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1.
Mutat Res ; 823: 111758, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34333390

RESUMO

Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma.


Assuntos
Reparo do DNA , Epigênese Genética/efeitos da radiação , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , Dano ao DNA , Bases de Dados Genéticas , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Genoma Humano/efeitos da radiação , Heterocromatina/química , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Histonas/genética , Histonas/metabolismo , Humanos , Melanoma/etiologia , Melanoma/metabolismo , Melanoma/patologia , Mutagênese , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Cultura Primária de Células , Dímeros de Pirimidina/agonistas , Dímeros de Pirimidina/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
2.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299263

RESUMO

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA/efeitos da radiação , Radioterapia com Íons Pesados/efeitos adversos , Técnicas de Cultura de Células , Análise por Conglomerados , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Eucromatina/genética , Eucromatina/efeitos da radiação , Fibroblastos , Radioterapia com Íons Pesados/métodos , Íons Pesados/efeitos adversos , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/efeitos da radiação , Transferência Linear de Energia/efeitos da radiação , Microscopia Eletrônica/métodos , Radiação Ionizante
3.
Nat Commun ; 12(1): 2428, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893291

RESUMO

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.


Assuntos
Dano ao DNA , Reparo do DNA , DNA/genética , Heterocromatina/genética , Animais , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/efeitos da radiação , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Metilação , Camundongos , Células NIH 3T3 , Raios Ultravioleta
4.
Cells ; 9(5)2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32397212

RESUMO

Compact chromatin is linked to a poor tumour prognosis and resistance to radiotherapy from photons. We investigated DNA damage induction and repair in the context of chromatin structure for densely ionising alpha radiation as well as its therapeutic potential. Chromatin opening by histone deacetylase inhibitor trichostatin A (TSA) pretreatment reduced clonogenic survival and increased γH2AX foci in MDA-MB-231 cells, indicative of increased damage induction by free radicals using gamma radiation. In contrast, TSA pretreatment tended to improve survival after alpha radiation while γH2AX foci were similar or lower; therefore, an increased DNA repair is suggested due to increased access of repair proteins. MDA-MB-231 cells exposed to fractionated gamma radiation (2 Gy × 6) expressed high levels of stem cell markers, elevated heterochromatin H3K9me3 marker, and a trend towards reduced clonogenic survival in response to alpha radiation. There was a higher level of H3K9me3 at baseline, and the ratio of DNA damage induced by alpha vs. gamma radiation was higher in the aggressive MDA-MB-231 cells compared to hormone receptor-positive MCF7 cells. We demonstrate that heterochromatin structure and stemness properties are induced by fractionated radiation exposure. Gamma radiation-exposed cells may be targeted using alpha radiation, and we provide a mechanistic basis for the involvement of chromatin in these effects.


Assuntos
Partículas alfa , Neoplasias da Mama/metabolismo , Raios gama , Heterocromatina/efeitos da radiação , Acetilação , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Clonais , Feminino , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Lisina/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Exposição à Radiação , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Esferoides Celulares/efeitos da radiação
5.
Biochemistry ; 57(19): 2756-2761, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29658277

RESUMO

One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.


Assuntos
Imunoprecipitação da Cromatina/métodos , Fragmentação do DNA/efeitos da radiação , DNA/genética , Sonicação/métodos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/efeitos da radiação , DNA/química , DNA/efeitos da radiação , Eucromatina/efeitos da radiação , Heterocromatina/efeitos da radiação , Camundongos , Nanopartículas/química , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética
6.
J Invest Dermatol ; 138(2): 405-412, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28951242

RESUMO

Exposure to solar UVB radiation leads to the formation of the highly mutagenic cyclobutane pyrimidine dimers (CPDs), the DNA damage responsible for mutations found in skin cancer. The frequency of CPD formation and the repair rate of those lesions are two important parameters to determine the probability of UVR-induced mutations. Previous work has shown that chronic irradiation with sublethal doses of UVB radiation (chronic low-dose UVB radiation) leads to the accumulation of residual CPD that persists over time. We have thus investigated the persistence, localization, and consequences on genome stability of those chronic low-dose UVB radiation-induced residual CPDs. We show that chronic low-dose UVB radiation-induced residual CPDs persist on DNA and are diluted via semiconservative replication. They are overrepresented in the heterochromatin and at the TT dipyrimidine sites, and they catalyze the incidence of sister chromatin exchange. Our results shed some light on the impact of chronic UVB radiation exposure on DNA, with a focus on residual CPDs, their distribution, and consequences.


Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Genoma Humano/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , Adulto , Biópsia , Reparo do DNA/genética , Replicação do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Fibroblastos , Genoma Humano/genética , Instabilidade Genômica/genética , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Humanos , Cultura Primária de Células , Dímeros de Pirimidina/efeitos da radiação , Pele/citologia , Pele/patologia , Pele/efeitos da radiação , Adulto Jovem
7.
Biochem J ; 474(2): 281-300, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27760841

RESUMO

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 B
8.
Int J Mol Sci ; 17(1)2016 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-26729112

RESUMO

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.


Assuntos
Cromatina/ultraestrutura , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Células-Tronco Embrionárias Humanas/efeitos da radiação , Diferenciação Celular , Linhagem Celular , Cromatina/efeitos da radiação , Heterocromatina/efeitos da radiação , Heterocromatina/ultraestrutura , Células-Tronco Embrionárias Humanas/citologia , Humanos , Radiação Ionizante
9.
Genetics ; 201(2): 563-72, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26294667

RESUMO

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.


Assuntos
Centrômero/genética , Aberrações Cromossômicas/efeitos da radiação , Heterocromatina/genética , Cromossomo X/genética , Animais , Drosophila melanogaster , Heterocromatina/efeitos da radiação , Larva/genética , Larva/efeitos da radiação , Masculino , Meiose/genética , Mitose/genética , Cromossomo X/efeitos da radiação
10.
Mutat Res Rev Mutat Res ; 764: 108-17, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26041270

RESUMO

Non-coding DNA comprises a very large proportion of the total genomic content in higher organisms, but its function remains largely unclear. Non-coding DNA sequences constitute the majority of peripheral heterochromatin, which has been hypothesized to be the genome's 'bodyguard' against DNA damage from chemicals and radiation for almost four decades. The bodyguard protective function of peripheral heterochromatin in genome defense has been strengthened by the results from numerous recent studies, which are summarized in this review. These data have suggested that cells and/or organisms with a higher level of heterochromatin and more non-coding DNA sequences, including longer telomeric DNA and rDNAs, exhibit a lower frequency of DNA damage, higher radioresistance and longer lifespan after IR exposure. In addition, the majority of heterochromatin is peripherally located in the three-dimensional structure of genome organization. Therefore, the peripheral heterochromatin with non-coding DNA could play a protective role in genome defense against DNA damage from ionizing radiation by both absorbing the radicals from water radiolysis in the cytosol and reducing the energy of IR. However, the bodyguard protection by heterochromatin has been challenged by the observation that DNA damage is less frequently detected in peripheral heterochromatin than in euchromatin, which is inconsistent with the expectation and simulation results. Previous studies have also shown that the DNA damage in peripheral heterochromatin is rarely repaired and moves more quickly, broadly and outwardly to approach the nuclear pore complex (NPC). Additionally, it has been shown that extrachromosomal circular DNAs (eccDNAs) are formed in the nucleus, highly detectable in the cytoplasm (particularly under stress conditions) and shuttle between the nucleus and the cytoplasm. Based on these studies, this review speculates that the sites of DNA damage in peripheral heterochromatin could occur more frequently and may be removed by repetitive elements in non-coding DNA through the formation of eccDNAs and expelled out of the nucleus to the cytoplasm via the NPC. Therefore, this review proposes that the genome and central protein-coding sequences are doubly protected by non-coding DNA in peripheral heterochromatin against DNA damage from radiation, which may be a novel protective role of non-coding DNA in genome defense.


Assuntos
DNA/metabolismo , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Núcleo Celular/genética , DNA/classificação , Dano ao DNA , Eucromatina/genética , Eucromatina/efeitos da radiação , Genoma , Radiação Ionizante
11.
Proc Natl Acad Sci U S A ; 112(21): E2836-44, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25964332

RESUMO

The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Transdução de Sinal Luminoso , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Cotilédone/efeitos da radiação , Metilação de DNA , Inativação Gênica , Genes de Plantas , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular , Transdução de Sinal Luminoso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , RNA Polimerase II/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
12.
Acta Biochim Pol ; 62(2): 173-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26015993

RESUMO

Histone modifications are involved in the DNA damage response (DDR). Here, by utilizing an ELISA immunoassay we assessed the methylation at H3K9 (H3K9me2 and H3K9me3) in two cell lines with differential sensitivity to radiation-induced apoptosis, HeLa (sensitive) and MCF-7 (resistant). We found that DNA damage induction by γ-irradiation leads to considerable accumulation (up to 5-fold) of H3K9me2 and H3K9me3, but not of H4K20me3 (control modification) in MCF-7 cells (p<0.05). Interestingly, a lower dose (2 Gy) was more effective than 5 Gy. In HeLa cells a smaller effect (approx. 1.5-1.8-fold) was evident only at 5 Gy. In conclusion, our findings reveal that DNA damage leads to specific accumulation of H3K9me2 and H3K9me3 in a cell-type specific manner.


Assuntos
Heterocromatina/metabolismo , Histonas/metabolismo , Radiação Ionizante , Dano ao DNA/fisiologia , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Heterocromatina/efeitos da radiação , Humanos , Lisina/metabolismo , Células MCF-7/metabolismo , Células MCF-7/efeitos da radiação , Metilação , Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-25813721

RESUMO

The purpose of this work is to test the hypothesis that kinetics of double strand breaks (DSB) repair is governed by complexity of DSB. To test the hypothesis we used our recent published mechanistic mathematical model of DSB repair for DSB induced by selected protons, deuterons, and helium ions of different energies representing radiations of different qualities. In light of recent advances in experimental and computational techniques, the most appropriate method to study cellular responses in radiation therapy, and exposures to low doses of ionizing radiations is using mechanistic approaches. To this end, we proposed a 'bottom-up' approach to study cellular response that starts with the DNA damage. Monte Carlo track structure method was employed to simulate initial damage induced in the genomic DNA by direct and indirect effects. Among the different types of DNA damage, DSB are known to be induced in simple and complex forms. The DSB repair model in G1 and early S phases of the cell cycle was employed to calculate the repair kinetics. The model considers the repair of simple and complex DSB, and the DSB produced in the heterochromatin. The inverse sampling method was used to calculate the repair kinetics for each individual DSB. The overall repair kinetics for 500 DSB induced by single tracks of the radiation under test were compared with experimental results. The results show that the model is capable of predicting the repair kinetics for the DSB induced by radiations of different qualities within an accepted range of uncertainty.


Assuntos
Ciclo Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fase G1/efeitos da radiação , Radiação Ionizante , Fase S/efeitos da radiação , Animais , Linhagem Celular , Cricetinae , Reparo do DNA/efeitos da radiação , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Modelos Moleculares , Método de Monte Carlo
14.
Mutat Res ; 750(1-2): 56-66, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23958412

RESUMO

Repair of double strand breaks (DSBs) is essential for cell survival and genome integrity. While much is known about the molecular mechanisms involved in DSB repair and checkpoint activation, the roles of nuclear dynamics of radiation-induced foci (RIF) in DNA repair are just beginning to emerge. Here, we summarize results from recent studies that point to distinct features of these dynamics in two different chromatin environments: heterochromatin and euchromatin. We also discuss how nuclear architecture and chromatin components might control these dynamics, and the need of novel quantification methods for a better description and interpretation of these phenomena. These studies are expected to provide new biomarkers for radiation risk and new strategies for cancer detection and treatment.


Assuntos
Núcleo Celular/efeitos da radiação , Eucromatina/efeitos da radiação , Heterocromatina/efeitos da radiação , Neoplasias Induzidas por Radiação/genética , Animais , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Eucromatina/genética , Heterocromatina/genética , Humanos , Cinética , Neoplasias Induzidas por Radiação/patologia
15.
Int J Radiat Biol ; 89(11): 898-906, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23692433

RESUMO

PURPOSE: With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation. MATERIALS AND METHODS: HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1-4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured. RESULTS: Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated. CONCLUSION: Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.


Assuntos
Adaptação Fisiológica/efeitos da radiação , Linfócitos B/efeitos da radiação , Metilação de DNA/efeitos da radiação , Raios gama/efeitos adversos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Heterocromatina/efeitos dos fármacos , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Humanos , Tolerância a Radiação/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 110(3): E212-20, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23267107

RESUMO

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.


