Kyoko Kano-Tanaka

Proliferation and synapse formation of neuroblastoma × glioma hybrid cells: Effect of glia maturation factor

Glia maturation factor (GMF), extracted from bovine brain, stimulated DNA synthesis and proliferation of glioma cells and hybrid cells derived from glioma and neuroblastoma cells (NG108-15), but had no effect on neuroblastoma cells. The synapse formation of NG108-15 cells with rat striated myotubes was lower in the presence of GMF than the control and also lower after treatment with prostaglandin E1 (PGE1) plus theophylline, indicating that GMF did not induce functional differentiation of NG108-15 cells. The results show that expression of mitogenic action for GMF in the hybrid cells is a property derived from the glioma parent, and that NG108-15 is therefore an excellent model for studying glial-neuronal interactions.

Identification of growth-promoting factors from various glioma cell lines and partial purification of the factor from C6 solid tumor.

Growth-promoting factors in the extracts of various glioma cell lines (C6, LRM55 and 354A) were investigated. The cell extracts of astrocytoma (C6) and mixed glioma (LRM55) showed a high mitogenic activity to normal glioblasts. With its low content of intracellular growth-promoting factor, rat peripheral glioma (354A) exhibited a high proliferative response to C6 cell extracts. The factor which was partially purified from C6 solid tumor by ion exchange and gel filtration column chromatographies had two forms of different molecular weights (150,000 Mr and 35,000 Mr) and the low molecular weight form was further split into two acidic proteins (pl 5.0 and pl 6.0) by isoelectric focusing. The mitogenic activity of the factor was susceptible to heat and to proteases, and the factor showed no esteropeptidase activity. These physicochemical properties closely resemble those of glia maturation factor from porcine brains.

Detection and partial purification of glial growth inhibitory factor(GGIF) in the conditioned medium of neuroblastoma cells

Glial growth inhibitory factor (GGIF) was detected in the culture medium of mouse neuroblastoma cells. The partial purification and characterization of GGIF revealed that it was an acidic and heat-unstable protein and separated into two molecular weight forms (GGIF1: 33.0 to 40.5 K Mr; and GGIF2: 31.0 to 34.0 K Mr) on DEAE. Sephacel column chromatography. GGIF inhibited the growth of neoplastic glial cells as well as normal glioblasts in monolayer culture. The inhibitory effect of GGIF on proliferation of glioblasts appeared even after the incubation for less than 1 h, and retained thereafter unless the factor was removed. Immobilized GGIF exposed to glioblasts was reusable, suggesting the existence of a certain component or receptor mediating GGIF action in the plasma membrane.

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.

Tumorigenicity, major histocompatibility antigens, and karyotypes of interspecific hybrids between mouse neuroblastoma and rat glioma or liver cells

Five interspecific hybrids of mouse neuroblastoma with rat glioma (NG108-15, 140-3, and 141-B) or with nontransformed rat liver cells (NBr-10A and NBr-20A) were examined for major histocompatibility (MHC) antigens and tumorigenicity in comparison with their karyotypes. Both mouse and rat MHC antigens were present in each hybrid population, as determined by a simple cytotoxicity test. All five hybrid cell lines produced tumors in athymic nude mice with varied take incidences. Four hybrid cells, NG108-15, 140-3, NBr-10A, and NBr-20A, were highly tumorigenic. Their karyotypes were characterized by a higher modal chromosome numbers than would be expected from the fusion of parent cells in which at least one parent contained an increased number of chromosomes. In contrast, 141-B cells, with massive loss of chromosomes from both malignant parents, were weakly tumorigenic. The results suggest that the retention of marker chromosomes as well as double minutes (DMs) or microchromosomes of neuroblastoma origin may be required for expression of malignancy in these hybrid cells. The survival time of tumor-bearing mice also varied within the five cell lines, but it was significantly short in NG108-15, which yielded lung metastases in the host animals.

Chromosome studies in mouse neuroblastoma cells.

Chromosome studies were carried out in cultured cells from a mouse neuroblastoma. C1300 tissue and in three clones established from this tumor. They possessed characteristic karyotypes with remarkable markers. Double minutes (DMs) were demonstrated in all cell lines, in addition to some other chromosomes aberrations, such as microchromosomes and chromosome pulverization.