RESUMEN
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.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Serina/metabolismo , Homólogo de la Proteína Chromobox 5 , Daño del ADN , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Imagen Óptica , Fosforilación , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Senescencia Celular/fisiología , Replicación del ADN/fisiología , Línea Celular Tumoral , Humanos , Lamina Tipo A/metabolismo , Células MCF-7 , Mutación , Proteína p53 Supresora de Tumor/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
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.
Asunto(s)
Cromatina/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/farmacología , Congelación/efectos adversos , Fase S/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromatina/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Dimetilsulfóxido/farmacología , Fibroblastos , Humanos , Células MCF-7 , Piel/citologíaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Núcleo Celular/fisiología , Modelos Teóricos , Tamaño de los Orgánulos/fisiología , Análisis de la Célula Individual/métodos , Núcleo Celular/ultraestructura , Cromatina/química , Cromatina/metabolismo , Cromatina/ultraestructura , Empaquetamiento del ADN , Células Hep G2 , Humanos , Imagenología Tridimensional , Cinética , Microscopía Confocal , Imagen de Lapso de Tiempo/métodosRESUMEN
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.
RESUMEN
Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1-deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro-differentiation was almost suppressed. Neuro-differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia-like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia-like brains that were treated with the cannabinoid receptor-1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co-regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro-differentiation as well as the pathophysiology of a schizophrenia-like phenotype.
Asunto(s)
Encéfalo/enzimología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Neurogénesis , Neuronas/enzimología , Esquizofrenia/enzimología , Acetilación , Animales , Antipsicóticos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/patología , Antagonistas de Receptores de Cannabinoides/farmacología , Modelos Animales de Enfermedad , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Acetato de Metilazoximetanol , Ratones Endogámicos C57BL , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Procesamiento Proteico-Postraduccional , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Esquizofrenia/inducido químicamente , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Transducción de Señal , Factores de TiempoRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Histonas/química , Transferencia Lineal de Energía , Microscopía Confocal , Proteína 1 de Unión al Supresor Tumoral P53/química , Apoptosis , Células Cultivadas , Reparación del ADN , Fibroblastos/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Humanos , Fosforilación , Radiación IonizanteRESUMEN
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.
Asunto(s)
Daño del ADN , Mutación , Proteínas Nucleares/deficiencia , Transactivadores/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Rayos Ultravioleta , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Regulación hacia Abajo , Humanos , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.
Asunto(s)
Cromatina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Núcleo Celular/metabolismo , Cromatina/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Fase G2/genética , Fase G2/fisiología , Antígeno Nuclear de Célula en Proliferación/genética , Fase S/genética , Fase S/fisiologíaRESUMEN
This review focuses on the function of heterochromatin protein HP1 in response to DNA damage. We specifically outline the regulatory mechanisms in which HP1 and its interacting partners are involved. HP1 protein subtypes (HP1α, HP1ß, and HP1γ) are the main components of constitutive heterochromatin, and HP1α and HP1ß in particular are responsible for heterochromatin maintenance. The recruitment of these proteins to DNA lesions is also important from the perspective of proper DNA repair mechanisms. For example, HP1α is necessary for the binding of the main DNA damage-related protein 53BP1 at DNA repair foci, which are positive not only for the HP1α protein but also for the RAD51 protein, a component of DNA repair machinery. The HP1ß protein also appears in monomeric form in DNA lesions together with the evolutionarily well-conserved protein called proliferating cell nuclear antigen (PCNA). The role of HP1 in DNA lesions is also mediated via the Kap1 transcription repressor. Taken together, these results indicate that the function of HP1 after DNA injury depends strongly on the kinetics of other DNA repair-related factors and their post-translational modifications, such as the phosphorylation of Kap-1.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Represoras/metabolismo , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Humanos , Procesamiento Proteico-Postraduccional , Proteína 28 que Contiene Motivos TripartitoRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Heterocromatina/metabolismo , Lamina Tipo B/genética , Osteoblastos/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Línea Celular Tumoral , Senescencia Celular/efectos de la radiación , Centrómero/metabolismo , Centrómero/efectos de la radiación , Centrómero/ultraestructura , Rayos gamma , Heterocromatina/efectos de la radiación , Heterocromatina/ultraestructura , Humanos , Lamina Tipo B/metabolismo , Células MCF-7 , Membrana Nuclear/metabolismo , Membrana Nuclear/efectos de la radiación , Membrana Nuclear/ultraestructura , Osteoblastos/patología , Osteoblastos/efectos de la radiación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Receptor de Lamina BRESUMEN
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.
