Serum-free Mouse Embryo Cells Research Articles

Increased sphingoid base‐1‐phosphates and failure of neural tube closure after exposure to fumonisin or FTY720

Fumonisin B(1) (FB(1)) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB(1)-contaminated food is associated with increased risk for neural tube defects (NTDs). FB(1) induces NTDs in inbred LM/Bc mice. FB(1) inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. Pregnant SWV and LM/Bc mice were treated with FB(1) (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5-8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5-8.5). LC/MS was used to detect sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB(1) (40 μM for 24 hr) and LC/MS was used to detect sphingoid base-1-phosphates. FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB(1) or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB(1), and Sa1P was higher in MEFs generated from the FB(1)-NTD-susceptible LM/Bc strain. Elevated sphingoid base-1-P after FB(1) or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB(1) or FTY720. Sa1P may represent a biomarker for FB(1)-NTD risk assessment.

The neuroprotective effects of electrolyzed reduced water and its model water containing molecular hydrogen and Pt nanoparticles

Human brain is the biggest energy consuming tissue in human body. Although it only represents 2% of the body weight, it receives 20% of total body oxygen consumption and 25% of total body glucose utilization. For that reason, brain is considered to be the most vulnerable part of human body against the reactive oxygen species (ROS), a by-product of aerobic respiration. Oxidative stress is directly related to a series of brain dysfunctional disease such as Alzheimer’ sd isease, Parkinson’s disease etc. Electrolyzed reduced water (ERW) is a functional drinking water containing a lot of molecular hydrogen and a small amount of platinum nanoparticles (Pt NPs, Table 1). ERW is known to scavenge ROS and protect DNA from oxidative damage [1]. We previously showed that ERW was capable of extending lifespan of Caenorhabditis elegans by scavenging ROS [2]. Molecular hydrogen could scavenge ROS and protected brain from oxidative stress [3]. Pt NPs are also a new type of multi-functional ROS scavenger [4]. Materials and methods In this research, we used TI-200S ERW derived from 2 mM NaOH solution produced by a batch type electrolysis device and model waters containing molecular hydrogen and synthetic Pt NPs of 2-3 nm sizes as research models of ERW to examine the anti-oxidant capabilities of ERW on several kinds of neural cells such as PC12, N1E115, and serum free mouse embryo (SFME) cells. We pretreated the ERW and 200 μM H2O2 and examined the neuroprotective effects of ERW on PC12, N1E115 and SFME cells, using WST-8 method. We also examined the intracellular ROS scavenging effects of ERW on N1E115 cells after pretreated cells with ERW and H2O2 using DCFH-DA. We checked the protective effects of ERW on mitochondria and cytoplasm by Rh123 and Fuo-3 AM stain. We also examined the ATP production of SFME cells after pretreated with ERW and H2O2 by Bioluminescence Assay Kit. Finally, we used dissolved hydrogen (DH) and Pt NPs as research models to examine their neuroprotective effects. Results

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

BackgroundThe Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.ResultsIn addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.ConclusionsThe mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor beta (TGF-beta) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-beta family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-beta-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial beta-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase‐9 in ras/myc‐transformed serum‐free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.

L-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nomega-nitro-L-arginine methyl ester (L-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the L-NAME containing water (4.24+/-0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the L-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-gamma; 100 U/ml) and lipopolysaccharide (LPS; 0.5 microg/ml) significantly enhanced NO production, and the presence of L-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-gamma and/or LPS treatments, not to mention by the L-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of L-NAME. Production of TNF-alpha by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of L-NAME. These results indicate that the inhibitory effects of L-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-alpha from macrophages (Mol Cell Biochem, 2007).

Differentiation of Serum-Free Mouse Embryo Cells into an Astrocytic Lineage is Associated with the Asymmetric Production of Early Neural, Neuronal and Glial Markers

Serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system (CNS), were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenic protein 2 (BMP2) to induce differentiation, and expression of cell-type specific markers. Nestin, a marker of early neural lineage, betaIII-tubulin, a marker of neuronal lineage, oligodendrocyte marker O4 (O4), a marker of oligodendrocytic lineage and glial fibrillary acidic protein (GFAP), a marker of astrocytic lineage, were analyzed. Characteristics of SFME cells, as a CNS progenitor, were identified and a possible mechanism, underlying SFME cell specification into an astrocytic lineage upon differentiation, was investigated. These markers were present, both at the initial proliferative phase and after induction of differentiation. GFAP expression increased strongly upon differentiation, while expression of the other markers changed very little. These results indicate that astrocytic differentiation is associated with the asymmetric production of these markers, rather than through induction of astrocytic markers.

