Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods. Copyright 2007 S. Karger AG, Basel.

• MeSH
• dítě MeSH
• financování organizované MeSH
• hybridizace nukleových kyselin MeSH
• indoly MeSH
• krevní buňky cytologie   účinky léků   MeSH
• lidé MeSH
• metafáze účinky léků   MeSH
• neuroblastom genetika   patologie   MeSH
• odběr biologického vzorku metody   MeSH
• proteasy farmakologie   MeSH
• spektrální karyotypizace metody   MeSH
• stárnutí buněk účinky léků   MeSH
• Wilmsův nádor genetika   patologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH

BACKGROUND: Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. METHODS: Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. RESULTS: Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. CONCLUSION: Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential.

• MeSH
• cytoskelet chemie   ultrastruktura   MeSH
• financování organizované MeSH
• fluorescenční protilátková technika nepřímá MeSH
• glioblastom genetika   chemie   patologie   ultrastruktura   MeSH
• gliový fibrilární kyselý protein analýza   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteiny intermediálních filament analýza   MeSH
• proteiny nervové tkáně analýza   MeSH
• vimentin analýza   MeSH
• Check Tag
• lidé MeSH