Asunto(s)
Nucléolo Celular/genética , Roturas del ADN de Doble Cadena , Acetilación , Animales , Arginina/metabolismo , Línea Celular , Nucléolo Celular/ultraestructura , Reparación del ADN , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Genes de ARNr , Histona Desacetilasa 1/metabolismo , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Metilación , Ratones , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismoRESUMEN
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.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Rayos gamma/efectos adversos , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Western Blotting , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Técnica del Anticuerpo Fluorescente , Humanos , Cuerpos de Inclusión Intranucleares/patología , Cuerpos de Inclusión Intranucleares/efectos de la radiación , Leucemia Promielocítica Aguda/patología , Leucemia Promielocítica Aguda/radioterapia , Microscopía Confocal , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Gadolinio/toxicidad , Glioblastoma/metabolismo , Nanopartículas del Metal/toxicidad , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , HumanosRESUMEN
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.
Asunto(s)
Amifostina/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Protectores contra Radiación/farmacología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Amifostina/farmacocinética , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Rayos gamma , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7/efectos de los fármacos , Células MCF-7/efectos de la radiación , Mercaptoetilaminas/farmacocinética , Microscopía Fluorescente/métodos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Patología/métodos , Procesamiento de Imagen Asistido por Computador/tendencias , Microscopía/tendenciasRESUMEN
We studied the histone signature of embryonic and adult brains to strengthen existing evidence of the importance of the histone code in mouse brain development. We analyzed the levels and distribution patterns of H3K9me1, H3K9me2, H3K9me3, and HP1ß in both embryonic and adult brains. Western blotting showed that during mouse brain development, the levels of H3K9me1, H3K9me2, and HP1ß exhibited almost identical trends, with the highest protein levels occurring at E15 stage. These trends differed from the relatively stable level of H3K9me3 at developmental stages E8, E13, E15, and E18. Compared with embryonic brains, adult brains were characterized by very low levels of H3K9me1/me2/me3 and HP1ß. Manipulation of the embryonic epigenome through histone deacetylase inhibitor treatment did not affect the distribution patterns of the studied histone markers in embryonic ventricular ependyma. Similarly, Hdac3 depletion in adult animals had no effect on histone methylation in the adult hippocampus. Our results indicate that the distribution of HP1ß in the embryonic mouse brain is related to that of H3K9me1/me2 but not to that of H3K9me3. The unique status of H3K9me3 in the brain was confirmed by its pronounced accumulation in the granular layer of the adult olfactory bulb. Moreover, among the studied proteins, H3K9me3 was the only posttranslational histone modification that was highly abundant at clusters of centromeric heterochromatin, called chromocenters. When we focused on the hippocampus, we found this region to be rich in H3K9me1 and H3K9me3, whereas H3K9me2 and HP1ß were present at a very low level or even absent in the hippocampal blade. Taken together, these results revealed differences in the epigenome of the embryonic and adult mouse brain and showed that the adult hippocampus, the granular layer of the adult olfactory bulb, and the ventricular ependyma of the embryonic brain are colonized by specific epigenetic marks.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía FluorescenteRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/genética , Reparación del ADN/genética , ADN/genética , Histonas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilación/efectos de la radiación , Proteína 1 de Unión al Supresor Tumoral P53 , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Ciclo Celular/efectos de la radiación , Fase G2/efectos de la radiación , Rayos gamma/efectos adversos , Animales , Apoptosis/efectos de la radiación , Línea Celular , Línea Celular Tumoral , Nucléolo Celular/efectos de la radiación , Biología Computacional , Daño del ADN/efectos de la radiación , Ratones , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Transcripción Genética , Rayos UltravioletaRESUMEN
We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.