NANOG is a transcription factor involved in the regulation of pluripotency and stemness. The functional paralog of NANOG, NANOGP8, differs from NANOG in only three amino acids and exhibits similar reprogramming activity. Given the transcriptional regulatory role played by NANOG, the nuclear localization of NANOG/NANOGP8 has primarily been considered to date. In this study, we investigated the intriguing extranuclear localization of NANOG and demonstrated that a substantial pool of NANOG/NANOGP8 is localized at the centrosome. Using double immunofluorescence, the colocalization of NANOG protein with pericentrin was identified by two independent anti-NANOG antibodies among 11 tumor and non-tumor cell lines. The validity of these observations was confirmed by transient expression of GFP-tagged NANOG, which also colocalized with pericentrin. Mass spectrometry of the anti-NANOG immunoprecipitated samples verified the antibody specificity and revealed the expression of both NANOG and NANOGP8, which was further confirmed by real-time PCR. Using cell fractionation, we show that a considerable amount of NANOG protein is present in the cytoplasm of RD and NTERA-2 cells. Importantly, cytoplasmic NANOG was unevenly distributed at the centrosome pair during the cell cycle and colocalized with the distal region of the mother centriole, and its presence was markedly associated with centriole maturation. Along with the finding that the centrosomal localization of NANOG/NANOGP8 was detected in various tumor and non-tumor cell types, these results provide the first evidence suggesting a common centrosome-specific role of NANOG.

• Keywords
• NANOG, NANOGP8, centrosome, human, localization, mother centriole,
• MeSH
• Centrioles immunology   MeSH
• Centrosome immunology   MeSH
• Humans MeSH
• Nanog Homeobox Protein immunology   MeSH
• Cell Proliferation MeSH
• Transfection MeSH
• Transcription Factors MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NANOG protein, human MeSH Browser
• Nanog Homeobox Protein MeSH
• Transcription Factors MeSH

The p73 protein is a member of the p53 family, and this protein is known to be essential for the maintenance of genomic stability, DNA repair, and apoptosis regulation. Transcription from two promoters leads to two main N-terminal isoforms: the TAp73 isoform is reported to have tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The present study is focused on the investigation of a possible role of both these p73 N-terminal isoforms in the process of centrosome amplification. HGG-02 and GM7 glioblastoma cell lines and the Daoy medulloblastoma cell line were used in this study. The cells were analyzed using indirect immunofluorescence to determine TAp73 and ΔNp73 expression patterns and possible co-localization with the BubR1 protein, as well as the number of centrosomes. A transiently transfected GM7 cell line was used to verify the results concerning the N-terminal isoforms in relation to centrosome amplification. We found that increased immunoreactivity for the ΔNp73 isoform is associated with the occurrence of an abnormal number of centrosomes in particular cells. Using the transiently transfected GM7 cell line, we confirmed that centrosome amplification is present in cells with overexpression of the ΔNp73 isoform. In contrast, the immunoreactivity for the TAp73 isoform was weak or medium in most of the cells with an aberrant number of centrosomes. To determine the putative counterpart of the p73 N-terminal isoforms among spindle assembly checkpoint (SAC) proteins, we also evaluated possible interactions between the N-terminal isoforms and BubR1 protein, but no co-localization of these proteins was observed.

• Keywords
• BubR1, Centrosome amplification, Glioblastoma multiforme, Medulloblastoma, TAp73, ΔNp73,
• MeSH
• Gene Amplification * MeSH
• Centrosome physiology   MeSH
• Chromosome Aberrations * MeSH
• DNA-Binding Proteins genetics   MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Brain Neoplasms genetics   pathology   MeSH
• DNA Repair MeSH
• Promoter Regions, Genetic MeSH
• Protein Isoforms MeSH
• Tumor Protein p73 MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BUB1 protein, human MeSH Browser
• delta Np73 protein, human MeSH Browser
• DNA-Binding Proteins MeSH
• Nuclear Proteins MeSH
• Tumor Suppressor Proteins MeSH
• Protein Isoforms MeSH
• Tumor Protein p73 MeSH
• Protein Serine-Threonine Kinases MeSH
• TP73 protein, human MeSH Browser

CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

• MeSH
• AC133 Antigen MeSH
• Cell Nucleus metabolism   MeSH
• Antigens, CD immunology   metabolism   MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Glycoproteins immunology   metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal immunology   MeSH
• Biomarkers, Tumor metabolism   MeSH
• Cell Line, Tumor MeSH
• Peptides immunology   metabolism   MeSH
• Rhabdomyosarcoma metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• AC133 Antigen MeSH
• Antigens, CD MeSH
• Glycoproteins MeSH
• Antibodies, Monoclonal MeSH
• Biomarkers, Tumor MeSH
• Peptides MeSH
• PROM1 protein, human MeSH Browser
• Prom1 protein, mouse MeSH Browser

The crucial role of cancer stem cells (CSCs) in the pathology of malignant diseases has been extensively studied during the last decade. Nestin, a class VI intermediate filament protein, was originally detected in neural stem cells during development. Its expression has also been reported in different tissues under various pathological conditions. Specifically, nestin has been shown to be expressed in transformed cells of various human malignancies, and a correlation between its expression and the clinical course of some diseases has been proved. Furthermore, the coexpression of nestin with other stem cell markers was described as a CSC phenotype that was subsequently verified using tumorigenicity assays. The primary aim of this review is to summarize the recent findings regarding nestin expression in CSCs, its possible role in CSC phenotypes, particularly with respect to capacity for self-renewal, and its utility as a putative marker of CSCs.

• Keywords
• Cancer stem cells, cytoskeleton, intermediate filaments, nestin, tumor markers,
• MeSH
• Humans MeSH
• Biomarkers, Tumor metabolism   MeSH
• Neoplastic Stem Cells metabolism   MeSH
• Neoplasms metabolism   pathology   MeSH
• Nestin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Biomarkers, Tumor MeSH
• NES protein, human MeSH Browser
• Nestin MeSH

Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.

• MeSH
• Cell Nucleus chemistry   metabolism   ultrastructure   MeSH
• Child MeSH
• Fluorescent Antibody Technique MeSH
• Glioblastoma metabolism   ultrastructure   MeSH
• Immunohistochemistry MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Medulloblastoma metabolism   ultrastructure   MeSH
• Cell Line, Tumor MeSH
• Nestin MeSH
• Neuroblastoma metabolism   ultrastructure   MeSH
• Child, Preschool MeSH
• Intermediate Filament Proteins analysis   metabolism   MeSH
• Nerve Tissue Proteins analysis   metabolism   MeSH
• Aged MeSH
• Microscopy, Electron, Transmission MeSH
• Blotting, Western MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NES protein, human MeSH Browser
• Nestin MeSH
• Intermediate Filament Proteins MeSH
• Nerve Tissue Proteins MeSH

PURPOSE: The aim of this study was to perform a detailed cytogenetic and molecular genetic analysis of a tumor taken from a 14.5-year-old boy with glioblastoma multiforme who showed an atypical clinical course. METHODS: Formalin-fixed, paraffin embedded tumor tissue and the corresponding HGG-02 cell line derived from this tumor were analyzed using fluorescence in situ hybridization (FISH), G-banding, multiplex ligation-dependent probe amplification (MLPA), functional analysis of separated alleles in yeast (FASAY), immunohistochemistry (IHC), and immunocytochemistry (ICC). RESULTS: Mutation of the p53 gene and hypermethylation of the MLH1 gene were detected by FASAY and MLPA, respectively. Cytogenetic analysis showed a polyploid karyotype with extensive heterogeneity in chromosome number. Using FISH, we identified a very unusual genetic change - a loss of EGFR gene copy in both the tumor tissue and the HGG-02 cell line. In accordance with the cytogenetic findings, IHC and ICC did not demonstrate overexpression of EGFR in the tumor tissue or HGG-02 cells. CONCLUSIONS: Despite his very poor prognosis, the patient experienced 34 months of event-free survival after surgery and adjuvant radiotherapy and chemotherapy. The detected loss of the EGFR gene copy may contribute to the unusual biological features of this tumor, but the forthcoming detailed expression analysis of cancer regulatory pathways is necessary to better understand this tumor phenotype.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   MeSH
• ErbB Receptors genetics   MeSH
• Phenotype MeSH
• Gene Dosage * MeSH
• Genes, p53 MeSH
• Glioblastoma genetics   pathology   therapy   MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Adolescent MeSH
• Brain metabolism   pathology   MeSH
• Mutation MeSH
• MutL Protein Homolog 1 MeSH
• Cell Line, Tumor MeSH
• Brain Neoplasms genetics   pathology   therapy   MeSH
• Disease Progression MeSH
• Treatment Outcome MeSH
• Check Tag
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• EGFR protein, human MeSH Browser
• ErbB Receptors MeSH
• Nuclear Proteins MeSH
• MLH1 protein, human MeSH Browser
• MutL Protein Homolog 1 MeSH