Downregulation of RUNX3 and TES by hypermethylation in glioblastoma.
Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Duplicates of three short-term cultured glioblastomas were exposed to 5 microM 5-aza-dC for 96 h followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, reverse transcription-polymerase chain reaction (RT-PCR), bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor-suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis.
Mueller
W
W
Department of Pathology, Cancer Center and Neurosurgical Service, Massachusetts General Hospital, Boston, MA 02114, USA.
Nutt
C L
CL
Ehrich
M
M
Riemenschneider
M J
MJ
von Deimling
A
A
van den Boom
D
D
Louis
D N
DN
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics* / Q000378:metabolism; D003598:Cytoskeletal Proteins; D019175:DNA Methylation; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics* / Q000473:pathology; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D050677:Core Binding Factor Alpha 3 Subunit; D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D016601:RNA-Binding Proteins; C493729:Runx3 protein, human; C429688:TES protein, human; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D001374:Azacitidine