (discussed in this article) and how confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place the Omics data in the context of space and time (discussed in our other article in this issue, "Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part B--Structuromics"). Finally, we introduce a novel method of specific chromatin nanotargeting and speculate future perspectives, which may combine nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA , DNA/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Radiobiologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Cromatina/efeitos da radiação , Dano ao DNA/genética , Genoma/genética , Genoma/efeitos da radiação , Humanos , Radiação IonizanteRESUMO
Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.
Assuntos
Núcleo Celular/metabolismo , Corpos Enovelados/metabolismo , DNA/genética , DNA/efeitos da radiação , Raios gama/efeitos adversos , Proteínas Nucleares/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Corpos Enovelados/genética , Corpos Enovelados/efeitos da radiação , Fase G2/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Células K562 , Camundongos , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.
Assuntos
Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Epigênese Genética , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND INFORMATION: The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1ß, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. RESULTS: We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1ß, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1ß was stable. Fluorescence intensity of HP1ß and BMI1 in UVA-induced DNA lesions and level of HP1ß after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. CONCLUSIONS: We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1ß and BMI1.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/efeitos da radiação , Lamina Tipo A/metabolismo , Células 3T3 , Animais , Proteínas Cromossômicas não Histona/análise , DNA/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A/análise , Camundongos , Complexo Repressor Polycomb 1/análise , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Raios UltravioletaRESUMO
A- and C-type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C-type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A-type lamin channels interact with several nuclear components, including fibrillarin- and UBF-positive regions of nucleoli, foci of heterochromatin protein 1 ß, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C-type lamin channels and nuclear pores, lamin-associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C-type lamins had low levels of B-type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell-specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures.
Assuntos
Núcleo Celular/genética , Reparo do DNA/genética , Lamina Tipo A/ultraestrutura , Lamina Tipo B/ultraestrutura , Animais , Proteínas Cromossômicas não Histona/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , CamundongosRESUMO
According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure-the problematics addressed in this work. The consequences for the mechanism of chromosomal translocations are compared for gamma rays and proton beams.