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.     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Enhanced production and extracellular deposition of the endothelial- type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum- free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.     

Modulation of extracellular proteolytic activity and anchorage-independent growth of cultured cells by sarcoma cell-derived factors: relationships to transforming growth factor-beta

We have previously described a factor(s) produced by 8387 fibrosarcoma cells, which can affect plasminogen activator (PA) activity of cultured cells. Since then, transforming growth factor-beta (TGF beta) has been established as a major growth factor/growth inhibitor that regulates both the expression and activity of PAs and their endothelial-type inhibitor (PAI-1). The present study was undertaken to characterize the 8387 fibrosarcoma cell-derived factor(s) and to investigate its relationships to TGF beta by analysis of modulation of PA activity and cell growth. The fibrosarcoma cell-derived proteins were partially purified from serum-free conditioned culture medium using Bio-Gel P-10 chromatography. Two separate fractions with apparent molecular weights of 16,000 and 12,000 contained activities that both decreased the secretion of PA activity by human lung fibroblasts and inhibited the soft agar growth of A549 lung adenocarcinoma cells. Both factors affected similarly the production of urokinase-type PA and PAI-1 in various cell lines and enhanced anchorage-independent growth of murine AKR-2B fibroblasts. The effects of these factors thus resembled those of TGF beta. The immunological relationships between the Mr 16,000 and Mr 12,000 factors and TGF beta were therefore studied using neutralizing anti-TGF beta antibodies. The TGF beta antibodies efficiently inhibited the effects of the Mr 16,000 factor but not those of the Mr 12,000 factor in cell culture assays. The results suggest that 8387 fibrosarcoma cells produce two major growth inhibitors, one of which is closely related to TGF beta.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.     

Minactivin: a human monocyte product which specifically inactivates urokinase-type plasminogen activators

Culture supernatants from monolayers of human peripheral monocytes strongly inhibited colorimetric assays of urokinase in which plasmin was measured by esterolysis. This inhibitory activity of monocyte culture supernatant was enhanced after culture with muramyl dipeptide. Inhibition was specific for plasminogen activators of Mr 52 000 and 36 000, as shown by three methods: (1) inhibition of plasminogen-dependent fibrinolysis; (2) inhibition at the level of plasminogen activation in a colorimetric assay; (3) the irreversible loss of plasminogen-activating activity, as evidenced by electrophoresis, after preincubation with culture media. The factor responsible for this inactivation (which we propose to call minactivin) had an apparent Mr of 66 000 on Sephacryl S300 gel chromatography and interacted with enzyme in a biphasic manner: a rapid partial inhibition (reversible by sodium dodecyl sulphate) was followed by slow inactivation (irreversible by sodium dodecyl sulphate). It is proposed that secretion of minactivin by monocytes may contribute to regulation of extracellular proteolysis at sites of tissue injury.     

Growth control in cultured 3T3 fibroblasts. V. Purification of an Mr 13,000 polypeptide responsible for growth inhibitory activity

A growth regulatory factor, which reversibly inhibits DNA synthesis and proliferation of fibroblasts, has been isolated from medium conditioned by exposure to density-inhibited mouse 3T3 cells. This factor, termed FGR-s (13K), yielded a single polypeptide (Mr 13,000) when analyzed by SDS PAGE under both reducing and nonreducing conditions. The dose-response curve of growth inhibition by FGR-s (13K) showed that 50% inhibition of 3T3 cell proliferation was achieved at a concentration of approximately 3 ng/ml, corresponding to approximately 0.23 nM. The activity of FGR-s (13K) was depleted by passing the material over an affinity column containing the monoclonal antibody 2A4; this monoclonal antibody had been previously characterized to bind to the Mr 13,000 polypeptide. These results indicate that we have purified a growth regulatory factor that acts to inhibit the proliferation of cells in an autocrine pathway.     

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.     

Transforming growth factor-beta inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes.

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-beta (TGF-beta). In this paper, we show that TGF-beta inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-beta-treated chondrocytes show reduced 125I-IL-1 binding that returns to a normal level when TGF-beta is removed from the culture medium. The inhibitory effect of TGF-beta is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor-ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of Mr = 116 and 80 kDa and a minor band of Mr = 100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-beta treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-beta to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Possible involvement of a GTP-binding protein, the substrate of islet-activating protein, in receptor-mediated signaling responsible for cell proliferation

Serum-induced DNA synthesis, as measured by increases in [3H]thymidine incorporation, in Swiss mouse 3T3 fibroblasts was markedly inhibited by exposure of the cells to islet-activating protein (IAP), pertussis toxin. The inhibition was well correlated with the toxin-induced ADP-ribosylation of a membrane GTP-binding protein with Mr = 41,000. The IAP-induced inhibition of cell growth was characterized by the following two features. First, the inhibition was selective to certain growth factors. DNA synthesis in 3T3 cells was supported by a combination of one of the competence factors and a progression factor such as insulin or epidermal growth factor. IAP was inhibitory when thrombin, fibroblast growth factor, prostaglandin F2 alpha, or phosphatidic acid was employed as a competence factor, but was not inhibitory when DNA synthesis was induced by combined addition of cholera toxin or phorbol ester with insulin. Second, IAP-induced inhibition was still observed when the toxin was added to cell culture 1-6 h later than the addition of the IAP-sensitive competence factors, which triggered rapid cellular responses such as adenylate cyclase inhibition, releases of inositol trisphosphate and arachidonic acid, and 45Ca influx within several minutes (Murayama, T., and Ui, M. (1985) J. Biol. Chem. 260, 7226-7233; Murayama, T., and Ui, M. (1987) J. Biol. Chem. 262, 5522-5529). Thus, IAP substrate GTP-binding protein(s) appears to be involved in the duration of rapid signals or the occurrence of new slow signals which are responsible for growth factor-induced cell proliferation. The site of the involvement may be proximal to protein phosphorylation by phorbol ester-activated and cAMP-dependent kinases.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts

DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.     

Neurons differentiating from murine neural crest in culture exhibit sensory or sympathetic-like calcium currents.

The trunk neural crest gives rise to peripheral sensory and sympathetic neurons. In culture, neural crest cells can be induced to differentiate into either neuronal phenotype. Few studies have examined the differentiation of physiological properties in cultures of neural crest cells. Using whole-cell recordings, our study examined the effects of growth factors on high-voltage-activated calcium current profiles exhibited by neurons differentiating in culture. We compared these profiles with those exhibited by sensory and sympathetic neurons. Neural crest cells in culture gave rise to neurons with calcium current profiles identical to either sensory or sympathetic neurons, depending on the growth conditions. On average, the calcium current profile for sensory neurons was 23% (L), 51% (N), and 12% (P), while sympathetic neurons had a similar L-type current (20%), higher N-type (76%), and lower P-type (4%). Neural crest cells cultured with human leukemia inhibitory factor plus somite cells produced neurons with a sympathetic-like calcium current profile (L: 17%, N: 75%, and P: 4%). However, murine leukemia inhibitory factor (L: 25%, N: 52%, and P: 13%) and ciliary neurotrophic factor (L: 18%, N: 49%, and P: 9%) plus somite cells produced neurons with sensory-like calcium current profiles. These growth conditions did not modify the calcium current profiles of neurons cultured from embryonic and neonatal ganglia. Similarly, murine leukemia inhibitory factor produced a greater percentage of neurons (57%) with sensitivity to capsaicin (sensory phenotype) than human leukemia inhibitory factor (3%). Physiological traits can be a useful tool for the determination of neuronal phenotype in culture where other traits may be less stable.     

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 to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. 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.     

Hormonal induction of specific protein synthesis in murine Leydig tumor cells

The M5480A murine Leydig cell tumor was used to investigate the effects of three hormones, which produce distinct biochemical actions, on cytoplasmic protein synthesis. Human choriogonadotropin treatment of tumor-bearing mice induced the synthesis of six proteins with relative molecular weights (Mr) of 135K, 82K, 60K, 19K, 18.2K and 17.3K (K = kilodaltons). Diethylstilbestrol induced one protein peak in common with the gonadotropin Mr = 135K) and three additional proteins with Mr's of 120K, 50K and 36K. Epidermal growth factor induced one major protein with Mr = 33K, which is similar to that of a protein induced in murine epidermal cells by tumor promoters. These studies demonstrate the induction of specific gene products in a hormone-responsive tumor.     

Stimulation by glia maturation factor of Ca-dependent phosphorylation of Mr 100 k protein in rat glioblasts

When quiescent rat glioblasts were stimulated by glia maturation factor (GMF), their intrinsic Ca²⁺-dependent phosphorylation of proteins, especially that of M_r 100 k protein, increased. The phosphorylation of M_r 100 k protein in the homogenate started rising 13 h (S phase) after GMF stimulation and reached the maximal level (8-fold greater than the control) at 26 h. Phosphorylation was also detected in intact cells by the use of [³²P]orthophosphate. Calmodulin augmented and W-7 (calmodulin inhibitor) slightly inhibited the phosphorylation, suggesting that Ca²⁺/calmodulin-dependent protein kinase may partly be involved in phosphorylation of the M_r 100 k protein. Subcellular fractionation experiments revealed that both M_r 100 k protein and its kinase were localized exclusively in the cytosol. We also found marked phosphorylation of M_r 100 k protein in neural tumor cell lines, mouse neuroblastoma (Neuro2a and NAs-1) and glioma (C6 and 354A). Since the peptide maps of ³²P-labeled peptides obtained by chemical cleavage from M_r 100 k protein of the cells were identical to those of glioblasts, the M_r 100 k proteins, regardless of cell origin, may be closely related in structure. Growth inhibitors, W-7 (50 μM), puromucin (2 μM), spongoadenosine (50 μM), diphenylhydantoin (0.3 mM), -sialosyl cholesterol (20 μg/ml) and protein kinase inhibitor, K252a (50 nM), lowered the phosphorylation of the M_r 100 k protein in the cell homogenate derived from glioblasts pretreated with the drugs for 24 h.

M_r 100 k protein of glioblasts and C6 cells was immunoprecipitated by anti-elongation factor-2 (EF-2) antiserum indicating an identity or similarity in structure between the protein and EF-2. These findings provide a possibility that cell growth may be brought about through a phosphorylation of M_r 100 k protein as one of the signal transduction processes subsequent to a mitogen stimulation.     

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.     

Feeder layer- and serum-free culture of human embryonic stem cells

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.