Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.

• MeSH
• acetylace MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• biotest metody   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• oprava DNA MeSH
• osteosarkom genetika   metabolismus   patologie   MeSH
• poškození DNA MeSH
• proteinfosfatasa 2C genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The main objective of this study was to analyze changes in the antiproliferative effect of vitamin D3, in the form of calcitriol and calcidiol, via its combined application with all-trans retinoic acid (ATRA) in osteosarcoma cell lines. The response to treatment with calcitriol and calcidiol alone was specific for each cell line. Nevertheless, we observed an enhanced effect of combined treatment with ATRA and calcitriol in the majority of the cell lines. Although the levels of respective nuclear receptors did not correlate with the sensitivity of cells to these drugs, vitamin D receptor (VDR) upregulation induced by ATRA was found in cell lines that were the most sensitive to the combined treatment. In addition, all these cell lines showed high endogenous levels of retinoic acid receptor α (RARα). Our study confirmed that the combination of calcitriol and ATRA can achieve enhanced antiproliferative effects in human osteosarcoma cell lines in vitro. Moreover, we provide the first evidence that ATRA is able to upregulate VDR expression in human osteosarcoma cells. According to our results, the endogenous levels of RARα and VDR could be used as a predictor of possible synergy between ATRA and calcitriol in osteosarcoma cells.

• MeSH
• alfa receptor kyseliny retinové metabolismus   MeSH
• antitumorózní látky aplikace a dávkování   MeSH
• kalcifediol aplikace a dávkování   MeSH
• kalcitriol aplikace a dávkování   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• osteosarkom farmakoterapie   metabolismus   MeSH
• protokoly antitumorózní kombinované chemoterapie MeSH
• receptory kalcitriolu metabolismus   MeSH
• tretinoin aplikace a dávkování   MeSH
• vitaminy aplikace a dávkování   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Some epidemiological studies suggested caffeine consumption as the cause for bone mineral density loss. Certain genes involved in this process are regulated by vitamin D receptor (VDR). Therefore, we investigated if caffeine can affect inducible expression of VDR-regulated genes, some of them being involved in bone mineralization process. By employing reporter gene assay, polymerase chain reaction, and western blotting, we monitored the VDR activity and expression in cell cultures of intestinal (LS180), osteosarcoma (HOS), and normal human osteoblasts in vitro. While caffeine stimulated calcitriol-inducible VDR-dependent nanoluciferase activity in stable reporter cell line IZ-VDRE (derived from LS180), it rather modulated mRNA levels of target genes, like CYP24A1, BGLAP, SPP1, and TNSF11 in LS180 and HOS cells. However, caffeine significantly decreased calcitriol-inducible CYP24A1, TNSF11, and SPP1 transcripts in osteoblasts. This decrease had non-linear U-shaped profile. Our in vitro data demonstrate biphasic action of caffeine on the expression of certain calcitriol-inducible VDR-regulated genes in normal human osteoblasts.

• MeSH
• kalcitriol farmakologie   MeSH
• kofein farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• osteoblasty účinky léků   metabolismus   MeSH
• osteosarkom farmakoterapie   metabolismus   MeSH
• receptory kalcitriolu účinky léků   metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• vitamin D metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin β and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.

• MeSH
• 53BP1 genetika   metabolismus   MeSH
• aktivní transport - buněčné jádro MeSH
• arginin chemie   genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• jaderné lokalizační signály * MeSH
• karyoferiny genetika   metabolismus   MeSH
• lidé MeSH
• lysin chemie   genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• osteosarkom genetika   metabolismus   patologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The three most frequent pediatric sarcomas, i.e., Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas-ABCG2, CD133, and nestin-was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.

• MeSH
• ABC transportér z rodiny G, člen 2 metabolismus   MeSH
• antigen AC133 metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši MeSH
• nádorová transformace buněk metabolismus   patologie   MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• nádorové proteiny metabolismus   MeSH
• nestin metabolismus   MeSH
• osteosarkom metabolismus   patologie   MeSH
• předškolní dítě MeSH
• rhabdomyosarkom metabolismus   patologie   MeSH
• sarkom metabolismus   patologie   MeSH
• transkripční faktory SOXB1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• myši MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.

