Glial fibrillary acidic protein (GFAP) is a M(r) 50,000 intracytoplasmic filamentous
protein that constitutes a portion of, and is specific for the cytoskeleton of the astrocyte. GFAP appears to stabilize the astrocyte's cytoskeleton, and helps maintain normal astrocyte cell shape through complex interactions between it and the nuclear and plasma membranes. A critical observation from histopathological studies is that there is progressive loss of GFAP expression with increasing astrocytic
anaplasia. To determine how GFAP expression affects human malignant
astrocytoma cells that are GFAP-deficient, we have stably transfected GFAP-negative SF-126 human
astrocytoma cells with a eukaryotic expression vector into which a
complementary DNA for the entire coding sequence of the
human GFAP protein has been inserted. Positive transformants were selected for
neomycin resistance, and GFAP-positive
astrocytoma cells have been identified by indirect immunofluorescence using anti-GFAP
antibodies. Upon successful transfection, 5 stably transfected SF-126
astrocytoma cell clones were selected that produced GFAP, formed elongated processes in cell culture, and demonstrated decreased proliferation in a tritiated
thymidine uptake assay when compared to the control. In addition, the 5
astrocytoma cell clones demonstrated marked reduction in the number and growth of colonies in soft
agar when compared to the control. Southern analysis revealed extra copies of the GFAP
complementary DNA that had become integrated into the SF-126
astrocytoma cell genome. Northern analysis revealed variable expression of a 3.5-kilobase GFAP
mRNA transcript among the 5 cell clones. The amount of GFAP
mRNA expression and immunoreactivity by the stably transfected SF-126
astrocytoma cell clones was found to correlate inversely with
astrocytoma proliferation and growth in soft
agar. The restoration of GFAP expression to GFAP-negative human
astrocytoma cells has affected
tumor cell morphology, proliferation, and growth in soft
agar in this experimental cell system.