The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and β-catenin in cHL cells. Furthermore, LEF-1- and β-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• buněčná adheze genetika   MeSH
• buněčné linie MeSH
• chemokin CCL19 metabolismus   MeSH
• chemotaxe genetika   imunologie   MeSH
• endoteliální buňky metabolismus   MeSH
• Hodgkinova nemoc genetika   imunologie   metabolismus   patologie   MeSH
• lidé MeSH
• mezibuněčná komunikace * MeSH
• nádorové mikroprostředí * genetika   imunologie   MeSH
• patologická angiogeneze MeSH
• pohyb buněk genetika   MeSH
• signální dráha Wnt * MeSH
• transkripční faktor LEF-1 genetika   metabolismus   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• chemokin CCL19 MeSH
• transkripční faktor LEF-1 MeSH
• vaskulární endoteliální růstový faktor A MeSH

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

• MeSH
• biologické modely MeSH
• biopsie MeSH
• buněčná adheze genetika   MeSH
• dikobrazovití a urzonovití MeSH
• exprese genu MeSH
• frizzled receptory metabolismus   MeSH
• Hodgkinova nemoc diagnostické zobrazování   genetika   metabolismus   patologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• počítačová rentgenová tomografie MeSH
• pohyb buněk genetika   MeSH
• proliferace buněk MeSH
• protein dishevelled metabolismus   MeSH
• protein Wnt 5a genetika   metabolismus   MeSH
• rhoA protein vázající GTP metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• frizzled receptory MeSH
• FZD5 protein, human MeSH Prohlížeč
• protein dishevelled MeSH
• protein Wnt 5a MeSH
• rhoA protein vázající GTP MeSH

AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.

• MeSH
• B-lymfocyty metabolismus   MeSH
• CHO buňky MeSH
• chronická lymfatická leukemie metabolismus   MeSH
• Cricetulus MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• imunohistochemie MeSH
• konfokální mikroskopie MeSH
• křečci praví MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• posttranslační úpravy proteinů * účinky léků   MeSH
• průtoková cytometrie MeSH
• pseudopodia metabolismus   MeSH
• signální transdukce * účinky léků   MeSH
• sirotčí receptory podobné receptoru tyrosinkinasy chemie   genetika   metabolismus   MeSH
• transfekce MeSH
• transport proteinů MeSH
• ubikvitinace MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ROR1 protein, human MeSH Prohlížeč
• sirotčí receptory podobné receptoru tyrosinkinasy MeSH

Fibroblast growth factor 2 (FGF2) is one of the most studied growth factors to date. Most attention has been dedicated to the smallest, 18 kDa FGF2 variant that is released by cells and acts through activation of cell-surface FGF-receptor tyrosine kinases. There are, however, several higher molecular weight (HMW) variants of FGF2 that rarely leave their producing cells, are retained in the nucleus and act independently of FGF-receptors (FGFR). Despite significant evidence documenting the expression and intracellular trafficking of HMW FGF2, many important questions remain about the physiological roles and mechanisms of action of HMW FGF2. In this review, we summarize the current knowledge about the biology of HMW FGF2, its role in disease and areas for future investigation.

• MeSH
• fenotyp MeSH
• fibroblastový růstový faktor 2 genetika   metabolismus   MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U2 metabolismus   MeSH
• molekulová hmotnost MeSH
• protein - isoformy chemie   genetika   metabolismus   MeSH
• proteinový komplex SMN metabolismus   MeSH
• receptory fibroblastových růstových faktorů chemie   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• ribozomální proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• malý jaderný ribonukleoprotein U2 MeSH
• protein - isoformy MeSH
• proteinový komplex SMN MeSH
• receptory fibroblastových růstových faktorů MeSH
• ribosomal protein L6 MeSH Prohlížeč
• ribozomální proteiny MeSH
• splicing factor 3a MeSH Prohlížeč

OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND METHODS: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells. RESULTS AND CONCLUSIONS: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.

• MeSH
• biologické modely MeSH
• buněčná diferenciace * MeSH
• buněčný rodokmen * MeSH
• cyklin A metabolismus   MeSH
• cyklin D MeSH
• cyklin E metabolismus   MeSH
• cyklin-dependentní kinasa 4 metabolismus   MeSH
• cyklin-dependentní kinasa 6 metabolismus   MeSH
• cykliny metabolismus   MeSH
• embryo savčí cytologie   metabolismus   MeSH
• embryonální kmenové buňky metabolismus   MeSH
• G1 fáze MeSH
• inhibitor p27 cyklin-dependentní kinasy metabolismus   MeSH
• intracelulární prostor metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk MeSH
• S fáze MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK4 protein, human MeSH Prohlížeč
• cyklin A MeSH
• cyklin D MeSH
• cyklin E MeSH
• cyklin-dependentní kinasa 4 MeSH
• cyklin-dependentní kinasa 6 MeSH
• cykliny MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.

