Established cell lines of mouse marrow adherent cells producing differentiation factor(s) for the granulocyte-macrophage lineage

Summary Two continuous cell lines derived from long-term cultures of AKR mouse bone marrow adherent cells were isolated. These cell lines release colony stimulating activity (CSA), a factor that induces in vitro differentiation of granulocyte-macrophage progenitor cells. The colony forming cells and cluster forming cells in mouse marrow responsive to CSA from cell line conditioned medium were compared with those responsive to CSA from mouse lung conditioned medium (MLCM). Colony forming cells were characterized by analysis of their density distribution after equilibrium centrifugation in density gradient. Cluster forming cells were characterized by analyzing the progeny of individual clusters after transfer to fresh semisolid culture medium containing MLCM. The results obtained indicate that the CSA from cell line conditioned medium closely compares with the CSA from MLCM in terms of the populations of colony and cluster forming cells stimulated. This work was supported by a research grant from the Institut National de la Santé et de la Recherche Médicale (CRL 802620), Paris, France.     

Erythropoietic enhancing activity (EEA) secreted by the human cell line,GCT

Medium conditioned by the monocyte-like cell line GCT contains colony-stimulating activity (CSA), a mediator of in vitro granulopoiesis. Also, the conditioned medium (CM) contains erythroid-enhancing activity (EEA), which can be demonstrated in a system utilizing either nonadherent marrow or blood mononuclear cells, erythropoietin (1–2 units/ml), and 20 ml/dl fetal calf serum. Under these conditions, GCT CM enhances the growth of CFU-E and BFU-E. Attempts were made to characterize the molecular features of EEA. Serum-free GCT cell CM was fractionated on Sephacryl S200 and Ultrogel AcA54. EEA and CSA cochromatographed with apparent molecular weights of ～ 40,000 daltons on Sephacryl and ～ 30,000 daltons on Ultrogel. Fractionation on DEAE Sephacel led to an apparent separation of CSA from EEA; however, when diluted, the fractions containing CSA had EEA. Undiluted fractions containing potent CSA inhibited erythropoiesis; however, dilution of these fractions resulted in marked EEA. Diluted crude GCT CM and DEAE Sephacel fractions enriched in EEA were also capable of sustaining BFU-E in liquid culture and mediating erythropoietin-independent colony growth. CSA could not be unequivocally separated from EEA on concanavalin A-Sepharose, since the diluted void volume containing CSA also had EEA. EEA was present in CM boiled for 60 minutes, whereas CSA was markedly reduced but not abolished. The inverse relationship between CSA concentration and EEA mandates dilution of fractions when bioassayed for these two activities. Although CSA and EEA are similar in molecular weight, they appear to be partially separable by ion-exchange chromatography and heat stability.     

Helper (OKT4) T cells from human T cell colonies produce potent colony-stimulating activity

Confluent T cell colonies were grown by culturing blood mononuclear cells in double agar layers containing autologous plasma and phytohemagglutinin (PHA) for one week (37 degrees C, 5% CO2). The plates were then overlaid with serum-free alpha medium which was harvested after 24 h. This medium was demonstrated to have colony-stimulating activity (CSA) of greater potency than conventionally prepared PHA-leukocyte conditioned medium, which was prepared by incubating cells from the same donors. Removal of OKT4-positive cells using a monoclonal antibody and complement abolished CSA production by cells from T cell colonies while the removal of OKT8-positive cells had no effect.     

Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium

Spinal motoneurons from chick embryos were purified by retrograde transport and fluorescence-activated cell sorting. Growth conditions for motoneurons were studied, with experiments focused on the effects of conditioned media from chick myotubes, fibroblasts, and spinal cord dividing cells. Motoneurons rapidly extended neurites when plated onto polylysine-coated dishes that had been exposed to these conditioned media. Enzymatic analysis of the substratum-binding, neurite outgrowth-promoting activity from myotube-conditioned medium indicated that it contained heparan sulfate and protein. The neurite outgrowth-promoting activity sedimented as a peak centered at a density of 1.34 in associative cesium chloride gradients, and eluted near the void volume of a Sepharose CL-6B column. Inclusion of myotube conditioned medium in the culture medium of motoneurons also enhanced their survival over periods greater than 2 days in culture. This enhancement of survival could not be explained by myotube-conditioned medium providing motoneurons with a continuous supply of the neurite outgrowth-promoting activity. Media conditioned by spinal cord dividing cells and fibroblasts supported motoneuron survival to some extent, but this effect was not as great as that of myotube-conditioned medium.     

