V současnosti není znám klinický marker, který by umožňoval odlišit pacienty s nádory hlavy a krku, kteří budou více profitovat z konvenční léčby, od těch, kterým lépe poslouží chirurgický zákrok. V rámci projektu významně přispějeme k řešení tohoto "Dilematu první léčby" zavedením metody in vitro detekce zlomů DNA, jež zodpovídají za účinek konvenční (chemo)radioterapie. V našem výzkumu a v praxi tato metoda umožní velmi citlivě stanovit radiosensitivitu jednotlivých nádorů již před započetím léčby, spolehlivě odhalit pacienty hypersensitivní či rezistentní k záření a optimalizovat terapii. Takto vytipujeme nádory neodpovídající na současnou léčbu, u kterých budeme, opět s využitím zavedené metody, zkoumat účinnost alternativních režimů ozařování a nových terapií – indukce replikačního stresu a ozařování protony. Vůbec poprvé budeme uvedené problémy studovat na paralelních primokulturách různých buněčných typů vyizolovaných z nádoru. Hlavním cílem projektu však zůstává vývoj levné, rychlé, robustní a snadno proveditelné diagnostické metody s možnostmi automatizace.; Currently, there is no clinical marker which allows a pre-therapeutic selection of patients with head and neck tumors that would profit more from the conservative therapy or primary surgery, respectively. We meaningfully contribute to solution of this "First Therapy Dilemma" by developing a method for in vitro detection of DNA breaks mediating the effects of (chemo)radiotherapy. By this method, we will sensitively determine the radiosensitivity of tumors before beginning the treatment, identify patients hypersensitive or resistant to radiation, and help to optimize the therapy. The method also enables us to elucidate the effectiveness of alternative irradiation regimes, explore and develop new therapeutic approaches (the replication stress induction and proton irradiation) and categorize patients for the best therapy. For the first time in this context, we compare primocultures of different cell types isolated from the tumor. Though, the main aim of this project remains to develop a cost-efficient, fast, robust, and easy to perform diagnostic method with a large room for automation.

• MeSH
• diagnostické techniky molekulární metody   MeSH
• genetické testování metody   MeSH
• lidé MeSH
• nádorové biomarkery genetika   MeSH
• nádory hlavy a krku diagnóza   genetika   terapie   MeSH
• poškození DNA genetika   MeSH
• protonová terapie MeSH
• radioterapie metody   MeSH
• techniky in vitro MeSH
• tolerance záření MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• radiologie, nukleární medicína a zobrazovací metody
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Východiska: Navzdory pokroku v protinádorové terapii jsou spinocelulární karcinomy v oblasti hlavy a krku (head and neck squamous cell carcinoma – HNSCC) stále spojeny s nízkou mírou dlouhodobého přežívání pacientů. Nedávné studie poukazují na to, že nádorové stroma může hrát důležitou roli v patogenezi tohoto maligního onemocnění. Fibroblasty jsou hlavní složkou nádorového mikroprostředí a mohou významně ovlivňovat progresi HNSCC, protože přispívají k významným znakům onkogeneze, jako je zánět, neomezený růst, angiogeneze, invaze, vznik metastáz a rezistence k terapii. Je dobře známo, že nádorové buňky mohou modulovat fenotyp fibroblastů do podoby fibroblastů asociovaných s nádorem (cancer-associated fibroblast –CAF), které následně mohou podporovat růst a šíření nádorových buněk. CAF stimulují progresi nádoru prostřednictvím kontaktů mezi buňkami, remodelace extracelulární matrix a produkce velkého množství signálních molekul a matrixových metaloproteináz. Genetické a epigenetické změny v epiteliálních buňkách tedy pravděpodobně nejsou výlučným faktorem řídícím kancerogenezi HNSCC, jelikož se na tomto procesu mohou významně podílet i negenetické změny v buňkách stromatu. Signály vyvolané buněčným stresem narušují funkční program mnoha buněk, čímž se ve tkáni vytváří oblasti predisponované k maligní transformaci. Koncept "nádorového pole“ (field cancerization) představuje proces aktivního vývoje mezibuněčných interakcí a zpětnovazebných smyček mezi nádorovými a stromálními buňkami. Tento model se jeví jako velice slibný, otvírá nové způsoby studia nádorového onemocnění a může poskytnout nové terapeutické cíle. Cíl: V tomto přehledovém článku diskutujeme současné poznatky o CAF, jako je jejich buněčný původ, fenotypová plasticita a funkční heterogenita, a podtrhujeme jejich vliv na progresi HNSCC

