Glioblastoma Cultures Research Articles

Selective Antimitotic Effects of Estramustine Correlate with Its Antimicrotubule Properties on Glioblastoma and Astrocytes

Estramustine is an estradiol-based agent that accumulates in cells containing estramustine binding protein. Previous studies have shown that this binding site is expressed in human glioblastoma cells and that estramustine accumulates in glioma cells, resulting in a concentration-dependent inhibition of proliferation. We have shown that estramustine treatment results in a rapid inhibition of deoxyribonucleic acid synthesis (within 4 h) in human glioblastoma cells associated with an alteration of cell size and shape, consistent with its known antimicrotubule activity. To extend these findings, we performed an immunohistochemical analysis of microtubules with a monoclonal antibody to beta-tubulin, using a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to measure the antimitotic effects of estramustine on both human glioblastoma and astrocyte cultures. Within 4 hours, estramustine (10 mumol/L) caused a dramatic alteration in the tubulin staining in glioma cells, characterized by a disorganization in microtubules. Cell shape and microtubule staining in astrocytes were relatively preserved. Estramustine had a concentration-dependent cytotoxic effect in tumor cultures, whereas it had no effect on astrocyte viability at any concentration. Differences in the antimitotic effects do not appear to be related to variations in proliferation rates among these different types of cells. These data suggest that although estramustine is a potent inhibitor of proliferation in glioblastoma cells, it has modest antiproliferative effects on astrocytes and its selective activity is closely correlated with its antimicrotubule properties.

Responses of human glioblastoma cells to human natural tumor necrosis factor-?: Susceptibility, mechanism of resistance and cytokine production studies

Responses and susceptibility of 14 human glioblastoma cell lines to human natural tumor necrosis factor-alpha (TNF) were studied in vitro. Susceptibility of glioblastoma cells to TNF varied in experimental conditions applied. Most of glioblastoma cell lines were resistant to cytotoxic activity of TNF in a MTT assay at concentrations below 16 U/ml for 72 h exposure. However, TNF at higher dose, in prolonged exposure and against low density of target cells was antiproliferative for certain glioblastoma cultures. TNF exposure at 10 U/ml for 48 h suppressed DNA synthesis in 9 of 14 glioblastoma cultures, but increased in 3 cultures. In addition, colony forming assay showed anti-clonogenic activity of TNF in 5 of 6 glioblastoma cell lines tested. In spite of their low susceptibility to TNF, glioblastoma cells well responded to TNF stimulation at low dose (10 U/ml) for a short period in the absence of cell damage. Productions of Interleukin-6 (IL-6), IL-8-like activity, granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2 (PGE2) and manganous superoxide dismutase (Mn-SOD) were enhanced or induced by the low-dose TNF stimulation. Mn-SOD, a protein protective against oxidative cell damage, was well induced in time- and dose-dependent manner, however did not correlate with TNF resistance. Whereas the levels of PGE2 in TNF-susceptible cell lines, H-4 and SF-188, were higher than those of other lines. In conclusion, most of glioblastoma cells are resistant to TNF cytotoxic effects, but highly responsive to TNF stimulation. Its effect on glioblastoma cells appears to modulate cell differentiation rather than to kill the cells.

Estramustine and estrone analogs rapidly and reversibly inhibit deoxyribonucleic acid synthesis and alter morphology in cultured human glioblastoma cells.