Assuntos
Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , RNA/biossíntese , Sirtuína 1/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Células Cultivadas , Síndrome de Cockayne/complicações , Reparo do DNA , Heterocromatina/genética , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Humanos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/complicações , Proteína Grupo D do Xeroderma Pigmentoso/química
17.
PLoS One ; 7(5): e38165, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22666473

RESUMO

PURPOSE: DNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Eucromatina/genética , Eucromatina/ultraestrutura , Heterocromatina/genética , Heterocromatina/ultraestrutura , Microscopia Eletrônica de Transmissão , Animais , Córtex Cerebral/citologia , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Eucromatina/metabolismo , Eucromatina/efeitos da radiação , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Camundongos , Neurônios/citologia , Neurônios/efeitos da radiação , Neurônios/ultraestrutura , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
18.
Proc Natl Acad Sci U S A ; 108(29): E314-22, 2011 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-21715654

RESUMO

Prep1 is a homeodomain transcription factor that is essential in embryonic development and functions in the adult as a tumor suppressor. We show here that Prep1 is involved in maintaining genomic stability and preventing neoplastic transformation. Hypomorphic homozygous Prep1(i/i) fetal liver cells and mouse embryonic fibroblasts (MEFs) exhibit increased basal DNA damage and normal DNA damage response after γ-irradiation compared with WT. Cytogenetic analysis shows the presence of numerous chromosomal aberrations and aneuploidy in very early-passage Prep1(i/i) MEFs. In human fibroblasts, acute Prep1 down-regulation by siRNA induces DNA damage response, like in Prep1(i/i) MEFs, together with an increase in heterochromatin-associated modifications: rapid increase of histone methylation and decreased transcription of satellite DNA. Ectopic expression of Prep1 rescues DNA damage and heterochromatin methylation. Inhibition of Suv39 activity blocks the chromatin but not the DNA damage phenotype. Finally, Prep1 deficiency facilitates cell immortalization, escape from oncogene-induced senescence, and H-Ras(V12)-dependent transformation. Importantly, the latter can be partially rescued by restoration of Prep1 level. The results show that the tumor suppressor role of Prep1 is associated with the maintenance of genomic stability.


Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação da Expressão Gênica/fisiologia , Instabilidade Genômica/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Imunoprecipitação da Cromatina , Ensaio Cometa , Análise Citogenética , Dano ao DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Raios gama , Regulação da Expressão Gênica/genética , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Oligonucleotídeos/genética , Fatores de Transcrição/metabolismo
19.
DNA Repair (Amst) ; 10(2): 119-25, 2011 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-21130713

RESUMO

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA , Reparo do DNA , Raios Ultravioleta , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Genes p53 , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Histonas/química , Histonas/genética , Histonas/efeitos da radiação , Humanos , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismo
20.
J Zhejiang Univ Sci B ; 11(10): 801-5, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20872988

RESUMO

To investigate the influence of microwave radiation on the human fibroblast nuclei, the effects of three variants of electromagnetic wave polarization, linear and left-handed and right-handed elliptically polarized, were examined. Experimental conditions were: frequency (f) 36.65 GHz, power density (P) at the surface of exposed object 1, 10, 30, and 100 µW/cm(2), exposure time 10 s. Human fibroblasts growing in a monolayer on a cover slide were exposed to microwave electromagnetic radiation. The layer of medium that covered cells during microwave exposure was about 1 mm thick. Cells were stained immediately after irradiation by 2% (w/v) orcein solution in 45% (w/v) acetic acid. Experiments were made at room temperature (25 °C), and control cell samples were processed in the same conditions. We assessed heterochromatin granule quantity (HGQ) at 600× magnification. Microwave irradiation at the intensity of 1 µW/cm(2) produced no effect, and irradiation at the intensities of 10 and 100 µW/cm(2) induced an increase in HGQ. More intense irradiation induced more chromatin condensation. The right-handed elliptically polarized radiation revealed more biological activity than the left-handed polarized one.


Assuntos
Núcleo Celular/efeitos da radiação , Fibroblastos/efeitos da radiação , Micro-Ondas , Núcleo Celular/ultraestrutura , Células Cultivadas , Fibroblastos/ultraestrutura , Heterocromatina/efeitos da radiação , Humanos
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