Differentiation Of Glioma Cells Research Articles

DIFFERENTIATION OF GLIOMA-CELLS BY A PDGF-ANTAGONIST

We previously reported that trapidil, a Platelet-derived Growth Factor (PDGF) antagonist, can inhibit the proliferation of PDGF-dependent glioma cells. In the current study, we explored the effect of trapidil on the differentiation of glioma cells by observing the morphological changes in glioma cells in control and trapidil-treated cultures under a phase contrast microscope. Most cells in the control cultures were flat, large, and irregularly shaped. On the other hand, most cells treated with trapidil formed several long cytoplasmic processes and exhibited fibrous morphology. Western blots and immunocytochemical analysis of glial fibrillary acidic protein (GFAP)-stained, trapidil-treated cultures revealed an increase in GFAP content over the control cultures. From these results we propose that trapidil induces the differentiation of glioma cells.

Subclones of C6 rat glioma cells differing in intermediate filament protein expression

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Differential effects of tumor necrosis factor on the growth and differentiation of neuroblastoma and glioma cells

We have studied the effect of tumor necrosis factor (TNF-α) on transformed neural and glial-derived cell lines. TNF-α at physiological doses was able to arrest the growth and inhibit DNA synthesis of N103 neuroblastoma cells. This phenomenon was accompanied by a morphological cell differentiation characterized by the outgrowth of neurites. By contrast, TNF-α induced an increase in the growth rate of C6 glioma cells and upon cytokine addition a higher number of C6 cells were found in the S + G2 phase of the cell cycle. C6 cells did not show morphological changes under this treatment. Analogous results were obtained with IFN-γ. These neurotrophic and mitogenic effects of TNF-α suggest a putative role of this cytokine in the regeneration of brain tissue upon brain injury.

Influence of Extracellular Matrix on the Proliferation and Differentiation of Glioma Cells in Culture

The influence of extracellular matrix on the proliferation and differentiation of glioma cells in vitro was investigated by culturing glioma cells in collagen gel and on collagen and laminin films. Rat C-6 glioma cells extended thin cytoplasmic processes, proliferated, and differentiated in collagen gel and on collagen film. The cells were stellate with multiple thin processes like the astrocyte in vivo. The intracellular content of cyclic adenosine-monophosphate in rat C-6 glioma cells on collagen film increased approximately four fold over the control level. In laminin film culture, rat C-6 glioma cells extended thin cytoplasmic processes with many knotty structures and proliferated. These findings confirm that extracellular matrix induces the differentiation of glioma cells.

Induction of glutathione S-transferase, placental type in T9 glioma cells by dibutyryladenosine 3?,5?-cyclic monophosphate and modification of its expression by naturally occurring isothiocyanates

The effect of dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl cAMP) on the expression of glutathione S-transferase placental type (GST-P) was examined in rat glioma cell line using an immunohistochemical technique. Cultured T9 glioma cells were negative for GST-P activity under normal conditions. However, treatment with 1 mM dibutyryl cAMP produced GST-P expression in about 50% of the cells, as well as some morphological changes. The expression of GST-P was increased with addition of dibutyryl cAMP together with 1 microgram/ml allyl isothiocyanate (AITC) or 0.1 microgram/ml benzyl isothiocyanate (BITC). With these combinated treatments, almost all cultured cells showed a strong positive reaction for GST-P, although AITC or BITC alone elicited GST-P in only 5% of the cultured cells. The results of the present study indicate that dibutyryl cAMP causes functional as well as morphological differentiation of T9 glioma cells.

Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation.

We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms.