Estramustine is an estradiol-based agent that has been shown to accumulate in human glioma cells, resulting in a concentration-dependent alteration in cell size and shape within minutes and an inhibition of proliferation over 3 to 6 days. We evaluated human glioblastoma cultures with [3H]thymidine incorporation assays to determine estramustine's early effects on deoxyribonucleic acid synthesis in these tumors. Because estramustine shares a common structural motif with other antimicrotubule drugs, we synthesized four A-ring conjugates of estrone that contained a carbamate moiety but lacked nitrogen mustard. These analogs were examined by [3H]thymidine incorporation and compared with vinblastine. Greater than 70% inhibition of [3H]thymidine incorporation occurred within 1 hour of treatment with estramustine at 10(-5) mol/L, which increased to 80% inhibition at 4 hours. Ethyl carbamate JE208 was nearly as effective as estramustine in inhibiting deoxyribonucleic acid synthesis, and both were more effective than vinblastine. The inhibitory effects of estramustine and estrone analogs were reversible; vinblastine was not reversible. Although estramustine and JE208 induced similar antiproliferative and morphological changes in glioblastoma cells that persisted for at least 4 days, there was a modest recovery of morphology and thymidine incorporation with JE208 after prolonged treatment. The common findings with estramustine and JE208 suggest that these agents may have a similar mechanism of action and form the basis for the investigation of new agents that may rapidly and reversibly inhibit glioblastoma.

Estramustine and Estrone Analogs Rapidly and Reversibly Inhibit Deoxyribonucleic Acid Synthesis and Alter Morphology in Cultured Human Glioblastoma Cells

ESTRAMUSTINE IS AN estradiol-based agent that has been shown to accumulate in human glioma cells, resulting in a concentration-dependent alteration in cell size and shape within minutes and an inhibition of proliferation over 3 to 6 days. We evaluated human glioblastoma cultures with [3H]thymidine incorporation assays to determine estramustine's early effects on deoxyribonucleic acid synthesis in these tumors. Because estramustine shares a common structural motif with other antimicrotubule drugs, we synthesized four A-ring conjugates of estrone that contained a carbamate moiety but lacked nitrogen mustard. These analogs were examined by [3H]thymidine incorporation and compared with vinblastine. Greater than 70% inhibition of [3H]thymidine incorporation occurred within 1 hour of treatment with estramustine at 10-5 mol/L, which increased to 80% inhibition at 4 hours. Ethyl carbamate JE208 was nearly as effective as estramustine in inhibiting deoxyribonucleic acid synthesis, and both were more effective than vinblastine. The inhibitory effects of estramustine and estrone analogs were reversible; vinblastine was not reversible. Although estramustine and JE208 induced similar antiproliferative and morphological changes in glioblastoma cells that persisted for at least 4 days, there was a modest recovery of morphology and thymidine incorporation with JE208 after prolonged treatment. The common findings with estramustine and JE208 suggest that these agents may have a similar mechanism of action and form the basis for the investigation of new agents that may rapidly and reversibly inhibit glioblastoma.

Oral Liarozole as a Catabolic Inhibitor Potently Increases Retinoic Acid in vivo: First Experience from an Ongoing Therapeutic Trial in Highly Malignant Primary Brain Tumors

<i>Background: </i>Retinoic acid, the intracellular metabolite of vitamin A, induces differentiation in glioblastoma cultures and recognizes nuclear retinoic acid (RA) receptors in human glial cells. Its degradation can be inhibited by the oral steroid synthesis modulator liarozole or R75251 (5-[(3-chlorophenyl) (lH-imidazole-l-yl)methyl]-lH-benzimidazole HC1). Measured by ³H-thymidine incorporation, liarozole also exhibits an intrinsic attenuating effect on glioblastoma growth in vitro. <i>Patients and Method: </i>Ten patients with primary malignant brain tumors have entered an open oral therapeutic pilot study with 13-cis-retinoic acid (isotre-tinoin^INN; Roaccutan® Roche) and the catabolic inhibitor liarozole. <i>Results: </i>Side effects such as cheilitis, conjunctivitis, and desquamation dose-depend-ently correlated with plasma levels and could be regularly used for dose adjustments. The combined therapy produced and maintained elevated plasma concentrations and clinical retinoid effects with fractions of formerly necessary 13-cis-RA doses. In vivo, 75-150 mg liarozole combined with 5-20 mg 13-cis-RA daily increased plasma 13-cis-RA and 4-oxo-13-cis-RA levels more than threefold. Long-term all-trans-RA therapy without liarozole leads to a progressive downregulation of plasma RA levels. Such a decrease may be prevented by combining all-trans-RA with its catabolic inhibitor liarozole (Janssen). <i>Conclusion: </i>First clinical experience recommends liarozole for either 13-cis-or all-trans RA therapy in retinoid-sensitive malignancies such as neuro-ectodermal glioma or acute promyelocytic leukemia.

Localization of the peripheral-type benzodiazepine binding site to mitochondria of human glioma cells.

Subcellular fractionation was performed on human U251 glioblastoma cultures. In all subcellular fractions, the binding of the peripheral benzodiazepine ligand, [3H]PK 11195, correlated with the specific activity of monoamine oxidase (r = 0.95, p less than 0.001) and succinate dehydrogenase (r = 0.93, p less than 0.001), two mitochondrial enzymes. The specific activity of plasma membrane and nuclear markers correlated poorly with the presence of PK 11195 binding sites. These data support the mitochondrion as the primary location of peripheral-type benzodiazepine binding sites (PBBS) in human glioma cells. Mitochondria-rich preparations were then assayed for [3H]Ro5-4964 binding. Six nM [3H]Ro5-4964 failed to specifically bind to human U251 mitochondria, but bound vigorously to mitochondria from rat C6 glioma. These data indicate that the low affinity of Ro5-4864 for PBBS in human glioma cells compared to those in rat is due to interspecies receptor variation rather than impaired drug transport into human cells.

In vitro Prostaglandin E2 production by glioblastoma cells and its effect on interleukin-2 activation of oncolytic lymphocytes

Glioblastoma cells constitutively produce various amounts of PGE2 (prostaglandin E2) in vitro. The amounts of PGE2 found in the conditioned medium of the glioblastoma cultures (less than 5 ng/ml) were not enough to inhibit the IL-2 (interleukin-2) activation of peripheral blood lymphocytes. However the amount of PGE2 produced by approximately 1 x 10(7) of the glioblastoma cells can be assumed to suppress the generation of IL-2-induced killing activity against glioblastoma cells.

Antigenic staining patterns of human glioma cultures: primary cultures, long-term cultures and cell lines

The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at different in vitro ages for glial fibrillary acidic protein, fibronectin, galactocerebroside, HNK-1/Leu 7, A2B5, vimentin, factor VIII and A4. Histologically, the cases were composed of eight low-grade astrocytomas, 11 high-grade astrocytomas, four low-grade oligodendrogliomas, seven high-grade oligodendrogliomas and 29 glioblastomas. The 45 cases were analysed within the first 3 weeks of culture, many of them as primary cultures. In 11 cases stainings were performed repeatedly at intervals of up to 6 months. Glial fibrillary acidic protein staining was positive in most of the early cultures of astrocytomas (low and high grade) and glioblastomas; expression in more than 50% of the cells was found in 1 of 5 low-grade astrocytomas, 5 of 11 high-grade astrocytomas and 14 of 29 glioblastomas. Two of the high-grade astrocytomas were stained once more after 6 weeks in culture and were found to be only 1% positive for glial fibrillary acidic protein but strongly positive for fibronectin. The same was true for five of the glioblastoma cases. Two of these cases remained glial fibrillary acid protein positive and developed into stable permanent cell lines. Only one case started with 1% of glial fibrillary acidic protein positive cells and later developed into a 99% glial fibrillary acidic protein positive cell line. Neither HNK-1/Leu 7 expression nor A2B5 staining appeared to have a relationship to the glial fibrillary acidic protein staining. It was observed that glial fibrillary acidic protein and HNK-1/Leu 7 were both 100% in some cases but that later one of the two antigens disappeared but not the other. The amount of glial fibrillary acidic protein staining does not allow the prediction of A2B5 staining. The study shows that initiation of primary cultures on an extracellular matrix yields more glial fibrillary acidic protein positive cells in primary cultures than have been found in other studies. It is concluded that only a rigid standardization of culture conditions will ensure the validity of comparisons of in vitro data obtained in primary cultures.

Regulation of viral persistence in human glioblastoma and rhabdomyosarcoma cells infected with coronavirus OC43

Cultures of human rhabdomyosarcoma (RD) and human glioblastoma (U87-MG) were compared for their ability to sustain a persistent infection with coronavirus OC43. Within 28 days, infectious virus and hemagglutinin were being produced at high levels in both types of cells. Temperature sensitive plaque variants were recovered at 31 °C. In both cell types, the virus caused increased antigen synthesis and cell death, if the temperature was lowered to 31 °C. Infectious virus was lost if cells were treated with antiserum to whole virus or if the temperature was raised to 39.5 °C. Probing the cured cells with OC43-specific 32P-cDNA showed that cured cells contained no detectable viral RNA. The relative ease of establishment and cure of these persistent infectious makes them attractive as models to study coronavirus regulatory processes.

Focal chemotherapy of brain tumours using semipermeable membranes.

Semipermeable silastic rubber membranes can be used to diffuse focally a variety of chemicals including antitumour drugs. Of the eight drugs tested, in vitro tests showed the best diffusion with dactinomycin, mithramycin, and oncovin, and the poorest diffusion with bleomycin, fluorouracil, and thiotepa. Biological testing was performed with mithramycin, dactinomycin, and oncovin using tissue cultures of human glioblastoma and subcutaneous implants of mouse ependymoblastoma. All three drugs caused rapid tissue culture cell death with direct injection, and impeded tumour growth when given intraperitoneally. Dactinomycin by silastic diffusion proved more effective than mithramycin against tissue cultures, but neither drug had a significant effect against local tumours treated with implanted drug capsules. Silastic diffusion of oncovin reduced tumour sizes significantly ipsilateral to the implant, compared to contralateral implants and to untreated controls.

Ultrastructural study of plasma membrane GM1 in neuroectodermal cells using cholera-peroxidase

Cholera toxin was coupled to peroxidase to yield a highly specific marker for GM1 gangliosides. Study of embryonic brain cells in culture revealed intense binding of cholera-peroxidase by plasma membranes of both neurons and glial cells. In contrast, long-term monolayer glioblastoma cultures, including one producing C-type virus, revealed virtually no labelling of their plasma membranes. Such cells were shown to be capable of incorporating exogenously applied GM1 into their plasma membranes. Studies with fixed brain and synaptosomal fractions were in accord with results on embryonic brain cells in culture, and autoradiographic findings with 125I cholera supported observations made utilizing cholera-peroxidase. From our studies there is some indication that long-term propagation in vitro alters the plasma membrane GM1.

Glucose metabolism in mixed glioblastoma and neuroblastoma cultures

When glioblastoma and neuroblastoma cells are mixed, an inhibition of 14CO 2 evolution from [1- 14C]-glucose occurs. This does not occur when Hela and Glioblastoma cells or Hela and neuroblastoma cells are mixed. Mixing the cells has no effect on the incorporation of [1- 14C] glucose.

Effects of dexamethasone and methylprednisolone on cell cultures of human glioblastomas.

✓ Eight biopsied glioblastomas were propagated in vitro through multiple, serial cell cultures which were exposed to dexamethasone and methylprednisolone in concentrations ranging from 0.25 to 400 µg/ml. The higher concentrations of both steroids produced inhibition of culture growth and cytotoxic damage which appeared relatively nonspecific. Although the responses observed were dose-dependent, the sensitivity among glioma cultures of different origins varied. Of the two steroids, Methylprednisolone was more injurious to glioma cells in vitro and was cytolytic in doses of 400 µg/ml. Growth inhibition was demonstrated after a 1-day exposure to this corticoid, but this effect was usually transient at lower doses. Thereafter, surviving resistant cells resumed their natural rate of growth in the continued presence of the steroid as well as under standard conditions of culture. Potential clinical applications of the antineoplastic properties of corticosteroids against the gliomas are discussed, emphasizing the need to investigate additional, possibly more efficacious compounds.