Differences in the in vitro growth pattern of fresh and cryopreserved granulo-monopoietic precursors

A study of the in vitro growth model of human granulo-monopoietic precursors (CFU-GM) before and after cryopreservation using both leukocyte feeder layers and GCT conditioned medium as the source of colony stimulating activity (CSA) is reported. The number of colonies produced with fresh cells was linearly related to the amount of marrow seeded with both CSA sources, whereas after cryopreservation this was true with feeder layers, and with GCT only at relatively high cell concentrations. This might indicate the production of granulopoietic stimulators on the part of a second population that is at least partly resistant to freezing. It seems more likely, however, that these results depend mainly on a sublethal damage to CFU-GM induced by freezing, thus making the cells hyporesponsive to some forms of CSA, such as those contained in GCT conditioned medium.     

Preparation of colony stimulating activity from large batches of human urine and production of antisera against it.

In vitro induction of myelopoetic colonies from mouse bone marrow has been used for measurement of leucopoetic colony stimulating activity (CSA) isolated from large batches of human urine. After high flow dialysis in artificial kidneys and immediate adsorption to DEAE-Cellulose, followed by purification on Con A-Sepharose, treatment with insoluble Papain and gelfiltration on Sephadex G 100, enrichment of CSA was about 6,000-fold. An important step of the enrichment procedure was the separation from a CSA-inhibiting protein, probably combining with CSA. Specific activity was further increased by preparative polyacrylamide gel electrophoresis to 5.3 X 10(6) units per mg protein. The total enrichment exceeded 25,000-fold. The final purification product consisted of a group of closely related proteins with high specific activity. Antisera raised with one of the electrophoretic fractions suppressed bioactivity in each of the different purification steps including the final CSA fractions differing in electrophoretic mobility. The antisera furthermore inhibited CSA in human lung and monocyte conditioned media but had only very little effect on partially purified CSA from stimulated human lymphocytes as well as CSA derived from mouse lung conditioned medium.     

Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line

24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.     

Conditioned medium from cultured rat hepatocytes completely blocks induction of glutamine synthetase by dexamethasone in several rat liver epithelial cell lines

A variant RL-ET-1G of a rat liver epithelial cell line (RL-ET-1) characterized by a very high inducibility for glutamine synthetase (GS) in response to dexamethasone was established by cultivation in glutamine-free, glutamate-supplemented culture medium. Using this cell line, conditioned medium produced by periportal hepatocytes in primary culture was found to suppress this induction, acting with a lag-phase of about 8 h irrespective whether the GS activity was basal or preinduced. Analysis of the response of several epithelial cell lines to the conditioned medium showed a reciprocal relationship between the dexamethasone-dependent induction and the residual activity after exposure to the conditioned medium, indicating that a hypothetical factor in the conditioned medium was interfering with the induction process but not with the basal GS level of these cells. Careful analysis revealed that the effect of the conditioned medium was neither due to deficiency of a component used up by the hepatocytes, nor due to glutamine or ammonia, both of which affected GS activity at concentrations above 0.5 mmol/L. The hypothetical factor was found to be quite small (molecular mass range 100–500 Da), heat and acid stable, as well as highly water soluble. Most interestingly, the conditioned medium did not suppress GS induction in astroglial cells and in the two hepatoma cell lines C2 and FAO, but strongly diminished the spontaneous induction of GS in cocultured pig hepatocytes, suggesting that the hypothetical factor acts primarily on normal nontransformed liver-derived cell populations.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

Isolation of a teratocarcinoma stem cell lectin implicated in intercellular adhesion

We have previously identified a cell surface teratocarcinoma stem cell lectin with a fucan/mannan specificity. We now report the purification of the hemagglutinin (lectin) from stem cell conditioned medium by exclusion on a Sepharose 2B column, followed by elution with 0.5M NaCl from DEAE-cellulose, providing an overall purification of about 90-fold. When this material was analyzed, by SDS-polyacrylamide gel electrophoresis, a major band of Mr 56000 was consistently observed. Hemagglutination activity was renatured from the gels and localized exclusively to a region of the gel that, as detected by fluorography, contains only the 56-kDa component. This suggested that this polypeptide comprises the lectin.     

Fetal rat lung fibroblasts produce a TGF beta homolog that blocks alveolar type II cell maturation

Normal growth and differentiation of the lung depends upon mesenchymal-epithelial interactions during development. Recombination experiments using immature (Day 17) and mature (Day 21) fetal rat lung fibroblasts (FRLF) revealed that the stimulatory effect of mature fibroblasts on fetal type II epithelial cells is blocked by immature fibroblasts. Similarly, conditioned medium from Day 17 FRLFs blocks the stimulatory effect (fibroblast-pneumonocyte factor) of Day 21 conditioned medium on type II epithelial cells. This blocking activity is nondialyzable, trypsin sensitive, and heat stable. Its activity is neutralized by an antibody to TGF beta, in both conditioned media and recombined cell studies, and its activity is mimicked by TGF beta. Developmentally, TGF beta-like activity is present in conditioned medium from 15- to 19-day FRLF, decreasing precipitously between 19 and 21 days gestation. Northern blot analysis of mRNAs from fetal rat lung fibroblasts on Days 17, 19, and 21 revealed expression of TGF beta at all three stages of development.     

