Medulloblastoma, the most prevalent brain tumor among children, requires a comprehensive understanding of its cellular characteristics for effective research and treatment. In this study, we focused on DAOY, a permanent cell line of medulloblastoma, and investigated the unique properties of DAOY cells when cultured as floating multicellular aggregates called spheres, as opposed to adherent monolayers. Through our comprehensive analysis, we identified distinct characteristics associated with DAOY spheres. Our findings demonstrate that DAOY spheres express markers for both neural stem cells, such as CD133 (PROM1), and differentiated neurons, exemplified by MAP2. Additionally, our investigation revealed that spheres-derived cells exhibit heightened resistance to ionizing radiation compared to adherent cells. Consequently, our results indicate that caution is advised when interpreting experimental results obtained from adherent cell cultures and extrapolating them to in vivo situations.

• Publikační typ
• časopisecké články MeSH

Prominin-1 (CD133) is a cholesterol-binding membrane glycoprotein selectively associated with highly curved and prominent membrane structures. It is widely recognized as an antigenic marker of stem cells and cancer stem cells and is frequently used to isolate them from biological and clinical samples. Recent progress in understanding various aspects of CD133 biology in different cell types has revealed the involvement of CD133 in the architecture and dynamics of plasma membrane protrusions, such as microvilli and cilia, including the release of extracellular vesicles, as well as in various signaling pathways, which may be regulated in part by posttranslational modifications of CD133 and its interactions with a variety of proteins and lipids. Hence, CD133 appears to be a master regulator of cell signaling as its engagement in PI3K/Akt, Src-FAK, Wnt/β-catenin, TGF-β/Smad and MAPK/ERK pathways may explain its broad action in many cellular processes, including cell proliferation, differentiation, and migration or intercellular communication. Here, we summarize early studies on CD133, as they are essential to grasp its novel features, and describe recent evidence demonstrating that this unique molecule is involved in membrane dynamics and molecular signaling that affects various facets of tissue homeostasis and cancer development. We hope this review will provide an informative resource for future efforts to elucidate the details of CD133's molecular function in health and disease.

• Klíčová slova
• CD133, Cancer, Cancer stem cell, Cell signaling, Cilium, Exosome, Lipid raft, Microvillus, Prominin-1, Stem cell,
• MeSH
• antigen AC133 metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fosfatidylinositol-3-kinasy * metabolismus   MeSH
• nádorové kmenové buňky metabolismus   MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antigen AC133 MeSH
• fosfatidylinositol-3-kinasy * MeSH

The heterogeneity of the glioma subtype glioblastoma multiforme (GBM) challenges effective neuropathological treatment. The reliance on in vitro studies and xenografted animal models to simulate human GBM has proven ineffective. Currently, a dearth of knowledge exists regarding the applicability of cell line biomolecules to the realm of GBM pathogenesis. Our study's objectives were to address this preclinical issue and assess prominin-1, ICAM-1, PARTICLE and GAS5 as potential GBM diagnostic targets. The methodologies included haemoxylin and eosin staining, immunofluorescence, in situ hybridization and quantitative PCR. The findings identified that morphology correlates with malignancy in GBM patient pathology. Immunofluorescence confocal microscopy revealed prominin-1 in pseudo-palisades adjacent to necrotic foci in both animal and human GBM. Evidence is presented for an ICAM-1 association with degenerating vasculature. Significantly elevated nuclear PARTICLE expression from in situ hybridization and quantitative PCR reflected its role as a tumor activator. GAS5 identified within necrotic GBM validated this potential prognostic biomolecule with extended survival. Here we present evidence for the stem cell marker prominin-1 and the chemotherapeutic target ICAM-1 in a glioma animal model and GBM pathology sections from patients that elicited alternative responses to adjuvant chemotherapy. This foremost study introduces the long non-coding RNA PARTICLE into the context of human GBM pathogenesis while substantiating the role of GAS5 as a tumor suppressor. The validation of GBM biomarkers from cellular models contributes to the advancement towards superior detection, therapeutic responders and the ultimate attainment of promising prognoses for this currently incurable brain cancer.

• Klíčová slova
• Biomarker, Eker rats, GAS5, GBM, Glioma, ICAM-1, PARTICLE, U87MG, lncRNA, prominin-1,
• Publikační typ
• časopisecké články MeSH

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

• MeSH
• lidé MeSH
• meduloblastom * genetika   MeSH
• myši MeSH
• nádory mozečku * genetika   MeSH
• nádory mozku * MeSH
• proteiny nervové tkáně MeSH
• proteiny vázající RNA genetika   MeSH
• proteomika MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• MSI1 protein, human MeSH Prohlížeč
• proteiny nervové tkáně MeSH
• proteiny vázající RNA MeSH

The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.

• Klíčová slova
• ER stress, UPR, mRNA translation, p47, p53,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

PURPOSE: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic-MD; enzymatic-ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. METHODS: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. RESULTS: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. CONCLUSIONS: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation.

• Klíčová slova
• cell isolation, glioblastoma multiforme, resistance, temozolomide,
• MeSH
• alkylační protinádorové látky farmakologie   MeSH
• chemorezistence MeSH
• glioblastom farmakoterapie   genetika   patologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory mozku farmakoterapie   genetika   patologie   MeSH
• pohyb buněk * účinky léků   MeSH
• proliferace buněk * účinky léků   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• screeningové testy protinádorových léčiv MeSH
• separace buněk metody   MeSH
• temozolomid farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkylační protinádorové látky MeSH
• temozolomid MeSH

In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CatK) are expressed in hypoxic GSC niches around arterioles in five human glioblastoma samples. In HSC niches, HSCs are retained by binding of SDF-1α and OPN to their receptors CXCR4 and CD44, respectively. Protease CatK cleaves SDF-1α to release HSCs out of niches. The aim of the present study was to reproduce the immunohistochemical localization of these GSC markers in 16 human glioblastoma samples with the addition of three novel markers. Furthermore, we assessed the type of blood vessels associated with GSC niches. In total, we found seven GSC niches containing CD133-positive and nestin-positive GSCs as a single-cell layer exclusively around the tunica adventitia of 2% of the CD31-positive and SMA-positive arterioles and not around capillaries and venules. Niches expressed SDF-1α, CXCR4, CatK, OPN, CD44, hypoxia-inducible factor-1α, and vascular endothelial growth factor. In conclusion, we show that GSC niches are present around arterioles and express bone marrow HSC niche proteins.

• Klíčová slova
• arterioles, blood vessels, bone marrow niches, glioma stem cell, hematopoietic stem cell, niches,
• MeSH
• antigeny CD44 analýza   MeSH
• arterioly patologie   MeSH
• barvení a značení metody   MeSH
• chemokin CXCL12 analýza   MeSH
• dospělí MeSH
• glioblastom krevní zásobení   patologie   MeSH
• hematopoetické kmenové buňky patologie   MeSH
• imunohistochemie metody   MeSH
• kathepsin K analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové kmenové buňky patologie   MeSH
• nádory mozku krevní zásobení   patologie   MeSH
• nika kmenových buněk * MeSH
• osteopontin analýza   MeSH
• receptory CXCR4 analýza   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD44 MeSH
• chemokin CXCL12 MeSH
• CXCR4 protein, human MeSH Prohlížeč
• kathepsin K MeSH
• osteopontin MeSH
• receptory CXCR4 MeSH
• SPP1 protein, human MeSH Prohlížeč

Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas.

• Klíčová slova
• cancer stem cells, drug discovery, genetic alterations, glioblastoma, primary cell culture,
• MeSH
• apoptóza MeSH
• buněčné kultury metody   MeSH
• glioblastom farmakoterapie   metabolismus   patologie   MeSH
• kultivační média bez séra farmakologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• myši MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádorové kmenové buňky účinky léků   metabolismus   patologie   MeSH
• nádory mozku farmakoterapie   metabolismus   patologie   MeSH
• prognóza MeSH
• proliferace buněk MeSH
• protinádorové látky farmakologie   MeSH
• techniky in vitro MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média bez séra MeSH
• nádorové biomarkery MeSH
• protinádorové látky MeSH

Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through its action as a histone deacetylase inhibitor. CD133 is considered to be a cancer stem cell marker in several tumors including neuroblastoma. CD133 transcription is strictly regulated by epigenetic modifications. We evaluated the epigenetic effects of treatment with 1mM VPA and its influence on the expression of CD133 in four human neuroblastoma cell lines. Chemoresistance and cell cycle of CD133+ and CD133- populations were examined by flow cytometry. We performed bisulfite conversion followed by methylation-sensitive high resolution melting analysis to assess the methylation status of CD133 promoters P1 and P3. Our results revealed that VPA induced CD133 expression that was associated with increased acetylation of histones H3 and H4. On treatment with VPA and cytostatics, CD133+ cells were mainly detected in the S and G2/M phases of the cell cycle and they showed less activated caspase-3 compared to CD133- cells. UKF-NB-3 neuroblastoma cells which express CD133 displayed higher colony and neurosphere formation capacities when treated with VPA, unlike IMR-32 which lacks for CD133 protein. Induction of CD133 in UKF-NB-3 was associated with increased expression of phosphorylated Akt and pluripotency transcription factors Nanog, Oct-4 and Sox2. VPA did not induce CD133 expression in cell lines with methylated P1 and P3 promoters, where the CD133 protein was not detected. Applying the demethylating agent 5-aza-2'-deoxycytidine to the cell lines with methylated promoters resulted in CD133 re-expression that was associated with a drop in P1 and P3 methylation level. In conclusion, CD133 expression in neuroblastoma can be regulated by histone acetylation and/or methylation of its CpG promoters. VPA can induce CD133+ cells which display high proliferation potential and low sensitivity to cytostatics in neuroblastoma. These results give new insight into the possible limitations to use VPA in cancer therapy.

• MeSH
• antigen AC133 metabolismus   MeSH
• buněčný cyklus účinky léků   MeSH
• cytostatické látky farmakologie   MeSH
• fluorescenční protilátková technika MeSH
• kaspasa 3 metabolismus   MeSH
• kyselina valproová farmakologie   MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buněčné linie MeSH
• neuroblastom farmakoterapie   MeSH
• protinádorové látky farmakologie   MeSH
• průtoková cytometrie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigen AC133 MeSH
• cytostatické látky MeSH
• kaspasa 3 MeSH
• kyselina valproová MeSH
• nádorové biomarkery MeSH
• protinádorové látky MeSH

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.

• MeSH
• buněčné sféroidy patologie   MeSH
• Cytomegalovirus fyziologie   MeSH
• dospělí MeSH
• fenotyp MeSH
• glioblastom mortalita   patologie   virologie   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorová transformace buněk metabolismus   MeSH
• nádorové kmenové buňky virologie   MeSH
• nádory mozku mortalita   patologie   virologie   MeSH
• přežití bez známek nemoci MeSH
• primární buněčná kultura MeSH
• prognóza MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH