Characterization of glial filament-cytoskeletal interactions in human astrocytomas: an immuno-ultrastructural analysis.
The role that glial filaments play in cells and tumors of glial origin is not well understood. We therefore undertook the present study to determine the relationships between glial and vimentin intermediate filaments (IFs), actin microfilaments, and CD44, a cell surface glycoprotein important in cell migration and invasion, in human astrocytoma cells. Three astrocytoma cell lines, U343 MG-A (U343), U251 MG (U251), and antisense GFAP-transfected U251 (asU251) were studied using immunofluorescence confocal and immunoelectron microscopy. Furthermore, we studied the phenotypic behaviour of these astrocytoma cell lines by analyzing their migration through Matrigel in vitro. U343 astrocytoma cells had the highest expression levels of glial fibrillary acidic protein (GFAP), whereas asU251 had virtually no expression of GFAP. Parental U251 cells had intermediate expression levels of GFAP. The elimination of GFAP expression in as U251 cells was accompanied by a marked increase in vimentin, actin microfilaments and CD44 levels. Gold labeling density counts of cytoskeletal and cell surface elements demonstrated that the differences between GFAP, actin, CD44 and vimentin levels in the different astrocytoma cell lines were statistically significant (p < 0.05). Results from the in vitro invasion assay revealed that U343 cells demonstrated the least invasive potential, whereas asU251 astrocytoma cells demonstrated the most. Our results show that elimination of GFAP expression by antisense leads to marked alterations in cell morphology and phenotypic behaviour. These data imply that GFAP may be linked spatially and functionally to cytoskeletal elements which may be altered when this IF is deleted in astrocytomas.
Division of Neurosurgery, The Hospital for Sick Children, The University of Toronto, Ontario, Canada. rutka@sickkids.on.ca
J T
JT
Rutka
C
C
Ackerley
S L
SL
Hubbard
A
A
Tilup
P B
PB
Dirks
S
S
Jung
S
S
Ivanchuk
M
M
Kurimoto
A
A
Tsugu
L E
LE
Becker
D001254:Astrocytoma; D001672:Biocompatible Materials; D015153:Blotting, Western; D003094:Collagen; D003599:Cytoskeleton / Q000378:metabolism*; D004338:Drug Combinations; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006801:Humans; D007797:Laminin; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009361:Neoplasm Invasiveness; D009457:Neuroglia / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011509:Proteoglycans; D014407:Tumor Cells, Cultured
D001672:Biocompatible Materials; D004338:Drug Combinations; D005904:Glial Fibrillary Acidic Protein; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen