• Publikační typ
• abstrakt z konference MeSH

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 (gammaH2AX)/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 (~135 keV mum-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 gammaH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET gamma-rays. Surprisingly, neon ions produced even more complex gammaH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to gamma-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.

• MeSH
• 53BP1 * chemie   MeSH
• apoptóza MeSH
• dvouřetězcové zlomy DNA * MeSH
• fibroblasty účinky záření   MeSH
• fluorescenční protilátková technika MeSH
• fosforylace MeSH
• histony * chemie   MeSH
• ionizující záření MeSH
• konfokální mikroskopie * MeSH
• kultivované buňky MeSH
• lidé MeSH
• lineární přenos energie * MeSH
• oprava DNA MeSH
• Check Tag
• lidé MeSH

Lymphocytes are among the most radiosensitive cells. After exposure of the organism to ionizing radiation, they promptly die by apoptosis at a rate proportional to the dose received. Because of this, they are frequently used in biodosimetry. We demonstrated that one hour after whole-body irradiation of rats, histone H2AX in the lymphocyte nuclei was quickly phosphorylated on serine 139, the phosphorylation process being directly dependent on the gamma radiation dose. In the work presented here, we studied the kinetics of lymphocyte depletion in the peripheral blood and phosphorylation of histone H2AX in the peripheral blood lymphocytes after local (thoracic) irradiation of rats. Twenty-four hours after whole-body irradiation of the rats at a dose of 5 Gy, the lymphocyte count declined to almost zero values, whereas after local irradiation of the thorax area, the counts of lymphocytes in the peripheral blood remained unaltered. The authors employed two methods (flow-cytometric and microscopic) for the gammaH2AX determination in the peripheral blood lymphocytes, 1 h after thoracic irradiation of rats. Flow cytometry revealed a dose dependence on the increase in gammaH2AX in a dose range of 10-30 Gy. The microscopic method was more sensitive in the case of lower radiation doses, the dependence on the dose being obvious from a dose as low as 5 Gy. The methods are able, in the dose range 5-30 Gy, to differentiate between the type of irradiation, i.e. the whole-body or local.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• buněčné jádro účinky záření   MeSH
• celotělové ozáření škodlivé účinky   MeSH
• financování organizované MeSH
• fosforylace účinky záření   MeSH
• histony účinky záření   MeSH
• hrudník cytologie   účinky záření   MeSH
• imunochemie metody   MeSH
• leukocyty mononukleární účinky záření   MeSH
• lymfocyty účinky léků   účinky záření   MeSH
• potkani Wistar MeSH
• průtoková cytometrie využití   MeSH
• radiometrie využití   MeSH
• statistika jako téma MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH

Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

• MeSH
• epigeneze genetická MeSH
• financování organizované MeSH
• histonlysin-N-methyltransferasa genetika   chemie   metabolismus   MeSH
• histony metabolismus   MeSH
• hybridizace genetická MeSH
• křížení genetické MeSH
• mapování chromozomů MeSH
• meióza MeSH
• metylace MeSH
• molekulární sekvence - údaje MeSH
• mužská infertilita genetika   MeSH
• myši inbrední C3H MeSH
• myši transgenní MeSH
• myši MeSH
• ovarium enzymologie   MeSH
• regulace genové exprese MeSH
• sekvence aminokyselin MeSH
• testis enzymologie   MeSH
• umělé bakteriální chromozomy MeSH
• vznik druhů (genetika) MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.

• MeSH
• buněčná smrt genetika   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• buněčný cyklus * genetika   MeSH
• CDC geny fyziologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• geminin MeSH
• histony genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mitóza genetika   MeSH
• nádorová transformace buněk genetika   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• nestabilita genomu * genetika   MeSH
• polyploidie MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• rekombinasa Rad51 MeSH
• replikace DNA * genetika   MeSH
• Check Tag
• lidé MeSH

Autosomal trisomies and monosomies bring serious threats to embryonic development through transcriptional disarray caused primarily by the dosage effect of the aneuploid part of the genome. The present study compared the effect of a mouse-viable 30-Mb segmental trisomy on the genome-wide transcriptional profile of somatic (liver) cells and male germ cells. Although the 1.6-fold change in expression of triplicated genes reflected the gene dosage in liver cells, the extra copy genes were compensated in early pachytene spermatocytes, showing 1.18-fold increase. Although more pronounced, the dosage compensation of trisomic genes was concordant with the incidence of HORMAD2 protein and histone gammaH2AX markers of unsynapsed chromatin. A possible explanation for this includes insufficient sensitivity to detect the meiotic silencing of unsynapsed chromatin markers in the 30-Mb region of the chromosome or an earlier silencing effect of another epigenetic factor. Taken together, our results indicate that the meiotic silencing of unsynapsed chromatin is the major, but most likely not the only, factor driving the dosage compensation of triplicated genes in primary spermatocytes.

• MeSH
• aneuploidie MeSH
• chromatin genetika   MeSH
• fenotyp MeSH
• genom * MeSH
• játra fyziologie   MeSH
• kompenzace dávky (genetika) * MeSH
• meióza genetika   MeSH
• mužská infertilita genetika   MeSH
• myši inbrední C57BL MeSH
• spermatocyty fyziologie   MeSH
• synaptonemální komplex genetika   MeSH
• těhotenství MeSH
• tělesná hmotnost genetika   MeSH
• transkriptom MeSH
• translokace genetická genetika   MeSH
• trizomie genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/gammaH2AX(+) foci), enhanced senescence-associated beta-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

• MeSH
• bakteriální toxiny metabolismus   farmakologie   MeSH
• buněčný cyklus účinky léků   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   metabolismus   MeSH
• cytokiny metabolismus   MeSH
• fenotyp MeSH
• Haemophilus ducreyi metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie účinky léků   fyziologie   MeSH
• poškození DNA MeSH
• signální transdukce fyziologie   MeSH
• stárnutí buněk fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bohemine and roscovitine are the most important representatives of the group of compounds structurally derived from olomoucine. Biologically they function as inhibitors of cyclin-dependent kinases (CDKs), the key regulators of cell cycle, which is often disrupted in cancer cells resulting in uncontrollable proliferation. Bohemine and roscovitine have demonstrated their cytostatic and cytotoxic in vitro and also in vivo effects. Currently the phase II clinical trials for roscovitine are underway. The aim of the study was to evaluate the potential in vitro radiosensitising effect of bohemine (BOH) and roscovitine (ROS). Clonogenic survival assay and human lung adenocarcinoma cell line A549 were used. Tested schedules were: A-pretreatment, B-concomitant application and C-posttreatment. Concentrations corresponded to IC10, IC25 and IC50 for BOH/ROS (0.1-30 microM). The radiation doses were 1, 2 and 3 Gy. Flow cytometry and western blot analysis were used to characterize cell cycle distribution, BrdU incorporation and DNA repair processes. The highest in vitro radiosensitising effect of BOH/ROS was observed for Schedule A in all tested concentrations (SER(37%) 1.46-3.20). Cell cycle analysis showed an inclination towards G0/G1 delay 48 hours posttreatment and unaltered level of apoptosis. Changes in the DNA repair processes were observed - inhibition of DNA-PK kinase, inhibition of BrdU incorporation, strong and enduring induction of p21 protein and long-lasting phosphorylation of gammaH2AX(Ser139). Certain low concentration activities of BOH/ROS in monotherapy were detected, mainly the activation of DNA-PK kinase. The results demonstrated strong in vitro radiosensitising effect of BOH/ROS that is concentration and especially schedule dependent. The strong cytostatic effect of the pretreatment schedule is mediated through the inhibition/rearrangements of DNA repair processes.

• MeSH
• adenokarcinom farmakoterapie   patologie   radioterapie   MeSH
• apoptóza účinky léků   účinky záření   MeSH
• buněčný cyklus účinky léků   účinky záření   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   metabolismus   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kinetin farmakologie   MeSH
• lidé MeSH
• nádorové buňky kultivované účinky léků   MeSH
• nádory plic farmakoterapie   radioterapie   MeSH
• průtoková cytometrie MeSH
• puriny farmakologie   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• rentgenové záření MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11-RAD50-NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients' survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (gammaH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non-BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.

• MeSH
• DNA vazebné proteiny * analýza   genetika   MeSH
• enzymy opravy DNA * analýza   genetika   MeSH
• genetická predispozice k nemoci MeSH
• imunohistochemie MeSH
• lidé MeSH
• mutační analýza DNA MeSH
• nádory prsu * etiologie   genetika   patologie   MeSH
• poškození DNA * genetika   MeSH
• protein BRCA1 analýza   MeSH
• protein BRCA2 analýza   MeSH
• proteiny buněčného cyklu * analýza   genetika   MeSH
• zárodečné mutace * MeSH
• zdraví rodiny MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

The ATM kinase is a tumor suppressor and key regulator of biological responses to DNA damage. Cultured cells respond to genotoxic insults that induce DNA double-strand breaks by prompt activation of ATM through its autophosphorylation on serine 1981. However, whether ATM-S1981 becomes phosphorylated in vivo, for example during physiological processes that generate DSBs, is unknown. Here we produced phospho-specific monoclonal antibodies against S1981-phosphorylated ATM (pS-ATM), and applied them to immunohistochemical analyses of a wide range of normal human tissues and testicular tumors. Our data show that regardless of proliferation and differentiation, most human tissues contain only the S1981-nonphosphorylated, inactive form of ATM. In contrast, nuclear staining for pS-ATM was detected in subsets of bone-marrow lymphocytes and primary spermatocytes in the adult testes, cell types in which DSBs are generated during physiological V(D)J recombination and meiotic recombination, respectively. Among testicular germ-cell tumors, an aberrant constitutive pS-ATM was observed especially in embryonal carcinomas, less in seminomas, and only modestly in teratomas and the pre-invasive carcinoma-in-situ stage. Compared with pS-ATM, phosphorylated histone H2AX (gammaH2AX), another DNA damage marker and ATM substrate, was detected in a higher proportion of cancer cells, and also in normal fetal gonocytes, and a wider range of adult spermatocyte differentiation stages. Collectively, our results strongly support the physiological relevance of the recently proposed model of ATM autoactivation, and provide further evidence for constitutive activation of the DNA damage machinery during cancer development. The new tools characterized here should facilitate monitoring of ATM activation in clinical specimens, and help develop future treatment strategies.

• MeSH
• ATM protein MeSH
• cervix uteri cytologie   MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• epitelové buňky účinky záření   MeSH
• ezofágus cytologie   MeSH
• fibroblasty účinky záření   MeSH
• fosforylace MeSH
• germinální a embryonální nádory patologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny * metabolismus   MeSH
• orgánová specificita MeSH
• plod cytologie   MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• rekombinantní proteiny MeSH
• testikulární nádory * metabolismus   MeSH
• žaludek cytologie   MeSH
• zdraví MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH