Glial fibrillary acidic protein (GFAP) is an intermediate filament specifically expressed in glial cells which helps to maintain and stabilize the glial cytoskeleton. Interestingly, with increasing astrocytic anaplasia, there is typically progressive loss of GFAP expression. In in vitro model systems, most permanent glioma cell lines are GFAP-negative. 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 using 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 expression in glioma cell lines, we studied the methylation state of the GFAP gene in the same glioma cell lines using a methyl-sensitive restriction enzyme digest of tumour and control DNA. Our analysis revealed that the GFAP gene was hypermethylated in 2/2 GFAP- negative glioma cell lines but not in 4/4 GFAP-positive glioma cell lines. To determine if methylation of CpG islands contained within the GFAP promoter could repress GFAP transcription, we designed deletional constructs from a 6 kb fragment of the mouse GFAP promoter, methylated them using Msp I- and Hpa II-methylases, and tested their activity in a standard CAT assay. Our data suggest that methylation of a 2 kb segment of the mouse GFAP promoter is sufficient to inactivate GFAP transcription. Our results further imply that methylation-mediated repression of GFAP transcription may be one candidate mechanism to account for decreased GFAP expression in certain human malignant glioma cell lines.

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PubMedDate not supplied

The effect of pulsing electromagnetic field on bone ingrowth into a porous coated implant.

Electromagnetic stimulation is known to promote bone formation in cases of fracture and pseudoarthrosis. The possibility of using electromagnetic field to promote bone ingrowth into a porous coated implant was investigated in this study using Japanese albino rabbits. A titanium implant coated with beads of 250-300 microns in diameter around a stem of 1.7 mm in diameter was inserted into the humerus cavity of a Japanese albino rabbit. The humerus was stimulated by pulsing electromagnetic fields at 2 Gauss, 10Hz, 25 microseconds for 14 days. The circumference of grown bone into the implant was measured, and calculated in area equivalent for determination of the area of new bone. Bone ingrowth was significantly promoted by consecutive 14 days. The result suggests that electromagnetic stimulation is useful for achieving further bone ingrowth into a porous coated implant.

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