Hidehiko Tsunooka

Characterization and partial purification of neuroblastoma growth inhibitory factor from the culture medium of glioblasts.

Neuroblastoma growth inhibitory factor (NGIF) exists in the conditioned medium of normal rat glioblasts. When neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) were cultured in the presence of the factor, the cell growth rates and DNA synthesis were markedly inhibited and the morphological differentiation including neural process formation was induced. However, the factor neither altered the growth rate nor the morphology of non-neuronal cells such as glial cell lines (C6 and 354A) or fibroblast (3T3). The molecular weight of the factor was estimated to be 75,000 Mr by gel filtration with Bio-Gel P-200, and the isoelectric point was 5.8. The factor was devoid of esteropeptidase activity, and susceptible to protease and thermal treatment. The growth inhibitory action of the factor was unrelated to the intracellular contents of cyclic AMP and GMP. The ability of NGIF to suppress preferentially the neural growth suggests its regulatory role in normal brain development.

Inhibition of growth of mouse neuroblastoma cells by protein factor derived from rat glioblasts

Growth inhibitory factor for mouse neuroblastoma cells was detected in the culture medium of fetal rat glioblasts, and was partially purified and characterized. The factor had an apparent molecular weight of 75,000 and an isoelectric point of 5.8, and showed no esterase activity. It possessed the activity to promote morphological differentiation including the formation of neural processes and the inhibition of cell division when tested on mouse neuroblastoma cell lines (Neuro 2a, NS-20Y, and NIE-115). The activity was susceptible to protease digestion and heat treatment. The serum over 25% cancelled the inhibitory activity of this factor. The factor failed to increase the intracellular contents of cAMP and cGMP. It showed no effect on either morphological differentiation or proliferation of glial cell lines, suggesting that under physiological conditions the factor acts specifically on neuronal cells.

Glial cell growth-promoting factor in astrocytoma (C6) cell extracts

The presence of glia maturation factor (GMF)-like activity was demonstrated in rat astrocytoma cells (C6 cells). The extracts of C6 cells enhanced the DNA synthesis of cultured glioblasts 3-fold at the maximum, inducing such morphological changes as extrusion of processes. C6 cells also showed a proliferative response to the extracts, though the responsiveness in terms of effective concentration of C6 extracts was about a half of the glioblast responsiveness. The extracts lowered growth rate of neuroblastoma cells and especially decreased their DNA synthesis without a morphological differentiation.

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3?:5?-monophosphate(dibutyryl cAMP) and prostaglandin E(1) (PGE(1)). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE(1) (0.5 ?g/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 ?g/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE(1), and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine ?-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.

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.

Inhibition by neuroblastoma growth inhibitory factor of ascites-type neuroblastoma cell growth in coculture with normal glioblasts

A new ascites type neuroblastoma clone (NAs-1), which is characteristic both in anchorage-independent growth and catecholaminergic functions, attached on the monolayer culture of glioblasts and was subjected to morphological differentiation including the extrusion of neuronal processes. Other conventional neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) as well as NAs-1 in cocultured with normal glioblasts underwent a decrease in cell growth rates and DNA synthesis under the effect of the neuroblastoma growth inhibitory factor (NGIF) produced by glioblasts. After their NGIF production had been reduced by u.v. irradiation, glioblasts lost the growth-inhibitory and differentiation-promoting effects in coculture with NAs-1. The supplement of NGIF into u.v.-treated glioblasts restored the dose-dependent growth inhibition of NAs-1. The addition of nerve growth factor into the coculture system brought about neither the marked effect on growth inhibition of NAs-1 nor the morphological differentiation. The results imply a direct function of NGIF on the paracrine regulation of neuroblastoma cell growth in the coculture with normal glioblasts.

In vitro and in vivo growth characteristics of a new ascites-type neuroblastoma cell from mouse C1300 neuroblastoma.

A clonal ascited type cell, NAs-1, was obtained in culture from a mouse neuroblastoma C1300. The cells were adapted to anchorage-independently grow in the flask by the in vitro-in vivo alternate passage technique, and retained the ability of growing and producing ascites fluid when intraperitoneally injected into mice. Although the majority of growing cells in culture medium showed a small and round cell shape without any neuronal process, occasionally non-specific attachment onto the flask surface was observed, but devoid of the extrusion of processes. Karyotype analysis showed a homogeneous chromosome number, 40, with a marker chromosome [t(13:16)] and a minichromosome. Catecholamines, norepinephrine and dopamine, were found in the cell extracts and the contents of dopamine was particularly high as shown in another catecholaminergic neuroblastoma cell, N1E-115. Neuron specific enolase (?-subunit) was also detected. The treatment of the cells by dibutyryl cyclic AMP, prostaglandin E(1), or BL191 (phosphodiesterase inhibitor) induced the biochemical differentiation in terms of catecholamine and cyclic AMP contents, but failed to promote typical morphological differentiations including the extension of process or the significant promotion of adherence onto the flask surface.

Serum acute phase reactants in pediatric patients; especially in neonates.

We studied serum acute phase reactant (APR) levels in 45 pediatric surgical patients. Alpha 1-acid glycoprotein (α1-AG) showed a peak value on day 3 postoperatively (P.O.) and alpha 1-antitrypsin (α1-AT) showed a high value on days 3–5 P.O. These glycoproteins returned to normal levels by day 21 P.O., but the level of haptoglobin (Hp) remained high until day 21 P.O. The postoperative changes of α1-AG and α1-AT correlated with the process of recovery from inflammatory conditions, but C-reactive protein (CRP) reached a peak on days 1–2 P.O. and returned to normal limits by day 14 P.O. In patients with infection, CRP returned to the normal level rapidly before recovery from infection. Of the 3 glycoproteins, α1-AG seemed to be a valuable indicator of the pathological conditions. Postoperative changes in APR levels should be useful for early detection of postoperative complications. Persistent ileus led to an increase in APR levels, as a consequence of an inflammatory reaction due to breakdown of the intestinal mucosal barrier.

Biological functions of neuroblastoma growth inhibitory factor(NGIF) derived from glia cells in culture

Great emphasis is being placed on the identif ication of neurotransmitter systems involved in the symptomatic manifestations of endocrinological disorders. Compellin~ evidence suggests that hypothalamic donaminergic neurons, whose cell bodies lie in the tuberoinfundibulum, are selectively involved in the disorders of prolactin secretion. Our study provides evidence that functional linkage of the hypothalamic dopaminergic neurons with prolactin cells initiated during fetal develonment; dopamine seems to be involved in the regulation of proliferation of prolactin cells. Therefore, altered profiles of the dopaminergic system during pregnancy influence the proliferation of prolactin cells in offspring.