Glial cells release a factor into their culture medium that induces a high 4egree of morphological differentiation 'in neuroblastoma cells under normal growth conditions. This phenomenon is not correlated with a change; in intracellular adenosine 3': 5'-cyclic'monophosphate or in the rate of cell growth. Media from other cell lines tested induce less morphological differentiation or have no effect.Process formation by neuroblastoma cells in tissue culture has been termed morphological differentiation, and its importance as a model for neural differentiation has been pointed out by many authors (1-4). X-Irradiation (5), removal of serum (2), and addition of N6,02'-ibutyryl adenosine 3': 5'-cyclic monophosphate (But2cAMP) (3, 4) or 5-bromodeoxyuridine (BrdU) (6) can induce this process formation. All these treatments are unphysiological, even at low degree of morphological differentiation, and they all interfere with the growth rate of the cells. Considering the theoretical importance of cell to cell interactions in the central nervous system, we tried to influence process formation by using medium from glial cell cultures, We report that such a glial conditioned medium induces a high percentage of morphological differentiation under physiological conditions.
MATERIALS AND METHODSNeuroblastoma' cells, clone NB-2a, derived from the C-1300 tumour cell line (7), and glial cells, clone C-6, derived from a N-nitrosomethylurea-induced rat glial tumour (8) To determine the percentage of cells undergoing morphological differentiation, 50,000 trypsinized neuroblastoma cells were plated per 60-mm dish. After 16 hr of incubation, the medium was replaced by the medium to be tested, and 48 hr later all cells with and without processes were counted in at least five randomly selected areas of each culture plate. Cells having processes longer than the diameter of the cell body were considered morphologically differentiated. A total of 300-400 cells were counted per dish. The values reported here represent the percentage of differentiated cells determined in three parallel cultures.For intracellular cAMP determination, the cells were quickly washed with Dulbecco's phosphate buffered saline (pH 7.2, 37°) then fixed with 1 ml of 0.05 N HCl. They were scraped with a rubber policeman and the suspension was heated for 10 min in boiling water. The samples were then neutralized with 0.1 N NaOH to pH 7.3 and treated with 50 Ml of 0.17 M BaSO4 and 50 Mu of 0.15 M Ba(OH)2. The solution was clarified with a glass-fiber filter and cAMP was determined in triplicate by the method of Gilman (14). For the early time points the cells of two or three plates were extracted in the same 1 ml of 0.05 N HCl. At each time point, three parallel cultures were washed, then solubilized in 0.5% sodium dodecyl sulfate for protein determinations (15).
RESULTS AND DISCUSSIONThe induction of morphological differentiation of neuroblastoma cells by glial conditioned medium is illustrated by the micrographs in Fig. 1 (Table 1). Unlike those treatments, condi...
Synaptic membranes, highly enriched in nicotinic receptor, contain three 43 000 molecular weight (Mr) peripheral proteins (distinctive in their peptide mapping profiles and earlier designated v1, v2, and v 3) as well as the receptor alpha 2 beta gamma delta integral membrane subunits. Of the three proteins, only v1 is copurified with the membrane-bound receptor, while v2 and v3 are prominent cytosolic proteins, which are retained at significant levels in receptor-rich membranes during multistep centrifugation and affinity partitioning purification procedures [Gysin, R., Wirth, M., & Flanagan, S. D. (1981) J. Biol. Chem. 256, 11373-11376]. Peptide mapping analysis of Torpedo v3 and rabbit skeletal actin indicates that the two proteins are closely related. The enzymatic activity, creatine phosphokinase (EC 2.7.3.2), copurifies with v2 during chromatofocusing fractionation of the cytosol. The Torpedo electroplax form of creatine phosphokinase has an electrophoretic mobility identical with that of the mammalian skeletal muscle form of the enzyme. Upon release of the membrane-bound forms of v1, creatine phosphokinase, and actin by the action of mild alkali, v1 remains in a high molecular weight form. Dissociation of v1 into lower molecular weight species requires urea or sodium dodecyl sulfate (NaDodSO4). Preparation of essentially pure v1 was achieved by eluting the v1 protein spots directly from naDodSO4-isoelectric focusing gels loaded with alkali extracts derived from membranes highly enriched in nicotinic receptor. Amino acid compositions of the purified fractions indicate that v1 and Torpedo creatine phosphokinase have distinct amino acid compositions from each other and from that of actin.(ABSTRACT TRUNCATED AT 250 WORDS)
Progress in FGF-2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF-2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF-2 gene and mutation of cys-70 and cys-88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF-2 protein in mammalian cells using MLV-based vectors. Single or double mutations of cys-70 and cys-88 to ser-70 and asp-88, respectively, markedly increased the amounts of FGF-2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp-88 residue. Addition of BMP2/4 secretion signal increased FGF-2 secretion, but also suppressed FGF-2 biosynthesis. The combination of BMP2/4 secretion signal and double cys-70 and cys-88 mutations increased the total amount of secreted FGF-2 protein >60-fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF-2 protein was functionally as effective as the unmodified FGF-2. An ex vivo application of rat skin fibroblasts (RSF) transduced with the modified FGF-2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF-2 vector increased serum FGF-2 level >15-fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing beta-galactosidase- or wild-type FGF-2-transduced control cells. This modified vector may be useful in FGF-2 gene therapy investigations.
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