Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity.
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.
Rutka
J T
JT
Brain Tumour Research Laboratory, Hospital for Sick Children, Toronto, Ontario, Canada.
Smith
S L
SL
D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015139:Blotting, Southern; D002455:Cell Division; D004247:DNA / Q000032:analysis*; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D004247:DNA