We established and characterized five cell lines derived from human malignant gliomas (four glioblastomas multiforme and one highly anaplastic astrocytoma). All cell lines exhibited tumor cell morphology and growth kinetics, and anchorage-independent growth in soft agar. Cytogenetic analysis revealed significant aneuploidy in all five cases as well as clonal chromosomal alterations unique to each cell line. No cell line was tumorigenic in athymic mice. Two of the cell lines were sensitive to carmustine (BCNU) in monolayer and soft-agar cultures. Electron microscopy showed marked variability between cell lines in the number and structure of intracytoplasmic organelles; SF-126 formed collagen fibers in vitro. Immunohistochemical analysis of the surgical specimens showed variable expression of glial fibrillary acidic protein (GFAP) in malignant astrocytes; positive immunostaining for glycoproteins of the extracellular matrix was found predominantly in perivascular regions. In early-passage cultures, only cell line SF-295 expressed GFAP; at establishment, none of the cell lines expressed GFAF or glutamine synthetase. Fibronectin and laminin were expressed by all cell lines in early-passage culture, but expression of these glycoproteins at establishment was variable. Only SF-126 was positively identified by immunostains for procollagen III; this was also the only cell line in which DEAE-cellulose chromatography and SDS-PAGE demonstrated interstitial collagen synthesis. These well-characterized glioma-derived cell lines may now serve as useful tools with which to study the cell biology of gliomas. The synthesis of interstitial collagen by a glioma-derived cell line may suggest a derivation from vascular mesenchymal elements, either reactive or transformed, in the original heterogeneous malignant glioma, rather than from a glial precursor cell.

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We studied the effect of retinoids on the growth and differentiation of a cell line (U 343 MG-A) derived from a human malignant astrocytoma. Cultures treated with all-trans or 13-cis retinoic acid showed a dose-dependent inhibition of proliferation and a marked reduction in the mean cell number at the plateau phase of growth (3.5 x 10(6) vs. 1 x 10(7) cells/25 cm2) compared with untreated cultures. At confluence, cells treated with all-trans or 13-cis retinoic acid were contact-inhibited, whereas control cultures showed crowding, piling, and overgrowth. All-trans retinol or retinyl acetate did not inhibit growth. Astrocytoma cultures treated with all-trans retinoic acid (10(-6) M) for 5 days were modestly growth-inhibited but by day 16 had the same numbers of cells as controls; cultures that received all-trans retinoic acid for 9 days were markedly growth-inhibited for 7 days after the drug was removed. All-trans and 13-cis retinoic acid (10(-6) M) prevented the EDTA-induced cell detachment seen in control cultures. Strongly adherent all-trans retinoic-acid-treated astrocytoma cells grew at a slower rate than did readily detached all-trans retinoic-acid-treated or control cells. Cell spreading, an increased cytoplasmic:nuclear ratio, and greater numbers of broadly bipolar cells, some bearing thin cytoplasmic processes, were seen in cultures treated with 10(-6) M all-trans or 13-cis retinoic acid. Small tightly packed cuboidal cells and large broadly bipolar cells were seen in astrocytoma cultures from which all-trans retinoic acid was removed on days 5 and 9. Indirect immunofluorescence revealed more intense staining with antiserum to glial fibrillary acidic protein in cultures treated with 10(-6) M all-trans retinoic acid than in control cultures; electron-microscope examination of similarly treated cultures revealed more abundant 8-10 nm intermediate filaments than in control cultures. An enzyme-linked immunosorbent assay showed that all-trans or 13-cis retinoic acid caused a dose-dependent increase in the quantity of glial fibrillary acidic protein in the astrocytoma cells.

To determine whether tumor necrosis factor is of potential value for the treatment of human malignant gliomas, we studied the effects of human recombinant tumor necrosis factor (rTNF-alpha) on the morphology, incorporation of tritiated thymidine, and proliferation of 5 established cell lines derived from human malignant gliomas and 3 normal human brain cell cultures. A radioreceptor analysis for rTNF-alpha was performed on all cell lines and cultures. Two of the 5 human glioma cell lines (SF-188 and U 343 MG-A) demonstrated a marked decrease (60% or less of untreated controls) in the uptake of tritiated thymidine when treated with rTNF-alpha at a concentration of 40 U/ml; rTNF-alpha at 100 U/ml had antiproliferative and cytotoxic effects on both cell lines. The growth and proliferation of cell lines SF-126 and U 251 MG were not affected by rTNF-alpha even at high concentrations (5,000 U/ml). The growth and proliferation of SF-539 were affected to an intermediate degree. A colony-forming efficiency assay corroborated the results of the proliferation studies: SF-126 was relatively resistant (surviving fraction of 0.9 at 500 U/ml) and SF-188 was relatively sensitive (surviving fraction of 0.08 at 500 U/ml) to the cytotoxic effects of rTNF-alpha. Time-sequence electron microscopy showed that rTNF-alpha at a concentration of 500 U/ml caused ultrastructural changes in SF-188, including increased intracytoplasmic vesiculation, swelling and degeneration of mitochondria, loss of cell:cell junctional complexes, and fragmentation of the plasma membrane. Studies with 125I-rTNF-alpha showed a variable degree of binding in all cell lines and cultures. SF-188, a highly sensitive cell line, demonstrated the strongest binding of 125I-rTNF-alpha (3,400 receptors/cell with high affinity; kd = 0.27 nM), while SF-126, a highly resistant cell line, had the weakest binding (809 receptors/cell; kd = 0.25 nM). We conclude that there is a spectrum of antiproliferative and cytotoxic activity among glioma-derived tumor cell lines exposed to rTNF-alpha. An increased number of rTNF-alpha receptors appears to be a necessary but insufficient condition to explain the antiproliferative effects observed in some glioma-derived cell lines.