K novým poznatkům o vlivu různých druhů ionizujícího záření na buňky patří mikro- a nanodozimetrické aspekty poškození chromatinu. Fyzikální vlastnosti incidentního ionizujícího záření (fotonů gama, protonů a iontů s vysokým LET) souvisí s charakterem poškození chromatinu, možnostmi buňky opravit a přežít vytvořené léze DNA a rizikem genetických změn. Přestože výsledky jednoznačně potvrzují pozitivní korelaci mezi LET ionizujícího záření, komplexností indukovaných dvouřetězcových zlomů DNA (DSB) a biologickou účinností (RBE) záření, zároveň odhalují, že těmto vztahům ještě dostatečně nerozumíme. Příkladem budiž zjištění, že různé urychlené ionty s podobným LET mohou poškozovat DNA odlišným způsobem a zabíjet tak buňky s nestejnou účinností. Stále také neumíme vysvětlit mnoho aspektů reparace DSB, například co rozhoduje o aktivaci určité reparační dráhy v místě konkrétního DSB a jak je tento výběr ovlivněn použitým ionizujícím zářením a strukturou chromatinu. Diskutované výsledky mohou být mj. důležité z hlediska nově se rozvíjející hadronové terapie nádorových onemocnění a plánování pilotovaných meziplanetárních letů. Z metodického hlediska potom tato práce ilustruje obrovský pokrok, který se udál na poli optické mikroskopie a jejích výzkumných aplikací. Detailněji je představena metoda lokalizační mikroskopie s rozlišením jednotlivých molekul (SMLM – single-molecule localization microscopy).
The present work introduces new findings about the influence of different radiation types on the cells, with the concern on the micro- and nanodosimetric aspects of chromatin damage. Emphasized is the relationship between the physical parameters of the incident radiation (g-rays, protons and high-LET heavy ions), character of chromatin damage, ability of cells to repair and survive DNA damage, and risk of genetic changes. While confirming a positive correlation between the LET of ionizing radiation, complexity of induced DNA double-strand breaks (DSB), and biological effectiveness (RBE) of radiation, at the same time, we show that our understanding of this relationship is only incomplete. Our discovery that various accelerated ions with similar LET can damage DNA in different ways and kill cells with unequal efficiency, could serve as an example. In addition, many aspects of DSB repair remain to be explained, for instance, how the cell activates the particular repair pathway at sites of individual DSBs, and how it depends on the radiation used and the chromatin architecture. The discussed results may be important, above all, for newly developing hadron therapy and in the context of manned interstellar flights planning. From the methodological point of view, we point to a tremendous progress in the field of optical microscopy and its research applications. In more detail, we introduce single-molecule localization microscopy (SMLM).
- Klíčová slova
- reparační ohniska indukovaná ionizujícím zářením (IRIF),
- MeSH
- chromatin účinky záření MeSH
- chromozomální aberace účinky záření MeSH
- fosforylace MeSH
- ionizující záření * MeSH
- lidé MeSH
- mikroskopie metody MeSH
- oprava DNA * účinky záření MeSH
- poškození DNA * účinky záření MeSH
- radiační expozice MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.
- MeSH
- 53BP1 genetika metabolismus MeSH
- chromatin chemie účinky záření ultrastruktura MeSH
- DNA vazebné proteiny nedostatek genetika MeSH
- embryo savčí MeSH
- fibroblasty cytologie metabolismus účinky záření MeSH
- FRAP MeSH
- histony genetika metabolismus MeSH
- lamin typ A nedostatek genetika MeSH
- luminescentní proteiny genetika metabolismus MeSH
- membránové proteiny nedostatek genetika MeSH
- myši MeSH
- oprava DNA * MeSH
- poškození DNA MeSH
- regulace genové exprese MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- reportérové geny MeSH
- signální transdukce MeSH
- transformované buněčné linie MeSH
- ultrafialové záření MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)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 dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome 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. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone; it requires sophisticated structural probing and imaging. In the first part of this review, the article "Giving Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part A--Radiomics," we showed the development of different Omics solutions and how they are contributing to a better understanding of cellular radiation response. In this Part B we show how high-resolution confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place Omics data in the context of space and time. The dynamics of double-strand breaks during repair processes and chromosomal rearrangements at the microscale correlated to aberration induction are explained. For the first time we visualize pan-nuclear nucleosomal rearrangements and clustering at the nanoscale during repair processes. Finally, we introduce a novel method of specific chromatin nanotargeting based on a computer database search of uniquely binding oligonucleotide combinations (COMBO-FISH). With these challenging techniques on hand, we speculate future perspectives that may combine specific COMBO-FISH 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.
