The nucleolus represents a highly dynamic nuclear compartment of the interphase nucleus. It plays a key role in ribosome biogenesis. The number of nucleoli, their size, and their activity increase in exponentially growing cells; therefore these parameters reflect the proliferating activity of the cells. A variety of staining techniques have been employed to vizualize nucleolar changes in malignant cells. Staining of so-called nucleolar organizer regions (NORs), based upon a strong avidity of nucleolar proteins to bind silver ions, represents the technique most frequently used by pathologists. Nucleolar changes and pleomorphism associated with overt proliferation of tumor cells have also been documented by immunohistochemical and ultrastructural studies. Contrary to cell proliferation, cytostatics-induced changes of nucleolar phenotype in malignant cells point to a potential role of nucleolar components in the execution of active cell death. Recent studies have provided direct clues that so-called death domains and other apoptosis-related proteins are accumulated in nucleoli upon induction of active cell death. It can be concluded that the plurifunctionality of nucleoli regarding cell proliferation and apoptosis could open new vistas toward understanding dysregulation in malignant cells.

• MeSH
• apoptóza fyziologie   MeSH
• buněčné dělení MeSH
• buněčné jadérko patologie   fyziologie   ultrastruktura   MeSH
• lidé MeSH
• nádory patologie   MeSH
• signální transdukce fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.

• Klíčová slova
• Acute myeloid leukemia, Apoptosis, Nucleolin, Nucleophosmin, Phosphorylation, RITA, p53,
• MeSH
• akutní myeloidní leukemie * metabolismus   MeSH
• apoptóza MeSH
• buněčné jadérko metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• nádorový supresorový protein p53 * MeSH

The present experimental study was undertaken to provide information on nucleolar changes accompanying the apoptotic process in large or giant binucleate and multinucleate cells (LBMNCs). Such cells were present in a small but constant percentage in cultures of HL-60 cells. The apoptotic process was induced by photodynamic treatment (PDT) by means of 5-aminolaevulinic acid (ALA) as the precursor of the photosensitizer protoporphyrin IX and irradiation with broad spectrum blue light (BL). Nucleolar changes in LBMNCs were characterized by marked reduction or disappearance of silver stained particles representing AgNORs in nucleoli including the large ones. In addition, PDT also significantly reduced the number of nucleoli regardless of their size. These changes apparently reflected the decrease or cessation of nucleolar biosynthetic activities and resembled those which were previously observed in naturally maturing bone marrow megakaryocytes (Janoutová et al., 2001).

• MeSH
• apoptóza účinky léků   MeSH
• barvení stříbrem MeSH
• buněčné jadérko chemie   účinky léků   ultrastruktura   MeSH
• fotochemoterapie * MeSH
• fotosenzibilizující látky farmakologie   MeSH
• granulocyty účinky léků   patologie   MeSH
• HL-60 buňky účinky léků   patologie   MeSH
• kyselina aminolevulová farmakologie   MeSH
• leukemie farmakoterapie   patologie   MeSH
• lidé MeSH
• obrovské buňky účinky léků   ultrastruktura   MeSH
• organizátor jadérka chemie   účinky léků   ultrastruktura   MeSH
• RNA analýza   MeSH
• světlo MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• kyselina aminolevulová MeSH
• RNA MeSH

The present study was designed to provide complementary information on the effects of histone deacetylase inhibitors (HDACi's) such as trichostatin A (TSA) and sodium valproate (VAP) on nuclei and nucleoli of leukemic myeloblasts represented by cultured Kasumi-1 cells. The number of apoptotic cells and bodies with characteristic chromatin condensation and fragmentation was greater after TSA treatment. However, in contrast to TSA, myeloblasts treated with VPA recovered and started to proliferate again. TSA-treated myeloblasts with a fine chromatin structure exhibited an intense phagocytosis of cell fragments. The decreased number and translocation of silver-stained proteins of nucleolus organiser regions (AgNORs) in large nucleoli of myeloblasts treated with HDACi's indicated that these cells entered apoptosis and/or ageing without preceding terminal maturation. The nucleolar asynchrony observed in an increased number of treated cells with both HDACi's studied here possibly represented myeloblasts resistant to such treatment. In conclusion, this study demonstrates that the chromatin structure and nucleoli visualised by simple cytochemical procedures provides useful information on the effects of HDACi's on myeloblasts and facilitated detection of these effects at the single cell level.