Interrelationship between differentiation and malignancy-associated properties in glioma

The phenotypic expression of cells derived from human anaplastic astrocytomas, rat glioma, normal human adult and foetal brain tissue have been examined for differentiated and malignancy-associated properties. Glial fibrillary acidic protein (GFAP), high affinity glutamate and gamma-amino butyric acid (GABA) uptake and glutamine synthetase were used as indicators of astroglial differentiation. Plasminogen activator and tumour angiogenesis factor were the malignancy-associated markers. The normal adult brain-derived lines showed some differentiated astroglial features and expressed low levels of the malignancy-associated properties. The foetal cultures contained highly differentiated astroglia while the glioma lines showed considerable phenotypic heterogeneity from highly differentiated to undifferentiated. The least differentiated glioma cells exhibited the highest plasminogen activator activities. The density-dependent control of phenotypic expression was also investigated. High affinity GABA uptake, and GFAP in rat C6 glioma cultures, increased with increasing monolayer cell density, events probably mediated by an increase in the formation of cell-cell contacts at confluence. Plasminogen activator activity decreased with increasing cell density.

Preferential synthesis of differentiation related cytoplasmic proteins in low-serum adapted differentiated glioma cells

Rat glioma (C6) cells can be induced to morphological and biochemical differentiation by culturing in serum-deprived conditions. The induced differentiated cells were found to preferentially synthesize some distinct cytoplasmic proteins, and the rate of synthesis quantitatively correlated well with the degree of morphological differentiation, whereas the total mRNA synthesis was decreased in the differentiated cells. These findings suggest that reduced total mRNA syntheses and prefereential translation of differentiation-related mRNAs are parts of the biochemical events during the differentiation process of neuroglia.

Electron-microscopic visualization of binding of antibodies from sera of glioma patients on cultured glioma cells.

Several human cell lines (normal and neoplastic glia, cerebral metastases from adenocarcinoma, fibroblasts) were incubated with sera from patients with well and poorly differentiated glioma and with sera from healthy donors and then stained with PAP complex to define and localize the antibody reaction with cell surface antigens by means of electron microscopy. The sera of glioma patients proved to contain antibodies which bound the tumor-associated antigenic determinants on the cell membranes of gliomas and of cerebral metastases from adenocarcinoma in tissue cultures. Further, absorption testing of the reactive sera on normal brain, well-differentiated astrocytoma and cultured glioblastoma cells, together with cross-reactivity experiments suggests that at least two antigens or groups of antigens are expressed on the glioma cell surface: one shared by well and poorly differentiated glioma cells and the other by poorly differentiated glioma cells and the cells of cerebral metastases from adenocarcinoma.

Modulation in the levels and localization of plasminogen activator in differentiating neuroblastoma cells

Variations in the specific activity and localization of plasminogen activator (PA) were examined prior to and during the differentiation of mouse neuroblastoma and rat glioma cells in culture, in an attempt to reveal whether PA is actively involved in neurite extension and development in these cells. Following differentiation induced by dibutyryl cyclic AMP, the level of PA increased by 10 to 20-fold in neuroblastoma N18TG-2 cells and in the hybrid cell line NG108-15, obtained by fusion of mouse neuroblastoma N18TG-2 and rat glioma C6BU-1 cells, but was very low in the glioma parental cells throughout differentiation. Plasminogen-dependent proteolysis was found not to be an obligatory requirement for the differentiation of the hybrid cells in culture, since the extension of neurites, the appearance of toxin-activated Rb +-efflux and the eletrical properties were largely unaffected by eliminating and blocking this activity. Confluent cells from several clones derived from mouse neuroblastoma C1300 cells displayed significant intracellular and secreted PA activities, and the axon-minus neuroblastoma NIA-103 cells were found to secrete the highest amounts of PA. Upon gel electrophoresis, all mouse neuroblastoma cell lines examined displayed a major molecular form of active PA with an apparent molecular weight of 80,000 and a minor one of 50,000. Only the larger enzyme form was expressed by the rat glioma or the hybrid cells, and the electrophoretic migration pattern of the active enzyme species remained unchanged upon differentiation of the various cell lines. Distribution of PA activity on the surface of differentiating neuroblastoma N1E-115 cells and hybrid NG108-15 cells was visualized by high resolution autoradiography of [ 125I]fibrin degradation products, cross-linked to the cell surface. Particularly intense labeling was observed along membranes of cell bodies in both developing and mature cells, around branching zones, along individual neurite extensions and in part, but not all, of the growth cones. These findings suggest that PA plays a role in the metabolism of mature cells of neuronal origin, and imply its involvement in the development and/or the functions of specific neuronal processes and growth cones.

Proliferation of glial-derived cells in defined media.

A serum-free defined medium has been formulated that supports proliferation and morphologic differentiation of U-251 MGsp human and C6-2BD rat glioma cells. This defined medium consists of a basal medium supplemented with transferrin, fibroblast growth factor, hydrocortisone, selenium, biotin, and fibronectin (G2 medium). When U-251 cells were plated in G2 medium on poly-D-lysine precoated dishes, their growth rate was 77% and final cell density was 82% of serum-grown counterparts. The growth rate of C6 cells in G2 medium was 67% compared to cells cultured in serum supplemented medium. Although G2 medium supported the growth of human and rat glioma cells, LA-N-1 human neuroblastoma and WI-38 human fibroblast cells showed no increase in cell number when grown in G2 medium compared to basal medium. A similar formulation (G3 medium), lacking fibroblast growth factor and hydrocortisone, supported the proliferation of RN-22 rat schwannoma cells. Morphologic differences were observed between cells grown in the presence of serum and in defined media. All three glial cell lines changed from a flattened shape in serum supplemented medium to a more spherical appearance in defined medium. In addition, both U-251 and C6 cells developed numerous processes, some reaching several cell diameters in length. These defined media will facilitate studies of the growth and differentiation of glial-derived cells.

Evaluation of the drug-induced morphological differentiation of rat glioma cells (C-6) from the aspects of S-100 protein level and con a binding pattern

The intracellular content of the nervous system specific protein S-100 began to increase with 4 days latency following the morphological differentiation of cultured rat glioma cells (C-6) with 1 mM dibutyryl cyclic AMP (dbcAMP), rising to approximately 10-fold over the control level at 15 days after the treatment. The concanavalin A (Con A) binding pattern on the external cell surface of C-6 cells exposed to dbcAMP appeared as a smooth layer of 40–60 nm thickness whereas that of control cells was irregularly thick and patchy. The correlation between morphological and biochemical changes of C-6 cells after dbcAMP treatment is discussed in relation to the mechanism controlling the differentiation of glioma cells.

The effect of nerve growth factor on undifferentiated glioma cells

Nerve growth factor (NGF) treatment of rats bearing implanted cells of an anaplastic glioma clone (F-98) led to a decreased tumor growth rate and increased survival time. Similar, but less pronounced, effects were observed if the cells were pretreated with NGF for 24 h before implantation. NGF also reduced the in vitro growth rate of F-98 and contributed to cellular maturation. Several well differentiated cell lines and clones were not visibly affected by NGF using the above parameters. We concluded that NGF has little or no effect on differentiated glioma cells, but significantly affects undifferentiated glioma cells by retarding their growth and inducing maturation.

Characterization of cell cycle and biological parameters of transplantable glioma cell lines and clones

Aberrant biological characteristics of established cell lines and clones, derived from nitrosourea-induced gliomas in CDF rats were compared with normal rat glial cells. Despite their tumorigenicity and unrestrained proliferation due to inherent cytogenetic anomalies, these neoplastic cells retained certain normal glial attributes to a variable degree. Differentiated glioma cells resembled normal glial cells to a greater extent than they resembled anaplastic glioma cells. They were also less malignant upon implantation. Cell cycle analyses revealed that considerable variations not only for the G1 phase but also for the S period were demonstrated among different tumor cell types. Shortening of the G1 phase may dictate a shorter generation time (shorter doubling time) since a larger potential proliferative pool may overcome the effect of a prolonged generation period may constitute a faster proliferation rate. Although the cell generation period of neoplastic cells is not necessarily shorter than that of normal glial cells, the lower proportion of nonproliferative cells results in a much faster growth rate when compared wo non-neoplastic glial cells in culture.

