Vis A. Liepkalns

Cell cycle phases of a novel human neural cell line and the effect of exogenous gangliosides

Cell cycle phases of a new cell line (10-1) derived from a gemistocytic astrocytoma were analysed with computer-generated curves fit to the percentage of labeled metaphases at various times after a [3H]-thymidine pulse. These large slowly growing tumor cells in vitro had a mitotic index of 2.8%, a confluent density of 34400 cells/cm2 and an average DNA content of 7.91 pg/cell. Ganglioside treatment (50 uM in the culture medium) prolonged the generation time (26 hrs to 33 hrs) as well as all the phases of the cell cycle. The most noticeable effect of ganglioside exposure was to increase the G1 phase by 40%. Therefore we conclude that exogenously added membrane components, such as gangliosides, can lengthen the gemistocytic astrocytoma cell cycle in vitro and modulate the proliferation of these neural cells.

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12‐18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two‐ to threefold as cell density increased 25‐fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di‐ and trisialogangliosides (GD2+ GDlb+ GT1) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM4+ GM3+ GM2) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM4+ GM3+ GM2) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM3 (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GMt (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.

Growth Characteristics of Human Glioma-derived and Fetal Neural Cells in Culture

Growth characteristics of human fetal neural cells (CH) and human glioblastoma multiforme-derived cells (12–18) in culture were compared. Cells were grown to confluent densities of 38,000 to 42,500 cells/cm2 for CH and 85,800 to 87,100 for 12–18. Population doubling times were 40.0 ± 5.1 hr and 66.5 ± 9.8 hr for CH and 12–18 cells. respectively. The mean DNA content per cell of the glioma-derived cells was twice that of the fetal brain cells at sparse, log, and confluent cell densities. High concentrations (40%) of serum in growth medium increased DNA contents in confluent CH, but not 12–18, cells. The amount of protein per cell also was consistently higher in glioma cells than CH cells, but, as cell densities increased, protein contents decreased for both: 1200 to 700 pg/cell in glioma cells, and 840 to 560 pg/cell in CH cells. In each cell line, initial rates of [3H]ThdR incorporation into TCA precipitable material decreased as cell density increased, but confluent glioma-derived cells incorporated 10 times more [3H]ThdR than confluent fetal cells. Almost all CH cells had a normal diploid chromosome number of 46. A histogram showing the relative frequencies of chromosome numbers of glioma-derived cells had peaks of 52, 79, and 105 chromosomes per metaphase, indicating a haploid number of 26 for most cells. Lengths of cell cycle phases, determined using autoradiographic techniques, indicate that glioma-derived cells had a longer generation time and S period than fetal neural cells. These data demonstrate several biological differences between glioblastomaderived cells and non-neoplastic fetal neural cells, indicating that this system is of potential value for comparative studies on growth control and contact inhibition.

Natural killer activity against cultured human neural tumor and fetal brain cells

Ten human neural tumor lines and three established from normal human brain were analyzed for sensitivities to natural killer (NK) cytolysis. Compared to MOLT-4, fetal brain cells were sensitive, but those from adult brain and eight of ten neural tumor cell lines demonstrated marked NK resistance. The frequencies of target-binding cells (TBC) and single-cell lysis of glioma cells bound within tumor cell conjugates demonstrated that the resistance of two lines was explained either by a decrease in the frequencies of TBC or reduced ability of bound NK cells to lyse the tumor cell conjugates. A third resistant line demonstrated decreases in both TBC and tumor cell conjugate lysis. Two glioma lines with less NK resistance had greater frequencies of TBC or conjugate lysis than the resistant lines. Thus, NK resistance can result from decreased recognition of targets, diminished NK lysis of bound targets, or a combination of both.

Effects of cell density on the neutral glycolipid composition of cultured human brain and glioma cells

Density dependent chain elongation of neutral glycosphingolipids (NGSL) is associated with contact inhibition of mitosis in several normal cultured cell lines. Transformed non-neural cell lines which have impaired contact inhibition frequently lose this biochemical response. To determine if either of these phenomena occur in human neural cells we determined NGSL compositions of cultured glioblastoma multiforme and normal fetal brain cells. Fetal cells generally had more total NGSL than the tumor cells. As a percentage of total NGSL, both cell lines at higher cell densities had larger proportions of ceramide trihexoside and globoside, but smaller proportions of cerebroside. This decrease was mainly in nonhydroxy fatty acid cerebroside of glioma cells, but in hydroxy fatty acid cerebroside of normal fetal brain cells. These results demonstrate that although glioblastoma multiforme cells have markedly impaired growth control, they still preserve density dependent chain elongation of NGSL. A role for this phenomenon in normal cellular growth control has yet to be established.

Messenger RNA populations and their nuclear precursors in cultured human glioma and fetal brain cells.

Using labelled single copy DNA to cytoplasmic messenger RNA from a glioma cell line, it is shown by the excess RNA hybridization technique that a human glioma and a human fetal brain cell line both contain the mid and low abundancy classes of cytoplasmic messenger RNA. However, the high abundancy class present in the glioma cells is absent from the hybridization profile of the fetal cell line. Most of the nuclear RNA species complementary to this single copy DNA were present in the low abundancy class of both cell types; the mid-abundancy class was present in much lower concentration than in glioma cytoplasmic RNA and the high abundancy class was essentially absent. The extent of formation of S1-nuclease resistant hybrids indicated that some of the messengers which are present in the high abundancy class in the cytoplasm of glioma cells are present in the lower abundancy classes of fetal brain cells. Thus the glioma cells appear to exhibit a higher degree of specialization potential than the embryonic cells.