Nanolive je příkladem rozvíjející se technologie v oblasti nanoskopie, která kombinuje holografii a tomografii (Tomografická difrakční mikroskopie, TDM). Tato technologie nepoužívá žádné značení, proto můžeme pozorovat buňky v jejich přirozeném prostředí, aniž bychom je jakkoliv modifikovali nebo měnili pomocí chemických značek. Můžeme tak zaznamenat všechny důležité životní buněčné pochody a tímto unikátním způsobem lépe pochopit chování buněk. Digitální holografický tomograf 3D Cell Explorer vytváří kolekci 96 vrstev v ose Z, kdy každá vrstva je vlastně detailní mapou indexů lomu (Refractive Index, RI) intracelulárních struktur. Tato inovativní technologie dokáže rekonstruovat ve vysokém rozlišení (x, y:200nm; z:400nm; t:1.7sec) kvalitní trojrozměrný obraz transparentních neznačených vzorků. Buňky je možné umístit do malého inkubátoru, a tak lze monitorovat chování buněk v kontrolovaném prostředí (teplota, CO2, 02, vlhkost) po dobu hodin, dnů i týdnů. Díky fyzikální podstatě zaznamenávání indexů lomu, je možné provádět následnou kvantitativní analýzu dat. 3D Cell Explorer -fluo obohacuje tomografická data o možnost kombinace s fluorescenčními značkami a umožňuje tak přesnou identifikaci organel, proteinů nebo dalších látek značených flourescenčně.Tato neinvazivní inovativní technika otevírá možnosti pro nové fascinující aplikace pro studium buněčného cyklu/smrti, studium infekce uvnitř buněk, lokalizaci nanočástic a imuno -onkologii.
Nanolive technology is an example of emerging smart nanoscopic technique by combining holography with tomography (Tomographic Diffractive Microscopy, TDM). This technology is 100 label free, so we can observe cells in their real environment without any behaviour modifications nor alterations due to chemical markers and capture important events all along cells life time and understanding in an unique way how cells behave. Digital Holographic Tomograph, called 3D Cell Explorer, creates a collection of 96 Z -stacked layers, each layer being a detailed Refractive Index (RI) map of the cells observed. This innovation technique guarantees three -dimensional high spatio -temporal resolution imaging (x, y:200nm; z:400nm; t:1.7sec) of transparent unlabeled specimens. Cells can be cultured and monitored under controlled environment conditions in continuous time -lapse for days/weeks using on -stage incubator. Based on the quantitative nature of the refractive index measurements, the system enables for quantitative data analysis for single cells and tissues. Moreover, 3D -Cell Explorer‑fluo enriches high quality tomographic data with fluorescent markers and enables also precise intracellular detection of organels, proteins or drugs based on fluorescence staining. This not invasive and quantitative imaging technique permits new intriguing applications in the cell death/cell cycle analysis, intracellular infection, intracellular nanoparticles localization and immuno -oncology.
The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors.
- MeSH
- 2D gelová elektroforéza MeSH
- apoptóza účinky léků MeSH
- buněčná adheze účinky léků MeSH
- chronická myeloidní leukemie metabolismus MeSH
- fibronektiny metabolismus MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- paxilin metabolismus MeSH
- piperaziny farmakologie MeSH
- protinádorové látky farmakologie MeSH
- průtoková cytometrie MeSH
- pyrimidiny farmakologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tropomyosin metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate).
- MeSH
- antigeny CD29 MeSH
- apoptóza MeSH
- buněčná adheze účinky léků MeSH
- fibronektiny metabolismus MeSH
- kyseliny hydroxamové farmakologie MeSH
- leukemie metabolismus MeSH
- lidé MeSH
- lymfocyty metabolismus účinky léků MeSH
- nádorové buněčné linie MeSH
- paxilin metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protinádorové látky farmakologie MeSH
- průtoková cytometrie MeSH
- separace buněk MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways.
The present study was undertaken to provide more information on nuclear diameter in leukemic granulocytic early precursors myeloblasts. These cells represented by K562 myeloblasts originated from the blastic phase of the chronic myeloid leukaemia (CML) carry characteristic bcr-abl fusion gene. They represent a convenient model for in vitro studies of CML myeloblasts and are sensitive to various agents which may induce ageing, differentiation and cell death. Mean nuclear diameter (MNuD) and largest nuclear diameter (Mx NuD) in stained cytospins of these cells were measured at a high light microscopic magnification by means of computer programme. Starving cultures were used for induction of ageing without preceding differentiation, sodium butyrate was used as a cytostatic agent or differentiation inducer and imatinib mesylate represented a cytostatic agent for CML. The largest shift of MNuD to smaller values was noted in ageing cultures or cultures treated with butyrate. The decrease of MNuD was less apparent in resistant cells treated with imatinib. This drug, however, produced a very large incidence of necrotic or apoptotic cells or bodies. From the methodical point of view it should be mentioned that values of maximal nuclear diameter (MxNuD) followed similar trends as MNuD and thus provided similar information. The measurement of both nuclear diameters, i.e. MNuD and MxNuD might be a complementary and simple tool to evaluate the cell state in cytological preparations because of their decrease in ageing cells or cells treated with antiproliferative drugs of different mode of action.
K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.
- MeSH
- buněčná smrt účinky léků účinky záření MeSH
- buňky K562 patologie účinky léků účinky záření MeSH
- časové faktory MeSH
- cytoskelet patologie účinky léků účinky záření MeSH
- DNA vazebné proteiny metabolismus MeSH
- faloidin chemie MeSH
- financování organizované MeSH
- fluorescein-5-isothiokyanát MeSH
- fotosenzibilizující látky farmakologie MeSH
- kyselina aminolevulová farmakologie MeSH
- lidé MeSH
- Lim-kinasy MeSH
- messenger RNA metabolismus MeSH
- mikrofilamentové proteiny metabolismus MeSH
- molekulární chaperony metabolismus MeSH
- onkogenní proteiny metabolismus MeSH
- proteinkinasy fyziologie MeSH
- proteiny tepelného šoku HSP90 metabolismus MeSH
- regulace genové exprese MeSH
- světlo MeSH
- transkripční faktory MeSH
- transportní proteiny metabolismus MeSH
- tyrosinkinasy metabolismus účinky léků účinky záření MeSH
- Check Tag
- lidé MeSH
We investigated the effect of UVA-activated 8-methoxypsoralen (PUVA) on the cell line Karpas 299 derived from anaplastic large-cell lymphoma (ALCL) expressing chimeric fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM/ALK). NPM/ALK activates phosphatidylinositol 3 kinase (PI3K)/Akt pathway responsible for the cell protection from apoptosis. We found that PUVA treatment first induced G2/M cell cycle arrest resulting in a decrease in the cell proliferation rate. The mitochondrial apoptosis was triggered immediately following PUVA treatment, as we judged from the unmasking of mitochondrial membrane antigen 7A6. However, the mitochondrial membrane depolarization was not observed and caspase-3 was only slightly activated. The late apoptotic events were lacking: neither translocation of phosphatidylserine to the outer side of plasma membrane nor DNA fragmentation occurred. We revealed that PUVA enhanced the expression of peroxiredoxin, stress protein endoplasmin and galectin-3. Galectin-3 has been shown to protect mitochondrial membrane integrity and prevent cytochrome c release thereby blocking the effector stage of apoptosis. We suggest that the elevated level of this protein following PUVA treatment acts in synergy with the constitutively expressed chimeric kinase NPM/ALK to block the apoptosis.
- MeSH
- buněčné linie MeSH
- cytochromy c metabolismus MeSH
- financování organizované MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- G2 fáze účinky léků MeSH
- galektin 3 metabolismus MeSH
- kaspasa 3 metabolismus MeSH
- lymfom T-buněčný metabolismus patologie MeSH
- membránové glykoproteiny metabolismus MeSH
- membránové proteiny metabolismus MeSH
- methoxsalen farmakologie MeSH
- mitochondrie metabolismus MeSH
- peroxidasy metabolismus MeSH
- peroxiredoxiny MeSH
- proteiny buněčného cyklu účinky léků MeSH
- proteiny teplotního šoku metabolismus MeSH
- PUVA terapie škodlivé účinky MeSH
- tyrosinkinasy metabolismus MeSH
- ultrafialové záření MeSH
- MeSH
- buněčné jadérko fyziologie genetika MeSH
- buněčné jádro fyziologie genetika MeSH
- chromatin fyziologie izolace a purifikace MeSH
- histocytochemie metody využití MeSH
- karyometrie * metody využití MeSH
- krevní buňky * cytologie fyziologie MeSH
- lidé MeSH
- mikroskopie metody využití MeSH
- nádorové biomarkery izolace a purifikace MeSH
- počítačové zpracování obrazu metody využití MeSH
- RNA jaderná * izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.
- MeSH
- barvení stříbrem MeSH
- buněčné jadérko metabolismus MeSH
- buněčný cyklus fyziologie MeSH
- buňky K562 MeSH
- buňky kostní dřeně metabolismus MeSH
- DNA nádorová metabolismus MeSH
- financování organizované MeSH
- granulocyty metabolismus ultrastruktura MeSH
- HL-60 buňky MeSH
- leukemie metabolismus MeSH
- lidé MeSH
- organizátor jadérka metabolismus MeSH
- plazmatické buňky metabolismus ultrastruktura MeSH
- RNA nádorová metabolismus MeSH
- stárnutí buněk fyziologie MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Klíčová slova
- Imatinib mesylate,
- MeSH
- buňky K562 enzymologie účinky léků MeSH
- chronická myeloidní leukemie MeSH
- hybridizace in situ fluorescenční MeSH
- leukocyty mononukleární enzymologie účinky léků MeSH
- piperaziny terapeutické užití MeSH
- pyrimidiny terapeutické užití MeSH
- techniky in vitro MeSH
- tyrosinkinasy antagonisté a inhibitory MeSH
