Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.
Esophageal cancer is a malignant disease with poor prognosis, increasing incidence, and ineffective treatment options. MicroRNAs are post-transcriptional regulators of gene expression involved in many biological processes including carcinogenesis. We determined miR-205 expression levels in tumor/non-tumor tissues of 45 esophageal cancer patients using qPCR and found that decreased level of miR-205 in tumor tissue correlates with poor overall survival in esophageal adenocarcinoma patients. Further, we observed significantly higher levels of miR-205 in tumor tissue of esophageal squamous cell carcinoma. Ectopic overexpression of miR-205 in adenocarcinoma cell line SK-GT-4 led to decreased cell proliferation, cell cycle arrest in G1, and decreased migration ability. Conversely, in squamous cell line KYSE-150, same effects like inhibition of proliferation, migration, and colony-forming potential and cell cycle arrest in G2 were observed after silencing of miR-205. We performed global gene expression profiling and revealed that suppressive functioning of miR-205 in adenocarcinoma could be realized through regulation of epithelial-mesenchymal transition (EMT), whereas oncogenic in squamous cell carcinoma by regulation of metalloproteinase 10. Our results suggest that miR-205 could serve as biomarker in esophageal cancer and acts as a tumor suppressor in esophageal adenocarcinoma and oncogene in esophageal squamous cell carcinoma.
- MeSH
- adenokarcinom genetika metabolismus patologie MeSH
- apoptóza genetika MeSH
- buněčný cyklus genetika MeSH
- dospělí MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikro RNA fyziologie MeSH
- míra přežití MeSH
- nádorové buněčné linie MeSH
- nádory jícnu genetika metabolismus patologie MeSH
- onkogeny genetika MeSH
- pohyb buněk genetika MeSH
- proliferace buněk genetika MeSH
- regulace genové exprese u nádorů fyziologie MeSH
- senioři MeSH
- spinocelulární karcinom genetika metabolismus patologie MeSH
- stanovení celkové genové exprese MeSH
- tumor supresorové geny MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
In all eukaryotes, the highly repeated 35S ribosomal DNA (rDNA) sequences encoding 18S-5.8S-26S ribosomal RNA (rRNA) typically show high levels of intragenomic uniformity due to homogenisation processes, leading to concerted evolution of 35S rDNA repeats. Here, we compared 35S rDNA divergence in several seed plants using next generation sequencing and a range of molecular and cytogenetic approaches. Most species showed similar 35S rDNA homogeneity indicating concerted evolution. However, Cycas revoluta exhibits an extraordinary diversity of rDNA repeats (nucleotide sequence divergence of different copies averaging 12 %), influencing both the coding and non-coding rDNA regions nearly equally. In contrast, its rRNA transcriptome was highly homogeneous suggesting that only a minority of genes (<20 %) encode functional rRNA. The most common SNPs were C > T substitutions located in symmetrical CG and CHG contexts which were also highly methylated. Both functional genes and pseudogenes appear to cluster on chromosomes. The extraordinary high levels of 35S rDNA diversity in C. revoluta, and probably other species of cycads, indicate that the frequency of repeat homogenisation has been much lower in this lineage, compared with all other land plant lineages studied. This has led to the accumulation of methylation-driven mutations and pseudogenisation. Potentially, the reduced homology between paralogs prevented their elimination by homologous recombination, resulting in long-term retention of rDNA pseudogenes in the genome.
- MeSH
- Cycas genetika MeSH
- DNA rostlinná genetika MeSH
- genetická transkripce genetika MeSH
- hybridizace in situ fluorescenční MeSH
- jednonukleotidový polymorfismus genetika MeSH
- mezerníky ribozomální DNA genetika MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 18S genetika MeSH
- RNA ribozomální 5.8S genetika MeSH
- RNA ribozomální genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.
- MeSH
- acetylace MeSH
- arginin metabolismus MeSH
- buněčné jadérko genetika ultrastruktura MeSH
- buněčné linie MeSH
- dvouřetězcové zlomy DNA * MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- fosfoproteiny metabolismus MeSH
- geny rRNA MeSH
- histondeacetylasa 1 metabolismus MeSH
- histony metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- proteinarginin-N-methyltransferasy metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression (miR-155: median TTP 5.8 versus 12.8 months, miR-484: median TTP 5.8 versus 8.9 months). Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma.
- MeSH
- dospělí MeSH
- indoly aplikace a dávkování MeSH
- Kaplanův-Meierův odhad MeSH
- karcinom z renálních buněk farmakoterapie genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metastázy nádorů MeSH
- mikro RNA biosyntéza genetika MeSH
- nádorové biomarkery biosyntéza genetika MeSH
- patologická angiogeneze farmakoterapie genetika patologie MeSH
- progrese nemoci MeSH
- pyrroly aplikace a dávkování MeSH
- regulace genové exprese u nádorů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential. FINDINGS: Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients. CONCLUSIONS: Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4646922201567057.
- MeSH
- adenokarcinom diagnóza genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- nádorové biomarkery analýza genetika metabolismus MeSH
- nádory jícnu diagnóza genetika MeSH
- prognóza MeSH
- regulace genové exprese u nádorů genetika MeSH
- senioři MeSH
- spinocelulární karcinom diagnóza genetika MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MikroRNA (miRNA) jsou početnou skupinou krátkých nekódujících RNA (cca 18–25 nukleotidů), které tlumí translaci vazbou na cílové mRNA, případně vedou k jejich degradaci. Tyto endogenní molekuly RNA jsou vysoce sekvenčně konzervované, a to i napříč nepříbuznými organizmy, což poukazuje na jejich roli v základních biologických procesech, jako je vývoj, diferenciace, proliferace a apoptóza. Rovněž se podílejí na regulaci kmenových vlastností buněk, imunitního systému nebo nádorové transformaci. Tento přehled podává ucelené informace o miRNA u karcinomu jícnu, a to jak o jejich roli v základních pochodech vzniku a rozvoje nádorového onemocnění, tak o možnostech jejich využití v klinické praxi při zpřesnění diagnózy, stanovení prognózy onemocnění a predikci léčebné odpovědi. Díky regulaci klíčových signálních drah v maligní transformaci představují miRNA rovněž slibné terapeutické cíle.
MicroRNAs are an abundant class of non‑coding RNAs (approx. 18–25 nucleotides in length) that suppress translation through binding to their target mRNAs, eventually leading to mRNAs degradation. Sequences of these endogenous RNA molecules are highly conserved, even among unrelated species, indicating their involvement in basic biological processes, such as development, differentiation, proliferation or apoptosis. MiRNAs also participate on regulation of cancer stem cell functioning, immune system and malignant transformation. This review provides a comprehensive overview of miRNAs functions in esophageal cancer, their roles in key pathogenetic pathways and disease development, as well as their potential usage in clinical routine as biomarkers improving diagnosis, prognosis and prediction of therapeutic response. Through regulation of signaling pathways important in malignant transformation, miRNAs present also promising therapeutic targets.
- Klíčová slova
- karcinom jícnu,
- MeSH
- adenokarcinom * diagnóza genetika MeSH
- apoptóza genetika MeSH
- Barrettův syndrom genetika MeSH
- lidé MeSH
- metastázy nádorů genetika MeSH
- mikro RNA * genetika MeSH
- nádorová transformace buněk genetika MeSH
- nádorové biomarkery * genetika MeSH
- nádory jícnu * diagnóza genetika MeSH
- prognóza MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů MeSH
- RNA nádorová krev moč MeSH
- spinocelulární karcinom * diagnóza genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
All dogroses (Rosa sect. Caninae) are characterized by the peculiar canina meiosis in which genetic material is unevenly distributed between female and male gametes. The pan-canina rDNA family (termed beta) appears to be conserved in all dogroses analyzed so far. Here, we have studied rDNAs in experimental hybrids obtained from open pollination of F1 plants derived from 2 independent intersectional crosses between the pentaploid dogrose species (2n = 5x = 35) Rosa rubiginosa as female parent (producing 4x egg cells due to the unique asymmetrical canina meiosis) and the tetraploid (2n = 4x = 28) garden rose R. hybrida 'André Brichet' as male parent (producing 2x pollen after normal meiosis). We analyzed the structure of rDNA units by molecular methods [CAPS and extensive sequencing of internal transcribed spacers (ITS)] and determined the number of loci on chromosomes by FISH. FISH showed that R. rubiginosa and 'André Brichet' harbored 5 and 4 highly heteromorphic rDNA loci, respectively. In the second generation of hybrid lines, we observed a reduced number of loci (4 and 5 instead of the expected 6). In R. rubiginosa and 'André Brichet', 2-3 major ITS types were found which is consistent with a weak homogenization pressure maintaining high diversity of ITS types in this genus. In contrast to expectation (the null hypothesis of Mendelian inheritance of ITS families), we observed reduced ITS diversity in some individuals of the second generation which might derive from self-fertilization or from a backcross to R. rubiginosa. In these individuals, the pan-canina beta family appeared to be markedly enriched, while the paternal families were lost or diminished in copies. Although the mechanism of biased meiotic transmission of certain rDNA types is currently unknown, we speculate that the bivalent-forming chromosomes carrying the beta rDNA family exhibit extraordinary pairing efficiency and/or are subjected to strong selection in Caninae polyploids.
- Publikační typ
- abstrakt z konference MeSH
