The immunocytochemical demonstration of
intermediate filament proteins in three human
neuroepithelial tumor cell lines maintained in vitro on a three-dimensional matrix was correlated with the proportion of cells in S phase. The cell lines of a
medulloblastoma (D283 Med), a
retinoblastoma (WERI-Rb1), and an
astrocytic glioma (U-251 MG) were cultured in an organ culture system, pulse-fed with
bromodeoxyuridine, and double-labeled by immunoperoxidase and by the
avidin-
biotin peroxidase complex method for
bromodeoxyuridine and for
intermediate filament proteins [each triplet of
neurofilament proteins, as well as
vimentin and glial fibrillary acidic (
GFA) protein] using eight different
antibodies. The average percentages of
bromodeoxyuridine-labeled cells for the D283 Med, WERI-Rb1, and U-251 MG lines were respectively, 25, 32, and 12% 30 minutes after pulse labeling. In the D283 Med line, 15- greater than 95% of the cells were positive for each
neurofilament protein, and 80% of the cells were positive for
vimentin; less than 10% of the cells in S phase were positive with each of the five antineurofilament
protein monoclonal antibodies (Mabs), but 20% of the
vimentin-positive cells were in S phase. In the WERI-Rb1 line, 44 and greater than 96% of the cells were positive for the high-molecular-weight neurofilament subunit and high- and middle-molecular-weight neurofilament subunits
proteins, respectively, but only 5% of the high-molecular-weight neurofilament positive cells were in S phase. In the U-251 MG line, 37 and 98% of the cells were positive for
GFA protein and
vimentin, respectively; only 3% of the
GFA protein-positive cells, but 13% of the
vimentin-positive cells, were in S phase. The results indicate that, when maintained in a matrix culture system, most cells in S phase in these lines lack markers of neural differentiation.