Glioblastoma is the most common and the most aggressive type of brain cancer. Aberrations of the RTK/RAS/PI3K-, p53-, and RB cell signaling pathways were recognized as a core requirement for pathogenesis of glioblastoma. The p53 tumor suppressor functions as a transcription factor transactivating expression of its target genes in response to various stress stimuli. We determined the p53 status in 36 samples of glioblastoma by functional analyses FASAY and split assay. Seventeen p53 mutations were detected and further analyzed by cDNA and gDNA sequencing in 17 patients (47.2 %). Fifteen (88.2 %) of the mutations were missense mutations causing amino acid substitutions, seven of them exhibited temperature-sensitivity. Two mutations were determined as short deletions, one of them causing formation of premature termination codon in position 247. Fluorescent in situ hybridization revealed the loss of the p53-specific 17p13.3 locus in four of 33 analyzed samples (12 %). In 12 out of 30 samples (40 %), the p53 protein accumulation was shown by immunoblotting. There was high (80 %) concordance between the presence of the clonal p53 mutation and the p53 protein accumulation.

• MeSH
• Gene Deletion MeSH
• Genes, p53 * MeSH
• Glioblastoma genetics   metabolism   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Yeasts MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Brain Neoplasms genetics   metabolism   MeSH
• Organ Specificity MeSH
• Sequence Analysis, DNA MeSH
• Aged MeSH
• Temperature MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser

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

Tumor suppressor p53 is transcription factor that participates in control of many cellular functions. Somatic mutations of the p53 gene are frequently detected in human cancers. Several methods can be used for identification of p53 mutations, including FASAY - functional analysis of separated alleles in yeast. FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. The validity of the method depends on a low background level. There are several sources of background as PCR-induced point mutations, low quality of RNA and alternative splicing of intron 9 affecting the p53 carboxy-terminus. In the present work we show that FASAY can be successfully used for analysis of mRNA isolated from blood samples that were collected and stored for 24 hours at 0 degrees C without undesired increase of background. We also measured fidelity of several commonly used DNA polymerases and determined the most suitable kinds of Pfu DNA polymerases for FASAY. Reaction conditions described in this report allow routine analysis of p53 status in leukemic cells using FASAY.

• MeSH
• Alleles MeSH
• Alternative Splicing MeSH
• Point Mutation MeSH
• DNA Primers MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• Genetic Techniques MeSH
• Genes, p53 * MeSH
• Introns MeSH
• Leukemia genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Mutagenesis, Site-Directed MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Polymerase Chain Reaction methods   MeSH
• Reproducibility of Results MeSH
• RNA genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• DNA-Directed DNA Polymerase MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• RNA MeSH