Vývoj ultrastrukturálních metod a jejich použití pro studium buěčnénho jádra

Despite the capabilities of molecular-biological methods in deciphering the interplay of different biological molecules and molecular complexes, the understanding of respective functions in living cells requires application of in situ methods. Obviously, these methods should provide maximal resolution and the best possible preservation of the biological object in a native state, as well as correct statistical evaluation of the spatial characteristics of detected molecular players. Transmission electron microscopy provides the highest possible resolution for analysis of biological samples. The simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. To analyze this, we have developed a complex stereological method for statistical evaluation of immunogold clustering and colocalization patterns of antigens on ultrathin sections, including a user-friendly interface. Functional microarchitecture of DNA replication and transcription sites has been successfully characterized using the developed stereological tools. Our data demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view...SOUHRN Navzdory možnostem, které přinášejí molekulárně-biologické metody při odhalování souhry různých biologických molekul a molekulárních komplexů, pochopení jednotlivých funkcí v živých buňkách vyžaduje použití in situ metod. Je zřejmé, že tyto metody by měly zajistit maximální rozlišení a nejlepší možné zachování biologického objektu v přirozeném stavu, stejně jako správné statistické vyhodnocení prostorových charakteristik lokalizace detekovaných molekulárních komplexů. Transmisní elektronová mikroskopie poskytuje nejlepší možné rozlišení pro analýzu biologických vzorků. Současná detekce více biologických molekul metodou nepřímého imunoznačení je zdrojem cenných informací o jejich lokalizaci v buněčných kompartmentech a jejich potenciálních interakcí v makromolekulárních komplexech. Vyvinuli jsme komplexní stereologickou metodu pro statistické hodnocení klastrování a kolokalizace značených antigenů na ultratenkých řezech, včetně uživatelsky přívětivého softwarového rozhraní. Dále jsme charakterizovali funkční mikroarchitekturu replikačních a transkripčních domén pomocí vyvinutých algoritmů. Výsledky ukazují, že DNA replikace je v buněčném jádře kompartmentalizována na úrovni DNA ohnisek a nasvědčují modelu, ve kterém centra syntézy DNA jsou zakotvena a chromatinové smyčky se pohybují. V HeLa buňkách...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Development of ultrastructural methods and their application in studies on the cell nucleus

Despite the capabilities of molecular-biological methods in deciphering the interplay of different biological molecules and molecular complexes, the understanding of respective functions in living cells requires application of in situ methods. Obviously, these methods should provide maximal resolution and the best possible preservation of the biological object in a native state, as well as correct statistical evaluation of the spatial characteristics of detected molecular players. Transmission electron microscopy provides the highest possible resolution for analysis of biological samples. The simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. To analyze this, we have developed a complex stereological method for statistical evaluation of immunogold clustering and colocalization patterns of antigens on ultrathin sections, including a user-friendly interface. Functional microarchitecture of DNA replication and transcription sites has been successfully characterized using the developed stereological tools. Our data demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view..

Characterisation of mesothelioma-initiating cells and their susceptibility to anti-cancer agents

Malignant mesothelioma (MM) is an aggressive type of tumour causing high mortality. One reason for this paradigm may be the existence of a subpopulation of tumour-initiating cells (TICs) that endow MM with drug resistance and recurrence. The objective of this study was to identify and characterise a TIC subpopulation in MM cells, using spheroid cultures, mesospheres, as a model of MM TICs. Mesospheres, typified by the stemness markers CD24, ABCG2 and OCT4, initiated tumours in immunodeficient mice more efficiently than adherent cells. CD24 knock-down cells lost the sphere-forming capacity and featured lower tumorigenicity. Upon serial transplantation, mesospheres were gradually more efficiently tumrigenic with increased level of stem cell markers. We also show that mesospheres feature mitochondrial and metabolic properties similar to those of normal and cancer stem cells. Finally, we show that mesothelioma-initiating cells are highly susceptible to mitochondrially targeted vitamin E succinate. This study documents that mesospheres can be used as a plausible model of mesothelioma-initiating cells and that they can be utilised in the search for efficient agents against MM

Mesospheres form tumours in serial transplantations.

