title: Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas.
abstract: Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.
authors.affiliation: Division of Neurosurgery, Arthur and Sonia Labatt Brain Tumor Research Centre, Program in Cell Biology, Suite 1503, The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
authors.forename: Andres
authors.initials: A
authors.lastname: Restrepo
authors.forename: Christian A
authors.initials: CA
authors.lastname: Smith
authors.forename: Sameer
authors.initials: S
authors.lastname: Agnihotri
authors.forename: Maryam
authors.initials: M
authors.lastname: Shekarforoush
authors.forename: Paul N
authors.initials: PN
authors.lastname: Kongkham
authors.forename: Ho Jun
authors.initials: HJ
authors.lastname: Seol
authors.forename: Paul
authors.initials: P
authors.lastname: Northcott
authors.forename: James T
authors.initials: JT
authors.lastname: Rutka
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D057890:Epigenomics; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics* / Q000378:metabolism; D005910:Glioma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D008156:Luciferases