• MeSH
• antitumorózní látky farmakologie   MeSH
• biologické markery MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• exprese genu účinky léků   MeSH
• krevní proteiny farmakologie   MeSH
• kultivační média bez séra farmakologie   MeSH
• kultivované buňky MeSH
• myši MeSH
• neurony cytologie   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   fyziologie   MeSH
• responzivní elementy fyziologie   MeSH
• tretinoin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• biologické markery MeSH
• krevní proteiny MeSH
• kultivační média bez séra MeSH
• tretinoin MeSH

Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III beta-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes -fetoprotein and GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.

• MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• embryo savčí MeSH
• interleukin-6 farmakologie   MeSH
• leukemický inhibiční faktor MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• neurony cytologie   účinky léků   MeSH
• synergismus léků MeSH
• tretinoin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• interleukin-6 MeSH
• leukemický inhibiční faktor MeSH
• Lif protein, mouse MeSH Prohlížeč
• tretinoin MeSH

In mouse embryonic stem (mES) cells, the expression of p27 is elevated when differentiation is induced. Using mES cells lacking p27 we tested the importance of p27 for the regulation of three critical cellular processes: proliferation, differentiation, and apoptosis. Although cell cycle distribution, DNA synthesis, and the activity of key G1/S-regulating cyclin-dependent kinases remained unaltered in p27-deficient ES cells during retinoic acid-induced differentiation, the amounts of cyclin D2 and D3 in such cells were much lower compared with normal mES cells. The onset of differentiation induces apoptosis in p27-deficient cells, the extent of which can be reduced by artificially increasing the level of cyclin D3. We suggest that the role of p27 in at least some differentiation pathways of mES cells is to prevent apoptosis, and that it is not involved in slowing cell cycle progression. We also propose that the pro-survival function of p27 is realized via regulation of metabolism of D-type cyclin(s).

• MeSH
• apoptóza * MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• cyklin D2 MeSH
• cyklin D3 MeSH
• cykliny analýza   metabolismus   MeSH
• embryo savčí cytologie   MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH
• interfáze MeSH
• kmenové buňky cytologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové supresorové proteiny nedostatek   fyziologie   MeSH
• proteiny buněčného cyklu fyziologie   MeSH
• tretinoin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ccnd2 protein, mouse MeSH Prohlížeč
• Ccnd3 protein, mouse MeSH Prohlížeč
• Cdkn1b protein, mouse MeSH Prohlížeč
• cyklin D2 MeSH
• cyklin D3 MeSH
• cykliny MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH
• nádorové supresorové proteiny MeSH
• proteiny buněčného cyklu MeSH
• tretinoin MeSH

Electroporation represents a powerful technique for cell transfection; however, its efficiency in haemopoietic cells (approximately 1%) is largely unsatisfactory. Biological processes in haemopoietic cells are often studied using leukaemia cell line HL-60. For this reason we developed conditions for efficiently introducing plasmids to HL-60 cells by electroporation, as an alternative to other techniques. This technique employs the electric pulse (250-270 V; 1000 microF) followed by separation of living cells on a Ficoll-Paque discontinuous gradient. Using 10-20 micrograms of plasmid, we routinely achieve 12-14% of transfectants.

• MeSH
• elektroporace * MeSH
• HL-60 buňky MeSH
• lidé MeSH
• luminescentní proteiny metabolismus   MeSH
• plazmidy genetika   metabolismus   MeSH
• transfekce * MeSH
• viabilita buněk fyziologie   MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luminescentní proteiny MeSH
• zelené fluorescenční proteiny MeSH

The v-myb oncogene of avian myeloblastosis virus transforms myelomonocytic cells in vitro. The line of v-myb-transformed chicken monoblasts BM2 can be induced to terminal differentiation using phorbol esters. The fact that Jun proteins are up-regulated in the phorbol ester-treated BM2 cells prompted us to investigate the role of the Jun proteins in regulation of myeloid differentiation. We ectopically expressed v-jun and c-jun in BM2 cells and evaluated their effects on differentiation and proliferation. c-Jun up-regulated the transactivation activity of v-Myb and induced a proliferation block and differentiation of BM2 cells. In contrast, v-Jun down-regulated v-Myb transactivation causing no dramatic effects on BM2 cells. This confirms that there is no strong correlation between transcriptional activation and strength of oncogenic transformation by v-Myb. Both c-Jun and v-Jun proteins affected sensitivity of BM2 cells to retinoic acid and phorbol ester. Sensitivity of BM2 cells to retinoic acid was enhanced by both Jun proteins, while sensitivity to phorbol 12-myristate 13-acetate was reduced by v-Jun. These data suggest thate Jun plays a major role in macrophage differentiation.

• MeSH
• buněčné dělení fyziologie   MeSH
• buněčné linie MeSH
• buněčný cyklus fyziologie   MeSH
• časové faktory MeSH
• geny myb * MeSH
• kinetika MeSH
• kur domácí MeSH
• matrixové metaloproteinasy metabolismus   MeSH
• onkogenní protein p65(gag-jun) genetika   metabolismus   MeSH
• protoonkogenní proteiny c-jun genetika   metabolismus   MeSH
• respirační vzplanutí fyziologie   MeSH
• transformované buněčné linie * MeSH
• viabilita buněk fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• matrixové metaloproteinasy MeSH
• onkogenní protein p65(gag-jun) MeSH
• protoonkogenní proteiny c-jun MeSH