ANALYSIS OF GLIAL FIBRILLARY ACIDIC PROTEIN GENE METHYLATION IN HUMAN-MALIGNANT GLIOMAS

Glial fibrillary acidic protein (GFAP) is an intermediate filament spe cifically expressed in glial cells which helps to maintain and stabili ze the glial cytoskeleton. Interestingly, with increasing astrocytic a naplasia, there is typically progressive loss of GFAP expression. In i n vitro model systems, most permanent glioma cell lines are GFAP-negat ive. To determine the mechanism by which the transcription of the GFAP gene may be repressed in glioma cell lines, we initially performed a Southern analysis on a panel of human malignant glioma cell lines ruin g a human cDNA probe for GFAP. By this method no large rearrangements or deletions of the GFAP gene were found. Postulating that a change in methylation status of the GFAP gene could conceivably alter its expre ssion in glioma cell lines, we studied the methylation state of the GF AP gene in the same glioma cell lines using a methyl-sensitive restric tion enzyme digest of tumour and control DNA. Our analysis revealed th at the GFAP gene was hypermethylated in 2/2 GFAP-negative glioma cell lines brit not in 4/4 GFRP-positive glioma cell lines. To determine if methylation of CpG islands contained within the GFAP promoter could r epress GFAP transcription, we designed deletional constructs from a 6 kb fragment of the mouse GFAP promoter; methylated them using Msp I- a nd Hpa II-methylases, and tested their activity in a standard CAT assa y. Our data suggest that methylation of a 2 kb segment of the mouse GF AP promoter is sufficient to inactivate GFAP transcription. Our result s further imply that methylation-mediated repression of GFAP transcrip tion may be one candidate mechanism to account for dea eased GFRP expr ession in certain human malignant glioma cell lines.