• MeSH
• apoptóza MeSH
• chromatin genetika   MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• imunoenzymatické techniky MeSH
• imunoprecipitace MeSH
• interakční proteinové domény a motivy MeSH
• kur domácí genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• MCM komplex, komponenta 2 genetika   metabolismus   MeSH
• MCM komplex, komponenta 7 genetika   metabolismus   MeSH
• MCM proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• nádorové buňky kultivované MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• osteosarkom genetika   metabolismus   patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• povrchová plasmonová rezonance MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• replikace DNA * MeSH
• replikační počátek genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Nestin has been detected in various malignancies and its expression correlates with advanced grade in some neoplasms. The aim of this study was to examine nestin expression in high-grade osteosarcomas and to determine its prognostic value. Using immunohistochemistry and immunofluorescence, we evaluated nestin expression in tumor tissue samples from 45 patients with high-grade osteosarcomas. In both methods, the frequency of nestin-positive tumor cells was classified into three categories (1+, 2+ and 3+ for immunohistochemistry; 1F+, 2F+ and 3F+ for immunofluorescence) and clinicopathological correlations were statistically evaluated and analyzed. Nestin-positive tumor cells were detected in all of the examined osteosarcomas using both immunohistochemistry and immunofluorescence, although the proportion of undoubtedly positive neoplastic cells varied in individual samples from a few nestin-positive tumor cells to diffuse nestin positivity. High levels of nestin expression detected by immunofluorescence (2F+ and 3F+) were associated with worse clinical outcomes (OS, p=0.031; EFS, p<0.001). However, high levels of nestin expression as measured by immunohistochemistry trended towards shorter patient survival rates but did not reach statistical significance. Despite significantly shorter survival rates observed in patients with high levels of nestin expression assessed by immunofluorescence, nestin does not seem to represent a powerful prognostic marker that would be superior to conventional methods.

• MeSH
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádory kostí metabolismus   mortalita   patologie   MeSH
• osteosarkom metabolismus   mortalita   patologie   MeSH
• prognóza MeSH
• proteiny intermediálních filament genetika   metabolismus   MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stupeň nádoru MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.

• MeSH
• 2D gelová elektroforéza MeSH
• apoptóza fyziologie   MeSH
• dítě MeSH
• DNA vazebné proteiny metabolismus   MeSH
• dospělí MeSH
• fluorescenční protilátková technika MeSH
• imunohistochemie MeSH
• jaderné proteiny metabolismus   MeSH
• koncové značení zlomů DNA in situ MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• nádorový supresorový protein p53 nedostatek   MeSH
• nádory kostí * genetika   metabolismus   MeSH
• oprava DNA MeSH
• osteosarkom * genetika   metabolismus   MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• regulace genové exprese u nádorů * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• transkripční faktor E2F1 * genetika   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH

This study was designed to investigate the association of MTHFR C677T polymorphism and the risk of two common musculoskeletal sarcomas, osteosarcoma and chondrosarcoma. MTHFR genotypes were determined in 56 patients (44 osteosarcoma, 12 chondrosarcoma) and 44 controls using the PCR-RFLP technique. In the gender subgroup analysis, wild-type A allele frequency was higher in male osteosarcoma patients than in male control subjects (P = 0.064). Mutant V allele and mutant VV genotype were similar in the control group compared to the sarcoma groups (P > 0.05). No correlation could be proved between patient tumour site, presence of metastasis, and local tumour relapse and MTHFR polymorphism. The MTHFR C677T polymorphism may not be important in an individual's susceptibility to osteosarcoma and chondrosarcoma in Turkey and may not be a useful marker for identifying patients at high risk of developing sarcomas.

• MeSH
• alely MeSH
• chondrosarkom genetika   metabolismus   MeSH
• dospělí MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• methylentetrahydrofolátreduktasa (NADPH2) genetika   metabolismus   MeSH
• mladiství MeSH
• osteosarkom genetika   metabolismus   MeSH
• polymorfismus genetický MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.

• MeSH
• apoptóza MeSH
• Arabidopsis metabolismus   MeSH
• buněčný cyklus MeSH
• chronická myeloidní leukemie metabolismus   patologie   MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   metabolismus   MeSH
• cytokininy antagonisté a inhibitory   metabolismus   MeSH
• cytoskelet MeSH
• financování organizované MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory kostí metabolismus   patologie   MeSH
• nádory prsu metabolismus   patologie   MeSH
• osteosarkom metabolismus   patologie   MeSH
• proliferace buněk účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• průtoková cytometrie MeSH
• pyrimidiny farmakologie   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• transportní proteiny MeSH
• Check Tag
• lidé MeSH