Large scale purification and further characterization of a rat liver cell conditioned medium multiplication stimulating activity

The application of roller-bottle cell culture techniques and a relatively simple purification scheme has led to the isolation of milligram quantities of a polypeptide cell multiplication stimulating activity (MSA) from Buffalo rat liver cell conditioned medium. We have characterized the apparently homogeneous MSA with respect to its biological activity, its N-terminal amino acid residue, and its amino acid composition, and have tested the MSA for growth-promoting activity in a number of cell types.     

Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium.

A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.     

A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.     

The isolation of novel mesenchymal stromal cell chemotactic factors from the conditioned medium of tumor cells

Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important in understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Hypothalamic lesions stimulating growth hormone cell activity in the goldfish

Summary The cytology of the growth-hormone (GH) cells of the goldfish pituitary were examined following electrothermic lesions of the anterior praeoptic hypothalamus and telencephalon. Following lesions of the nucleus preopticus (NPO) light microscopy of the pituitary revealed a significant increase in the nuclear diameter and a degranulation of the GH cells. Lesions of the telencephalon anterior or dorsal to the NPO had no cytological effect on the GH cells. The ultrastructural appearance of the GH cells of NPO-lesioned fish was characterized by a marked degranulation of the cytoplasm and a proliferation of the rough endoplasmic reticulum indicative of enhanced secretory activity. The GH cells of the proximal pars distalis (PPD) are directly innervated by peptidergic (type A) and aminergic-like (type B) neurosecretory axons. Following lesions of the NPO, there was a marked reduction in the number of type A fibers in the PPD. These results suggest that the type A fibers innervating the GH cells originate in the NPO and act to inhibit the secretory activity of the GH cells.     

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.     

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.     

Effect of endothelial cell conditioned medium on the growth of human bone marrow fibroblasts

Both endothelial cells (EC) and fibroblasts, two discrete populations of hemopoietic stroma, are known to modulate the proliferation and differentiation of hemopoietic progenitors. Recent reports also demonstrated that EC stimulate the in vitro growth of fibroblasts via a soluble factor. This finding seems to support the hypothesis that EC may play a role in the pathogenesis of bone marrow fibrosis in myeloproliferative disorders (MD). We have studied the effects of the conditioned medium (CM) from human umbilical vein EC cultures, obtained in serum free conditions, on the growth of bone marrow fibroblasts from normal donors and from patients with MD. The results show that EC derived CM contains a factor which stimulates the proliferation of fibroblasts and that can act as an authentic growth factor by inducing "quiescent" fibroblasts to proliferate. Moreover, we found that this endothelial derived growth factor (EDGF) equally promotes the proliferation of both normal and pathological progenitors of bone marrow fibroblasts (CFU-F) by increasing both the number and the size of the colonies.     

Multiplication-stimulating activity for chicken embryo fibroblasts from rat liver cell conditioned medium: a family of small polypeptides

A partially purified multiplication-stimulating activity for chicken embryo fibroblasts in cell culture was isolated from rat liver cell conditioned medium (see preceding paper, Dulak and Temin, 1973). It has been analyzed by isoelectric focusing and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Multiplication-stimulating activity resided in a family of at least four polypeptides which were similar in apparent molecular size, but different in electrical charge. These polypeptides have a specific activity of about 50,000 with respect to serum. One of them has been purified on a small scale to apparent homogeneity in a sodium dodecyl sulfate polyacrylamide gel.     

Enhancement of erythroid progenitor cell growth in medium conditioned by a human cancer cell line, KONT

Erythroid-potentiating activity (EPA) was detected in culture medium conditioned by a human cancer cell line (KONT) that produces colony-stimulating activity (CSA), using erythroid colony formation in vitro. EPA in the medium conditioned by the KONT cells (KONT-CM) was markedly heat stable. After treating KONT-CM at 80 degrees C for 30 min, 30% EPA remained, while CSA was completely inactivated. Both EPA and CSA appeared in approximately the same fractions of the gel filtration, indicating a molecular weight of approximately 30,000 daltons. EPA bound partially to Concanavalin-A Sepharose, whereas CSA almost did not bind. Our results indicate that EPA can be separated from CSA based on heat stability and binding to Concanavalin-A Sepharose.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.     

Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium

Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm-associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage.     

UDP-galactose inhibition of BALB/3T12-3 cell growth : Requirement for medium galactosyltransferase activity

Incubation of BALB/3T12-3 cells with uridine diphosphate galactose (UDP-gal) resulted in a concentration-dependent inhibition of cell growth when cells were cultured in calf serum-supplemented Dulbecco's modified Eagle medium (CS-DMEM). Cell growth was completely inhibited by 5 mM UDP-gal with an ID₅₀ of 0.75 mM. This inhibitory effect was reversible. Other nucleotide-sugars, as well as galactose, glucose, and galactose-1-phosphate had no effect on cell growth. UDP-gal had no effect on cell growth when cells were cultured in heat-inactivated calf serum containing DMEM (HICS-DMEM) suggesting that a serum enzyme activity was responsible for the inhibition observed in CS-DMEM. No significant difference could be detected by descending chromatography in the degradation of UDP-gal during 96 h of incubation in CS-DMEM and in HICS-DMEM. Furthermore, the potential breakdown products of UDP-gal had no effect on cell growth when added directly to 3T12 cultures. When cells were incubated with 5 mM UDP-gal+5 mM CDP-choline (a potent inhibitor of pyrophosphatase activity), complete inhibition of cell growth was still observed. However, if cells were incubated with 5 mM UDP-gal+UDP (which inhibited calf serum galactosyltransferase activity), no inhibition of cell growth was observed over that found for UDP alone, suggesting that galactosyltransferase and not pyrophosphatase activity mediated the effect of UDP-gal on cell growth. A direct effect of UDP-gal on cells was suggested by (a) normal growth of cells in UDP-gal-conditioned medium (preincubated with UDP-gal for 24 h followed by dialysis to remove UDP-gal); (b) 3-fold greater incorporation of [³H]galactose from UDP-[³H]gal into cells grown in CS-DMEM than in HICS-DMEM. These studies suggest that the inhibition of 3T12 cell growth by exogenous UDP-gal may be due to alteration of cell surface glycoconjugates by extracellular galactosyltransferase activity.     

Megakaryocyte colony culture using a liver cell conditioned medium

Megakaryocyte colonies may now be grown in semi-solid agar, agarose, and plasma clot systems using a variety of conditioned media. These include mitogen activated mouse spleen cells, L-cells (fibroblasts), and a myelomonocytic cell line. To these is now added a conditioned medium from BRL-3A, a rat liver cell line. Megakaryocytes show full maturation to cytoplasmic fragmentation in this system. It is suggested that colony stimulating factor for megakaryocytes may come from a variety of sources; the relationship of any of these to the physiological regulator or regulators remains quite unknown.