The induction of glial proliferation by an astrocytoma-derived growth factor resembling glia maturation factor.

Glia maturation factor (GMF)-like activity which induces DNA synthesis and morphological differentiation of density-inhibited glioblasts was detected in various glial tumor cells. A polypeptide from C6 cells (rat astrocytoma) which has a molecular weight range of 40,000-50,000 showed the highest activity. This factor also induced DNA synthesis in glioma cells (354A and LRM55) and fibroblast (Swiss 3T3). The activity was susceptible to heat treatment at 70 degrees C for 5 min, or to proteases such as trypsin, chymotrypsin, papain, and subtilisin, but it was devoid of esteropeptidase activity. The isoelectric point was found to be 5.3. Subcellular fractionation localized the activity in cytosomal and microsomal fractions. These properties closely resemble those of GMF from pig and bovine brain.

Temperature dependence of morphological response of glioblasts to glia maturation factor.

Glioblasts respond to glia maturation factor (GMF) by outgrowth of cell processes. This morphological response takes place at 37 degrees C but not at 23 degrees C, suggesting the involvement of temperature-dependent chemical processes in the mechanism of action of GMF. The morphological response of glioblasts to GMF is correlated with the rearrangement of microtubules but not with any net change in tubulin content of the cells.

Enucleation blocks the morphological response of glioblasts to glia maturation factor.

Culture glioblasts obtained from rat fetuses were enucleated with cytochalasin B. The glia maturation factor stimulated the morphological differentiation of nucleated but not enucleated cells. In contrast, 8-bromo-cyclic AMP stimulated the morphological differentiation of both nucleated and enucleated cells. This distinction implies a difference in the mode of action of the two agents on cultured glioblasts, and suggests that an interaction between the nucleus and cytoplasm is required for expression of the morphological effect of the glia maturation factor.

Glia maturation factor promotes contact inhibition in cancer cells.

The effect of bovine glia maturation factor on the growth pattern of cancer cells was investigated in the rat glioma cell line 354A. When the cells were grown in the serum-free defined medium N2 in the absence of the factor, the cells proliferated with a doubling time of 24 hr without showing contact inhibition. After reaching confluency, the cell layer formed numerous foci from which heaps of cell colonies arose. The addition of glia maturation factor to the culture stimulated cell division in the logarithmic phase but prevented overgrowth once the cells arrived at confluency. The ability of glia maturation factor to restore contact inhibition suggests a regulatory role in normal and neoplastic cells.

Glia maturation factor in bovine brain: partial purification and physicochemical characterization.

Glia maturation factor (GMF) is partially purified from bovine brains by the following procedure: extraction at physiologic pH, dialysis and freeze-drying of the extract, ethanol washing of the dried powder and re-extraction of the ethanol-washed residue with Tris-buffered saline, ion-exchange chromatography with DEAE Sephadex and molecular sieving with Bio-gel P-200. The partially purified protein has an apparent molecular weight of 23,000 and an isoelectric point of 4.75, and retains both morphological transforming and mitogenic activities when tested on glioblasts. Both activities are susceptible to protease digestion and heat inactivation. The procedure results in a 400-fold purification of the morphological activity and a 1400-fold purification of the mitogenic activity. Both activities are detectable when GMF is used in nanogram quantities. The possibility that both functions are expressions of the same factor and the possible role of GMF in the differential or sequential stimulation of cell growth and maturation are discussed.

Biological effects of bovine glia maturation factor on glial cells in culture.

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4-8 days after subculture show the highest response t GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 h after stimulation; maximum incorporation of [methyl-3H]thymidine was detected at 18 h. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.

Distribution of glia maturation factor-like activity in organs and cells.

Glia maturation factor (GMF) is a protein first isolated from the adult pig brain. GMF-like activity can be demonstrated in rat organs, including brain, kidney and heart. The activity in these organs is low in newborn animals, but increases with development, reaching the adult level in 1 or 2 weeks. GMF-like activities in the various organs are similar in physicochemical properties, being heat-labile, susceptible to proteolytic enzymes, and are associated with an acidic molecule of large size. Cultured rat glioblasts and C6 glioma cells, but not their conditioned media, contain large amounts of endogenous GMF-like activity. GMF obtained from brains and cultured glial cells also possess mitogenic action. Subcellular fractionation localizes GMF-like activity in the cytosol and in microsomal and nerve ending fractions. GMF-like activity is also detectable in bovine, sheep, monkey and human brains. The results suggest that GMF is ubiquitous in distribution, and at least a portion of it may be associated with the structural components of the cells.

Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.

Glia maturation factor: effect on chemical differentiation of glioblasts in culture.

A protein factor from the adult brain increases the concentrations of adenosine 3',5'-monophosphate and S-100 protein in glioblasts in culture. Such changes are correlated with the outgrowth of cell processes.