Isolation of Ursolic Acid from Apple Peels and Its Specific Efficacy as a Potent Antitumor Agent

Dried apple peels were extracted with n-hexane, chloroform, and methanol successively. The portion of the chloroform extract that showed the strongest cytotoxic activity was purified by silica gel chromatography to isolate ursolic acid (UA). The amount of the isolated UA was 0.71% of the dried peels. Normal mouse embryo cells [serum-free mouse embryo (SFME) cells] and tumorigenic human c-Haras- and mouse c-myc-transformed SFME cells [r/m highly metastatic (HM)-SFME-1 cells] were treated with various concentrations of UA (2.5‐20µM) to investigate its effects on cell growth. UA at 10µ Ma ppeared very effective at suppressing the tumor cell growth, affecting more than 82% of r/mHM-SFME1 cells, while it inhibited cell growth in only about 7% of SFME cells .T umorigenic r/m HM-SFME-1 cells were also treated with various concentrations (2.5‐10µM) of epidermal growth factor (EGF) or aminoguanidine (AG) in the presence of UA (2.5‐ 10µM). Neither EGF nor AG seemed to have any effect on UA-inhibited cell growth. In the present study, it is revealed tha tU A could be av ery effective and promising agent for antitumor treatments,

Interferon‐gamma and lipopolysaccharide stimulation increases matrix metalloproteinase‐9 expression and enhances invasion activity in ras/myc‐transformed serum‐free mouse embryo cells

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma, 100 units/ml) and/or lipopolysaccharide (LPS, 0.5 microg/ml) for 24 h to investigate the effects of these ligands on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Aminoguanidine (AG, 1mM; a nitric oxide synthase [NOS] inhibitor) was also added along with IFN-gamma and LPS to analyze a possible association of NO with invasiveness. Treatment of cells with IFN-gamma alone did not alter MMP-9 mRNA expression or pro-MMP-9 production, but LPS alone and IFN-gamma+LPS co-treatment enhanced them significantly. TIMP-1 mRNA expression remained unchanged with or without treatment and the mRNA expression of MMP-9 exceeded that of TIMP-1 in LPS- or IFN-gamma+LPS-treated cells. Co-treatment of cells with IFN-gamma and LPS up-regulated invasiveness and indicated a possible involvement of NO in the enhancement of invasiveness. These results suggest that ras/myc SFME cells respond to inflammatory and infectious conditions and that they may possibly modulate their characteristics as cancer cells due to their increase in MMP-9 expression and invasion activity.

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-gamma, 100 units/ml) and lipopolysaccharide (LPS, 0.5 microg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-gamma and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 microM; an NO donor) and/or (+/-)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 microM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-gamma and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells.

Serum‐free mouse embryo cells generate a self‐sustaining feedback loop for an astrocyte marker protein and respond to cytokines and bisphenol A in accordance with the subtle difference in their differentiation state

Serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system, generated a self-sustaining feedback loop for glial fibrillary acidic protein (GFAP) expression after a period of cell passages. The period required was about 150 days (30 passages). SFME and high-GFAP-expressing SFME (G-SFME) cells were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenetic protein 2 (BMP2) to induce differentiation and their responses to cytokine signals were analyzed. Although differentiation was significantly induced in both cell types, SFME cells showed more obvious responses to the cytokine signals. Various concentrations of bisphenol A (BPA) (0.1 pg/ml to 1 microg/ml) were added to determine its effects on cell differentiation. A completely serum-free culture was developed for effective differentiation of G-SFME cells with LIF and BMP2, and GFAP expression was significantly increased in the presence of 1-100 pg/ml BPA. These increases were attributed to excessive activation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (Smad1) by the low-level BPA. The data obtained in the present study revealed that the sensitivity of the cells to LIF, BMP2 and BPA could change upon cell differentiation, suggesting that the cells may possibly respond differently to cytokines and endocrine disruptors depending on subtle differences in their differentiation state.

Low-level bisphenol A increases production of glial fibrillary acidic protein in differentiating astrocyte progenitor cells through excessive STAT3 and Smad1 activation

The effects of bisphenol A (BPA) on the differentiation of serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system, were examined. SFME cells were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenetic protein 2 (BMP2) to increase glial fibrillary acidic protein (GFAP) expression and induce cell differentiation. Various concentrations of BPA (0.1 pg/ml–1 μg/ml) were then added to determine their effects on the cell differentiation. SFME cells were effectively differentiated by LIF and BMP2 in completely serum-free cultures. Cell proliferation following cell differentiation was not significantly affected by low-level BPA. However, GFAP expression was significantly increased in SFME cells in the presence of 1–100 pg/ml BPA. These increases were due to excessive activation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (Smad1) by the low-level BPA.

Increased synthesis of GFAP by TCDD in differentiation-disrupted SFME cells

The effects of 2,3,7,8-tetrachloro-dibenzo- p-dioxin (TCDD) on gene expression and synthesis of glial fibrillary acidic protein (GFAP) in differentiation-disrupted serum-free mouse embryo (SFME) cells were examined. SFME cells were exposed to fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) to induce differentiation and increase GFAP synthesis. Disruption of differentiation was caused by low-level toluene, significantly inhibiting GFAP synthesis. TCDD at 0.01, 0.1 and 1 pg/ml in the presence of low-level toluene increased GFAP synthesis in the SFME cells, while expression of GFAP mRNA showed no significant change. The TCDD-treated SFME cells detached from the culture substratum, indicating an apparent change in cell adhesion. These results suggest that low-level TCDD further disrupts differentiation of SFME cells in the presence of low-level toluene by upregulating GFAP synthesis and by altering the ability in cell adhesion and that GFAP synthesis is not disrupted at transcription but at translation.

Toluene at environmentally relevant low levels disrupts differentiation of astrocyte precursor cells.

Recent findings that describe endocrine disruption caused by exposure to low levels of certain chemicals in the environment have led to a paradigm shift in the way toxicology studies are designed. Toluene at high levels damages the human central nervous system; however, the effects of toluene at low levels have not been studied. The authors used serum-free mouse embryo cells-a precursor of astrocytes-to predict the effect of chemicals on developing brain cells. When serum-free mouse embryo cells were exposed to low levels of toluene, induction of glial fibrillary acidic protein was inhibited. This study demonstrated that environmentally relevant low levels of toluene could disrupt normal prenatal brain development.

Neural stem cells lose telomerase activity upon differentiating into astrocytes.

Serum-free mouse embryo (SFME) cells were established by D. Barnes et al., and are known to be a neural stem cell line, which differentiate into astrocytes upon treatment with TGF-beta. Therefore, SFME cells is thought to be a model well suited to analyze the differentiation mechanism of neural stem cells. Until now, we have investigated the regulation mechanisms of telomerase activity and telomere length in human cancer and normal cells. Telomerase is the enzyme responsible for the synthesis and maintenance of telomere repeats located at chromosomal ends and is normally expressed in embryonic and germline cells, but not in most normal cells. Here, using SFME cells, we attempted to analyze the regulation mechanism of telomerase activity in neural stem cells and to detect a change upon differentiation into astrocytes. When SFME cells were cultured in the presence of TGF-beta, cells showed anelongated morphology and decreased its growth to 50% of control culture. Cells also expressed the glial fibrillary acidic protein (GFAP), a marker for astrocytes,indicating that TGF-beta induced differentiation in SFME cells from neural stem cells into astrocytes. At the same time,TGF-beta also inhibited telomerase activity and repressed the expression of the mouse telomerase reverse transcriptase(mTERT), demonstrating that SFME cells was vested with a finite replicative life span upon treatment with TGF-beta. To understand the mechanisms regulating mTERT levels during differentiation into astrocytes, we have estimated the expression level of c-myc, which is known to be a key molecule in activating the TERT promoter. As a result, TGF-beta-treated SFME cells were shown to repress the expression of c-myc. Furthermore, promoter analysis, using the 5'-region of the mTERT gene, which possess two E-box elements bound to c-Myc/Max, demonstrated that mTERT promoter activity greatly decreased in TGF-beta-treated SFME cells as compared to non-treated SFME cells. These suggest that c-myc might play a critical role in the expression of mTERT, and that down-regulation of c-myc dependent upon the astrocytic differentiation in SFME cells might cause the repression of mTERT in TGF-beta-treated SFME cells.

Simian virus 40 large T antigen J domain and Rb-binding motif are sufficient to block apoptosis induced by growth factor withdrawal in a neural stem cell line.

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation.

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.

The Human Urinary Epidermal Growth Factor (EGF) Precursor: ISOLATION OF A BIOLOGICALLY ACTIVE 160-KILODALTON HEPARIN-BINDING PRO-EGF WITH A TRUNCATED CARBOXYL TERMINUS

In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal growth factor (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues 21-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human fibroblasts, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a growth factor for serum-free mouse embryo cells.

Differentiation of serum‐free mouse embryo cells into astrocytes is accompanied by induction of glutamine synthetase activity

Serum-free mouse embryo (SFME) cells derived in a defined serum-free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serum-free medium. Exposure of SFME cells to transforming growth factor beta (TGF-beta) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF-beta or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TFG-beta or serum. The induction of GS activity was dose- and time-dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF-beta or serum.

Down regulation of nestin by TGF-beta or serum in SFME cells accompanies differentiation into astrocytes.

The serum-free mouse embryo (SFME) cell line, derived from 16-day-old mouse embryos in medium in which serum was replaced by growth factors and other supplements, has been cultured for more than 200 generations. SFME cells are nontumorigenic, lack gross chromosomal abnormalities, and display characteristics of CNS progenitor cells. SFME cells show reversible induction of the astrocyte-specific marker glial fibrillary acidic protein when cultured in the presence of TGF-beta or serum. In order to determine if SFME cells exhibit further characteristics of CNS progenitor cells we investigated the expression of the gene encoding nestin, an intermediate filament protein expressed by neuroepithelial stem cells of the CNS. SFME cells express nestin in serum-free medium, and nestin expression is reversibly down-regulated by TGF-beta or serum. These results demonstrate that nestin expression is regulated by factors present in serum and support the hypothesis that SFME cells represent a CNS progenitor cell.