Morphological characterization of nitrosourea-induced glioma cell lines and clones.

A morphological characterization of cell lines and clones derived from nitrosourea-induced gliomas in CDF rats was performed and compared with normal rat glial cells. The glioma cell lines and clones proved to be morphologically stable. They retained individual characteristics and their state of differentiation despite repeated propagations in vitro. There are no indications suggesting that different cell types are transformed to a common phenotype. Different glioma cell lines maintain their individual morphological distinctiveness, however, normal glial cells give consistent morphological features and growth patterns irrespective of the variation of animal resource and passage level. The criss-cross or streaming cellular alignment and the multilayeredness make glioma cultures morphologically distinguishable from normal glial cultures which are characterized by monolayered epithelioid configuration. Furthermore, an elevated mitotic index and an increased nuclear-cytoplasmic ratio are observed consistently with glioma cells in culture, irrespective of the degree of anaplasia. Differentiated glioma cells preserve certain normal traits of glial components (i.e. morphological differentiation, cytoskeletal elaboration, etc.), yet express various neoplastic phenotypes (i.e., autonomous growth behaviors, tumorigenicity upon transplantation, etc.), though to a lesser extent than glioma cells of anaplastic variety. Despite the intimate interdigitations between apposed plasmalemmae of adjacent tumor cells, no recognizable intercellular junctional devices are present. The in vitro glioma cell system may serve as an excellent model for investigating the mechanisms for autonomous biological behavior and proliferation of neoplastic cells and for therapeutic screening.

PROCEEDINGS OF THE FIFTY‐FOURTH MEETING OF THE BRITISH NEUROPATHOLOGICAL SOCIETY HELD AT THE NATIONAL HOSPITAL AND THE INSTITUTE OF NEUROLOGY, LONDON, 13–14 JANUARY 1978

Ethylnitrosourea‐induced brain carcinogenesis: an investigation using cultures derived during the latent periodEarly stages of the development of experimental gliomas: a light and electron microscope studyCyclic AMP and the differentiation of malignant glioma cells in cultureGlial fibrillary acidic protein in human intracranial tumours. Immunohistochemical studies with quantitation by radioimmunoassayThe influence of different blood‐glucose levels on the brain damage in rats after cerebral ischaemiaThe effects of hypoxia combined with bilateral common carotid artery occlusion in the brainPathogenesis of idiopathic faecal incontinenceUltrastructural changes in polymyositisMuscle lesions in a hereditary disease of lambs (‘daft lamb disease’)Pietrain Creeper sydrome: A primary myopathy of the pig?Spinal ataxia in horses associated with ectopic lymphoid tissueThe cervical cord in multiple sclerosisAgeing in the pigmented nerve cells of the human CNSAn immunofluorescence study of the passive Arthus reaction in rat sciatic nerveThe influence of neural structures on the maintenance of perineurial organizationEffect of postnatal lead exposure on Purkinje cell dendritic development in the ratEffects on the developing rat cerebellum of refeeding after neonatal undernutritionSome observations on the cerebellar external granular layerUltrastructural and immunohistochemical identification of cell types isolated from developing rat cerebellumChanges in the cellularity of the rat subependymal plate after local X‐irradiationTargeting of lesions and variation in agent concentration in different areas of the brain in scrapieSpinal cord lesions in the cat induced by local injection of lysolecithinImpairment of the metabolic homeostatic function of the muscles of mice with muscular dystrophy (dy/dy)Characterization of acid phosphatase in regenerating muscle fibresBehaviour of normal and dystrophic human cells in cultureElement analysis in human neuromuscular disorders using X‐ray fluorescence