Background: Despite progress in anticancer therapies, head and neck squamous cell carcinoma (HNSCC) has still a low survival rate. Recent studies have shown that tumour stroma may play an important role in the pathogenesis of this malignant disease. Fibroblasts are a major component of the tumour microenvironment and may significantly influence HNSCC progression as indicated by the contribution they make to important hallmarks of cancer, such as inflammation, non-restricted growth, angiogenesis, invasion, metastasis, and therapy resistance. It is well known that tumour cells can confer a cancer-associated fibroblast (CAF) phenotype that supports the growth and dissemination of cancer cells. CAFs can stimulate cancer progression through cell-cell contacts and communication, remodelling of extracellular matrix, and production of many signal molecules and matrix metalloproteinases. Consequently, genetic changes in epithelial cells are probably not the only factor that drives HNSCC carcinogenesis. Non-genetic changes in the tumour stroma can also be significantly involved. Stress-induced signals can induce a multicellular program, creating a field of tissue that is predisposed to malignant transformation. The "field cancerization" concept represents a process of active evolution of intercellular interactions and feedback loops between tumour and stromal cells. This model paves the way to study cancer from a new perspective and identify new therapeutic targets. Purpose: In this review, we discuss current knowledge about CAFs, such as their cellular origin, phenotypical plasticity and functional heterogeneity, and stress their contribution to HNSCC progression.

• MeSH
• dlaždicobuněčné karcinomy hlavy a krku * etiologie   patofyziologie   MeSH
• extracelulární matrix MeSH
• fibroblasty asociované s nádorem MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové mikroprostředí MeSH
• patologická angiogeneze MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Due to the close relationship between carcinogenesis and human papillomavirus (HPV), and since they are transmitted via huge number of asymptomatic carriers, the detection of HPV is really needed to reduce the risk of developing cancer. According to the best of our knowledge, our study provides the very first method for one-step detection of viral infection and if it has initiated the subsequent cancer proliferation. The proposed novel nanosystem consists of magnetic glass particles (MGPs), which were attached with DNA probe on their surface to hybridize with target DNAs. The MGP-probe-DNA hybrid was finally conjugated with CdTe/ZnSe core/shell quantum dots (QDs). The proposed detection system is based on a novel mechanism in which the MGPs separate out the target DNAs from different biological samples using external magnetic field for better and clear detection and the QDs give different fluorescent maxima for different target DNAs due to their ability to interact differently with different nucleotides. Firstly, the method was optimized using HPV genes cloned into synthetic plasmids. Then it was applied directly on the samples from normal and cancerous cells. After that, the real hospital samples of head and neck squamous cell carcinoma (HNSCC) with or without the infection of HPV were also analyzed. Our novel nano-system is proved successful in detecting and distinguishing between the patients suffering by HPV infection with or without subsequent cancer having detection limit estimated as 1.0 x 109 (GEq/mL). The proposed methodology is faster and cost-effective, which can be applied at the clinical level to help the doctors to decide the strategy of medication that may save the life of the patients with an early treatment.