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

Bovine endothelial cell plasminogen activator inhibitor. Purification and heat activation

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant. The yield was 2.2 mg from 1.61 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 kDa. The amino-terminal 40-amino-acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI-1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI-1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 X 10(5) U/mg) and 4 M guanidine-HCl (1.5 X 10(5) U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100 degrees C for 2.5 min, generating the specific activity of 1.0 X 10(6) U/mg. The heat-activated PAI lost its activity during incubation at 56 degrees C for 30 min, but repeated heat at 100 degrees C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37 degrees C, 56 degrees C and 80 degrees C, however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat-induced, specific conformational change, but tends to be masked again during renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form.     

A protein from Daudi cell line conditioned medium induces ovarian dysgenesis in Drosophila melanogaster.

From a medium in which Daudi cells had been grown, we isolated by HPLC a protein that caused ovarian abnormalities in adult females of Drosophila melanogaster when injected into preblastoderm embryos. This protein, whose apparent M(r) is between 30,000 and 50,000, was found to be a moderately polar compound which is heat stable and whose activity is destroyed by acidification. The protein is characteristic of medium conditioned from Daudi cells.     

Adhesion-promoting factor from embryonic fibroblasts for normal liver epithelial cells

In the present study we show that adhesion of normal rat liver epithelial cells (RL34) to substratum coated with type I collagen (collagen substratum) is promoted by a factor involved in 80% ammonium sulfate precipitated proteins from serum-free conditioned medium (PCM) of rat embryo fibroblasts. Adhesion of RL34 cells to collagen substratum was promoted dose dependently by whole PCM and the maximum effects on adhesion could be achieved by 200 micrograms/ml whole PCM. Kinetics studies with 100 micrograms/ml whole PCM showed that adhesion proceeded very slowly, taking 16 h to reach a plateau. Adhesion-promoting activity in whole PCM was sensitive to treatments with trypsin, acid, and heat but stable to dithiothreitol treatment. Further purification of whole PCM was performed using a combination of chromatography on blue Sepharose column, gel filtration column and heparin Sepharose column. The partially purified proteins, referred to as heparin PCM, are not bound or only weakly bound to heparin under physiological ion strength and pH, and the apparent molecular weight (Mr) range is estimated to be 40,000 to 60,000 from gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. When whole PCM or heparin PCM was used for coating on plastic or collagen substratum, they no longer exerted the promoting activity.     

Peptide amidation: evidence for multiple molecular forms of the amidating enzyme

Amidating enzyme extracted from porcine pituitary was separated into glycosylated and non-glycosylated forms by fractionation on a column of Concanavalin-A Sepharose. The molecular weights of the species present were assessed by HPLC gel exclusion chromatography, which demonstrated that both the glycosylated and the non-glycosylated forms of the enzyme comprise multiple components. The apparent molecular weights of the non-glycosylated forms ranged from approximately 35 kDa to 100 kDa; the glycosylated enzyme contained species with molecular weights ranging from 65 kDa to 135 kDa. Similar proportions of glycosylated to non-glycosylated enzyme (approximately 1:4) were found in the anterior and posterior regions of the pituitary; higher proportions (approximately 1:1) were observed in the thyroid, adrenals and pancreas. The glycosylated forms of the amidating enzyme were shown to exhibit the same mandatory requirement for copper as the non-glycosylated forms, and no differences were seen in respect of their stimulation by dopamine or their pH optima. Both forms catalysed the hydroxylation of glyoxylic acid phenylhydrazone, indicating a common mechanism of action. By these criteria, glycosylation does not affect the activity of the amidating enzyme.     

Isolation and characterization of a neu protein-specific activating factor from human ATL-2 cell conditioned medium.

The rat neu gene product is a 185 kD membrane bound tyrosine kinase that is closely related to, yet distinct from the epidermal growth factor receptor. The biochemical and cellular effects of a neu protein-specific activating factor (NAF) detected in human ATL-2 cell conditioned medium were recently described (1). To further characterize NAF, some of its physicochemical properties were examined and a method for purifying this factor from ATL-2 cell conditioned medium was developed. In these studies NAF was found to be heat stable and sensitive to the protease chymotrypsin. In addition, a method for purifying this activity was developed using a quantifiable, in vitro autophosphorylation assay system to measure NAF activity in fractions following ion-exchange and then reverse-phase HPLC.