• MeSH
• apoptóza účinky léků   MeSH
• buněčné jádro účinky léků   enzymologie   MeSH
• histondeacetylasy metabolismus   MeSH
• inhibitory histondeacetylas * MeSH
• inhibitory proteas farmakologie   MeSH
• kyselina valproová farmakologie   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• myeloidní leukemie enzymologie   patologie   MeSH
• nádorové buněčné linie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histondeacetylasy MeSH
• inhibitory histondeacetylas * MeSH
• inhibitory proteas MeSH
• kyselina valproová MeSH
• kyseliny hydroxamové MeSH
• trichostatin A MeSH Prohlížeč

The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis.

• MeSH
• acetylace MeSH
• aktivace enzymů MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• buněčné jadérko účinky léků   metabolismus   MeSH
• buněčný cyklus účinky léků   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• frakcionace buněk MeSH
• histony metabolismus   MeSH
• imunohistochemie MeSH
• kaspasy metabolismus   MeSH
• koncové značení zlomů DNA in situ MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory prsu metabolismus   patologie   MeSH
• nukleolin MeSH
• organizátor jadérka patologie   MeSH
• poly(ADP-ribosa)-polymerasy metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• puriny farmakologie   MeSH
• roskovitin MeSH
• velikost buňky účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• cyklin-dependentní kinasy MeSH
• fosfoproteiny MeSH
• histony MeSH
• kaspasy MeSH
• nádorové proteiny MeSH
• nádorový supresorový protein p53 MeSH
• poly(ADP-ribosa)-polymerasy MeSH
• proteiny vázající RNA MeSH
• puriny MeSH
• roskovitin MeSH

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

• Klíčová slova
• DNA damage, UBF1, live cells, nucleolus, nuncleoli tracking,
• MeSH
• apoptóza účinky záření   MeSH
• buněčné jadérko účinky záření   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky záření   MeSH
• G2 fáze účinky záření   MeSH
• genetická transkripce MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• poškození DNA účinky záření   MeSH
• transkripční iniciační komplex Pol1 - proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• výpočetní biologie MeSH
• záření gama škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

• Klíčová slova
• ACTINOMYCIN D, APOPTOSIS, LEUKEMIA, MUTATED NUCLEOPHOSMIN, P53-DEFICIENCY,
• MeSH
• apoptóza MeSH
• buněčné jadérko metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• daktinomycin farmakologie   MeSH
• HeLa buňky MeSH
• HL-60 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• leukemie genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• nukleofosmin MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• daktinomycin MeSH
• jaderné proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH

BACKGROUND: Since photodynamic treatment (PDT) induces apoptosis in individual HL-60 cells originating from early granulocytic precursors of acute myeloid leukemia, the present study was undertaken to provide information on such treatment of K562 cells, which originate from early granulocytic precursors of chronic myeloid leukemia (blastic phase) and carry the bcr/abl fusion gene with anti-apoptotic properties. MATERIAL/METHODS: PDT was based on the 5-aminolevulinic acid treatment of K562 cells, followed by blue light irradiation under conditions which in HL-60 cells induce an apoptotic process without previous terminal maturation. Nuclei and nucleoli were visualized by cytochemical procedures to demonstrate DNA, RNA and silver stained proteins of nucleolus organizer regions (AgNORs). TUNEL and propidium iodide assays were used for additional control of the incidence of apoptotic and necrotic cells. RESULTS: In contrast to HL-60 cells, PDT did not induce apoptosis in K562 cells. However, after PDT some K562 cells exhibited major alterations, expressed by nuclear and cell swelling, reflecting a necrotic process, confirmed by propidium iodide. In addition, PDT produced a reduction of AgNORs, though smaller than that previously described in apoptotic HL-60 cells. CONCLUSIONS: PDT produced only necrotic changes in some K562 cells under conditions which induced in apoptosis in HL-60 cells. Thus the induction of apoptosis, nuclear and nucleolar changes in individual cells does not depend on the inducer--PDT--but on the cell properties.

• MeSH
• apoptóza MeSH
• buněčné jadérko účinky léků   ultrastruktura   MeSH
• buněčné jádro účinky léků   ultrastruktura   MeSH
• buňky K562 MeSH
• chromatin ultrastruktura   MeSH
• chronická myeloidní leukemie farmakoterapie   patologie   MeSH
• fotosenzibilizující látky farmakologie   MeSH
• HL-60 buňky MeSH
• kmenové buňky účinky léků   MeSH
• kyselina aminolevulová farmakologie   MeSH
• lidé MeSH
• mitóza účinky léků   MeSH
• nekróza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• fotosenzibilizující látky MeSH
• kyselina aminolevulová MeSH

The present study was designed to provide more information on nucleoli in apoptotic cells,which were represented in the present study by cultured leukemic myeloblasts (Kasumi-1 cells). The apoptotic process in these cells was produced by trichostatin A (TSA) that is a histone deacetylase inhibitor with strong cytostatic effects. The selected TSA concentration added to cultures facilitated to study apoptotic and not-apoptotic cells in one and the same specimen. The nucleolar diameter and density were determined using computer assisted measurement and densitometry in specimens stained for RNA. In comparison with not-apoptotic cells, in apoptotic cells, nucleolar mean diameter did not change significantly and nucleolar RNA density was also not apparently different. On the other hand, the cytoplasmic RNA density in apoptotic cells was markedly reduced. Thus it seemed to be possible that the transcribed RNA remained "frozen"within the nucleolus but its transport to the cytoplasm decreased or stopped. However, the possibility of the RNA degradation in the cytoplasm of apoptotic cells based on the present study cannot be eliminated. At this occasion it should be added that AgNORs reflecting nucleolar biosynthetic and cell proliferation activity in apoptotic cells decreased in number or disappeared. The presented results also indicated that large nucleoli intensely stained for RNA need not be necessarily related to the high nucleolar biosynthetic or cell proliferation activity and may be also present in apoptotic cells responding to the cytostatic treatment.

• MeSH
• antigeny jaderné analýza   ultrastruktura   MeSH
• apoptóza * MeSH
• buněčné jadérko chemie   účinky léků   ultrastruktura   MeSH
• chronická myeloidní leukemie patologie   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• prekurzorové buňky granulocytů účinky léků   ultrastruktura   MeSH
• RNA analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny jaderné MeSH
• kyseliny hydroxamové MeSH
• nucleolar organizer region associated proteins MeSH Prohlížeč
• RNA MeSH
• trichostatin A MeSH Prohlížeč

The nuclear and nucleolar ultrastructure was studied by means of conventional transmission electron microscopy to provide more and complementary information on nucleolar changes accompanying the apoptotic process in leukaemic granulocytic precursors (HL-60 cells) produced by PDT without previous terminal differentiation. PDT induced the apoptotic process using BL irradiation and ALA as a precursor of the photosensitizer protoporphyrin IX. PDT produced marked changes of the nucleolar ultrastructure in apoptotic cells, such as reduction of the number and loss of fibrillar centres surrounding dense fibrillar components. Such nucleolar changes are known to reflect an alteration of nucleolar biosynthetic activities, which are believed to be located at the periphery of fibrillar centres. Some electron micrographs also indicated that fibrillar centres apparently migrated out from nucleolar bodies.

• MeSH
• apoptóza fyziologie   MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné jadérko účinky léků   metabolismus   ultrastruktura   MeSH
• fotosenzibilizující látky farmakologie   MeSH
• granulocyty cytologie   metabolismus   MeSH
• HL-60 buňky cytologie   účinky léků   MeSH
• kyselina aminolevulová farmakologie   MeSH
• lidé MeSH
• světlo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• kyselina aminolevulová MeSH