• MeSH
• biosenzitivní techniky metody   MeSH
• dlaždicobuněčné karcinomy hlavy a krku virologie   MeSH
• DNA sondy chemie   genetika   MeSH
• DNA virů krev   chemie   genetika   MeSH
• dospělí MeSH
• fluorescenční mikroskopie metody   MeSH
• fluorescenční spektrometrie metody   MeSH
• hybridizace nukleových kyselin MeSH
• infekce papilomavirem diagnóza   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• magnetické jevy MeSH
• nádorové buněčné linie MeSH
• Papillomaviridae chemie   MeSH
• senioři MeSH
• sklo chemie   MeSH
• sloučeniny kadmia chemie   MeSH
• telur chemie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

• MeSH
• dvouřetězcové zlomy DNA účinky záření   MeSH
• gadolinium chemie   MeSH
• HeLa buňky MeSH
• konfokální mikroskopie MeSH
• kovové nanočástice chemie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• poškození DNA účinky záření   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The mechanisms underlying cell protection from cryoinjury are not yet fully understood. Recent biological studies have addressed cryopreserved cell survival but have not correlated the cryoprotection effectiveness with the impact of cryoprotectants on the most important cell structure, the nucleus, and the freeze/thaw process. We identified changes of cell nuclei states caused by different types of cryoprotectants and associate them with alterations of the freeze/thaw process in cells. Namely, we investigated both higher-order chromatin structure and nuclear envelope integrity as possible markers of freezing and thawing processes. Moreover, we analyzed in detail the relationship between nuclear envelope integrity, chromatin condensation, freeze/thaw processes in cells, and cryopreservation efficiency for dimethyl sulfoxide, glycerol, trehalose, and antifreeze protein. Our interdisciplinary study reveals how changes in cell nuclei induced by cryoprotectants affect the ability of cells to withstand freezing and thawing and how nuclei changes correlate with processes during freezing and thawing. Our results contribute to the deeper fundamental understanding of the freezing processes, notably in the cell nucleus, which will expand the applications and lead to the rational design of cryoprotective materials and protocols.

• MeSH
• buněčné jádro * metabolismus   MeSH
• buněčné linie MeSH
• kryoprezervace * MeSH
• lidé MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

• MeSH
• 53BP1 metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• rekombinační oprava DNA * MeSH
• transport proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• chromatin účinky léků   genetika   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dvouřetězcové zlomy DNA účinky léků   MeSH
• fibroblasty MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• kůže cytologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• S fáze účinky léků   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zmrazování škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

This meta-analysis aims to evaluate the effects of high c-Met levels in head and neck squamous cell carcinomas (HNSCC) on survival and clinicopathological features. Publications concerned with the clinical significance of c-Met protein expression in HNSCC were identified from the Scopus and Web of Science database searches. To elucidate the relationship between c-Met expression and clinical outcomes, a meta-analysis of the selected articles was conducted. Seventeen publications involving a total of 1724 patients met the inclusion criteria. c-Met overexpression was significantly correlated with poor overall survival (hazard ratio (HR) = 2.19, 95% confidence interval (CI) = 1.55-3.10). c-Met immunohistochemical staining positivity was also associated with worse relapse-free survival (HR = 1.64, 95% CI = 1.24-2.17) and presence of regional lymph node metastases (odds ratio (OR) = 1.76, 95% CI = 1.26-2.45). High levels of c-Met expression in HNSCC predict unfavorable prognosis associated with common clinicopathological features.

• MeSH
• analýza přežití MeSH
• dlaždicobuněčné karcinomy hlavy a krku diagnóza   mortalita   patologie   MeSH
• lidé MeSH
• lymfatické metastázy MeSH
• prognóza MeSH
• protoonkogenní proteiny c-met metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• práce podpořená grantem 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

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90-, CD44-/CD90-, CD44+/CD90+, CD44-/CD90-). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90-) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44- subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44- subpopulations. Furthermore, co-cultivation experiments revealed that CD44-/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90-). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44-/CD90+, but this subpopulation had a low migratory capacity.

• Publikační typ
• časopisecké články MeSH