<p>(A) Ist-Mes-2 sphere cells (generation 1, G1) were subcutaneously grafted into Balb-c/nude mice at 10⁶ cells per animal. When tumours reached about 2,000 mm³, the mice were sacrificed, tumours excised and malignant cells grew <i>in vitro</i> as a cell line. The adherent cells were converted into spheres (G1) and these were grafted into Balb-c/nude mice to form tumours that were used for generation 2 (G2) spheres. This procedure was repeated two more times to derive G3 and G4 spheres. The inset in panel A shows the lag to tumour appearance following cell grafting for individual sphere cell generations. Cells of individual generations were evaluated for stemness markers as shown using qPCR (B) and WB (panel C shows the blots, panel D their densitometric evaluations) and for CS activity (E). (F) Tumours derived from adherent cells and spheres of individual generations were excised, paraffin-embedded and stained for the MM marker mesothelin (upper images show staining with the exclusion of the primary IgG) and with H&E. Data shown in panel A are derived from 5 animals and are mean values ± S.E.M, data in panels B and E are mean values from three independent experiments ± S.D. Images in panel C are representative of two individual experiments and their densitometric evaluations in panel D represent mean values with differences lower than 10%. Images in panel F were obtained using one tumour for each condition (generation). The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images are representative of three different tumours.</p

IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.

<p>^aNumbers in bold indicate significantly different data (<i>p</i><0.05) from adherent CD24^-and parental cells.</p><p>The data are mean values ± S.D. from three independent experiments.</p><p>IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.</p

CD24 supports initiation and progression of mesotheliomas.

<p>(A) Mock-transfected and CD24^- Ist-Mes-2 cells (10⁶ per animal) were grafted subcutaneously into NOD/SCID mice and tumour formation followed using USI. (B) CD24^- and mock-transfected Ist-Mes-2 cell-derived tumours were evaluated for CD24, CD47, EpCAM and Oct3/4 by western blotting using anti-actin IgG as a loading control, with panel C documenting densitometric evaluation of the blots. (D) A representative image of a mouse with CD24^- cell-derived tumour and parental cell-derived tumour is shown. (E) Representative USI images of a tumour derived from mock-transfected and CD24^- cells acquired on different days are shown, with the yellow arrows indicating the position of the tumours. Data in panel A are mean values ±S.E.M., and are derived from four animals. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel B are representative of two independent experiments with the densitometric evaluation showing average data with differences less than 10%.</p

Mesospheres derived from different tumour generations show different mitochondrial features.

<p>Sphere cells representing individual generations were evaluated for respiration using the routine protocol (A) and the protocol for permeabilised cells (B) by means of the Oxygraph 2k high resolution respirometer. The symbols GM_L, GM_P, GMS_P, GMS_E and S(Rot)_E represent routine respiration, respiration via complex I, respiration via complex I+II, uncoupled respiration and uncoupled respiration via CII, respectively. (C) Mitochondrial mass was evaluated using MitoTracker Green as detailed in the Methods section. Superoxide was evaluated using the fluorescent probe MitoSOX (D), glucose uptake using the fluorescent glucose analogue 2-NBDG (E), ATP levels by a luciferase-based assay (F), ΔΨ_m using the fluorescent probe TMRM (G), lactate levels using a commercial kit (H), for SDH (I) and SQR (J) activities using enzymatic assays, and hand-held cells counter for cell size (K). (L) TEM was performed on individual generation sphere cells as detailed in the Methods section. Data in panels A-K are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel L are representative of three independent experiments. The white bar in panel K in the upper images represents 500 nm, in the lower images 200 nm.</p

Sphere cells derived from MM cell lines show increased levels of ‘stemness’ markers.

<p>(A) Ist-Mes-2, Meso-2 and MM-BI cells were allowed to form spheres, after which these were placed in the complete medium to cause differentiation and re-adhesion of the cells. Relative levels <i>CD24</i>, <i>ABCG2</i> and <i>OCT4</i> mRNAs were assessed in the original adherent cells (set as 1), the spheres and the re-adherent cells. (B) Ist-Mes-2, Meso-2 and MM-BI cells spheres were tested for the level of <i>SOX4</i>, <i>C-MYC</i>, <i>ABCB5</i> and <i>KLF4</i> mRNA and their level expressed relative to that of adherent cells. The data are mean values ± S.D. derived from three independent experiments, the symbol ‘*’ stands significantly different data with <i>p</i><0.05, ‘**’ for those with <i>p</i><0.005.</p

Effect of CD24 knock-down on mitochondrial function.

<p>Parental and CD24^- adherent and sphere Ist-Mes-2, non-permeabilised cells were evaluated for routine respiration using the Oxygraph instrument (A). Panel B shows respiration related to the maximum respiratory capacity of the cells (ETC). The symbols in panel A stand for: R, routine, L, leak, E, ETC, netR, R-L, ROX, residual respiration. Parental, mock-transfected, CD24^- adherent and sphere Ist-Mes-2 cells were evaluated for ΔΨ_m using TMRM (C), superoxide generation using MitoSOX (D), lactate production using a commercial kit (E), citrate synthase (CS) activity (F), relative SDH (G) and SQR activity (H), glucose uptake (I) and ATP level (J). Panel K documents the level of <i>PCG1α</i>mRNA in parental, mock-transfected and CD24^- adherent and sphere Ist-Mes-2 cells. Data shown are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05.</p