- MeSH
- buněčné jádro účinky záření MeSH
- chromatin genetika účinky záření MeSH
- DNA účinky záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- genom genetika MeSH
- ionizující záření MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nestabilita genomu MeSH
- oprava DNA genetika MeSH
- translokace genetická genetika účinky záření MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
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.
- MeSH
- buněčné jádro genetika MeSH
- chromatin účinky záření MeSH
- DNA účinky záření MeSH
- genom genetika účinky záření MeSH
- ionizující záření MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nestabilita genomu účinky záření MeSH
- oprava DNA * MeSH
- poškození DNA genetika účinky záření MeSH
- radiobiologie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1β to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPβ to UV-irradiated chromatin.
- MeSH
- acetylace MeSH
- buněčné linie MeSH
- buňky 3T3 MeSH
- časosběrné zobrazování MeSH
- chromatin metabolismus účinky záření MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- FRAP MeSH
- histony metabolismus MeSH
- inhibitory histondeacetylas metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- konfokální mikroskopie metody MeSH
- kyseliny hydroxamové metabolismus MeSH
- lidé MeSH
- myši MeSH
- poškození DNA MeSH
- protoonkogenní proteiny genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- represorové proteiny genetika metabolismus MeSH
- ultrafialové záření MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.
Tumour cells are characterized by karyotype instability, which is accompanied by specific events in the chromatin structure and epigenetic patterns. Epigenetics involves heritable changes in the physical and biochemical state of chromatin, which have no effect on DNA sequences; therefore, changes in the nuclear radial arrangement of chromosomes can also be considered epigenetic events. Nuclear radial distributions of select genomic regions have been studied in many tumour cells and are not influenced by aberrations in chromosome number. On the other hand, genes involved in translocations take up new positions midway between the original coding sequences. The differentiation of leukaemia cells with clinically used agents is often accompanied by nuclear repositioning of tumour-related genes. However, the nuclear rearrangement is cell-type specific and not always associated with changes in the transcriptional activity. Similarly, cell type-specific chromatin structure is observed in tumour cells treated with select cytostatics and inhibitors of epigenetic processes, which have significant influences on the histone code. Chromatin structure and histone modifications were also affected by gamma radiation in leukaemia, multiple myeloma, and solid tumour cells. Interestingly, gamma radiation induced loci proximity, which has been suggested to increase the probability of exchange aberrations typically associated with tumour progression.
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné jádro metabolismus MeSH
- chromatin metabolismus účinky léků účinky záření MeSH
- chromozomální aberace MeSH
- epigeneze genetická MeSH
- financování organizované MeSH
- genetická transkripce MeSH
- histony metabolismus MeSH
- karyotypizace MeSH
- lidé MeSH
- nádory genetika terapie MeSH
- progrese nemoci MeSH
- Check Tag
- lidé MeSH
For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by gamma-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.
- MeSH
- apoptóza účinky záření MeSH
- buněčné jádro metabolismus MeSH
- chromatin účinky záření MeSH
- chromatinová imunoprecipitace MeSH
- DNA účinky záření MeSH
- fibroblasty cytologie metabolismus účinky záření MeSH
- fluorescenční protilátková technika MeSH
- G1 fáze fyziologie účinky záření MeSH
- histondeacetylasy metabolismus MeSH
- hybridizace in situ fluorescenční MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory histondeacetylas MeSH
- kůže cytologie metabolismus účinky záření MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- oprava DNA účinky záření MeSH
- poškození DNA účinky záření MeSH
- radioizotopy kobaltu MeSH
- regulace genové exprese účinky záření MeSH
- S fáze fyziologie účinky záření MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- ATM protein MeSH
- chromatin účinky záření MeSH
- dimerizace MeSH
- DNA vazebné proteiny MeSH
- fosforylace účinky záření MeSH
- ionizující záření MeSH
- kvarterní struktura proteinů účinky záření MeSH
- lidé MeSH
- nádorové supresorové proteiny MeSH
- poškození DNA * MeSH
- protein-serin-threoninkinasy * chemie metabolismus MeSH
- proteiny buněčného cyklu MeSH
- vazba proteinů účinky záření MeSH
- zlomy chromozomů MeSH
- Check Tag
- lidé MeSH
- MeSH
- buněčné jádro genetika účinky záření ultrastruktura MeSH
- buněčný cyklus fyziologie MeSH
- chromatin fyziologie účinky záření ultrastruktura MeSH
- fluorescenční mikroskopie metody statistika a číselné údaje MeSH
- hybridizace in situ fluorescenční metody statistika a číselné údaje MeSH
- lidé MeSH
- nádorové buňky kultivované fyziologie ultrastruktura MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
