Identification of a novel growth factor with transforming activity secreted by individual chick embryos

Development ◽  
1990 ◽  
Vol 109 (4) ◽  
pp. 905-910 ◽  
Author(s):  
J. Smith ◽  
J.C. McLachlan
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Chick Embryo ◽  
Primitive Streak ◽  
Soft Agar ◽  
Heat Stable ◽  
Stage Of Development ◽  
Transforming Activity ◽  
Embryo Tissue ◽  
Differential Responsiveness

Previously we have developed a microassay for anchorage independent growth (AIG) of fibroblasts in soft agar, which can detect very small quantities of transforming growth factors (TGFs). Using this assay, we have shown that small pieces of dissected chick embryo tissue will stimulate AIG of both NR6 and NRK 49f cells, and that this property can be used to map production of growth factors with transforming activity in individual early embryos. We now show that this activity can be transferred to conditioned medium (CM) prepared using chick embryo tissue. Using two cell lines with differential responsiveness to TGFs, and by coincubating normal and heat-treated CM with trypsin, Con-A and neutralising antibodies, we show that CM contains at least two different growth factors with transforming activity. One of these is heat-stable, and stimulates colony formation in NRK 49f cells in the presence of EGF, but not in its absence. This activity corresponds to a TGF beta-like molecule. The other component is a heat-labile glycoprotein, which has TGF alpha-like properties, but does not appear to behave like known TGFs with these properties. It therefore appears to be a novel growth factor. Both activities are present from the intermediate primitive streak stage of development.

scholarly journals Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor.

1986 ◽  
Vol 6 (3) ◽  
pp. 870-877 ◽  
Author(s):  
D F Stern ◽  
A B Roberts ◽  
N S Roche ◽  
M B Sporn ◽  
R A Weinberg
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Soft Agar ◽  
Tgf Beta ◽  
Transfected Cells ◽  
Exogenous Growth ◽  
Epidermal Growth ◽  
Ras Oncogenes ◽  
Exogenous Growth Factors

To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta). Control cells did not grow in soft agar under any conditions. ras-Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage-independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors.

scholarly journals Basic fibroblast growth factor in the chick embryo: immunolocalization to striated muscle cells and their precursors.

1989 ◽  
Vol 108 (6) ◽  
pp. 2459-2466 ◽  
Author(s):  
J Joseph-Silverstein ◽  
S A Consigli ◽  
K M Lyser ◽  
C Ver Pault
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Chick Embryo ◽  
Muscle Development ◽  
Striated Muscle ◽  
Muscle Cells ◽  
Limb Bud ◽  
Fibroblast Growth ◽  
Lack Of Information ◽  
Immunohistochemical Techniques

The identification of acidic and basic fibroblast growth factors (FGFs) in a number of embryonic tissue extracts has implicated these growth factors in the regulation of a variety of embryonic events including angiogenesis, eye development, and muscle differentiation. Lack of information concerning the cellular distribution of the growth factor within these tissues has made it extremely difficult to assign developmental roles to FGF. We have localized bFGF in the developing chick embryo using immunohistochemical techniques and our monospecific polyclonal rabbit anti-human bFGF IgG. The spatial pattern for bFGF localization was highly specific. The anti-human bFGF antibodies recognized striated muscle cells and their precursors in 2-6-d chick embryos. Myocardium, somite myotome, and limb bud muscle all stain positively for bFGF. In addition, the anti-human bFGF antibodies localized specifically to the cell, rather than to the extracellular matrix or nucleus of myotubes. The localization of bFGF demonstrated here provides further support for the hypothesis (Clegg et al., 1987; Seed et al., 1988) that this growth factor is involved in muscle development.

Isolation and partial purification of growth factors with TGF-like activity from human malignant gliomas

1989 ◽  
Vol 71 (6) ◽  
pp. 875-883 ◽  
Author(s):  
James T. Rutka ◽  
Mark L. Rosenblum ◽  
Robert Stern ◽  
Henry J. Ralston ◽  
Dolores Dougherty ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Growth Factors ◽  
Growth Factor ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Transforming Growth Factor ◽  
Malignant Gliomas ◽  
Soft Agar ◽  
Soft Agar Assay

✓ The effect of concentrated conditioned medium from each of eight human malignant glioma cell lines on the growth of indicator cells (normal rat kidney fibroblasts (NRK), clone 14) was determined in monolayer and in soft agar assay systems. The conditioned medium from all cell lines was mitogenic in the monolayer assay, but only SF-210, U-343 MG-A, and U-251 MG produced soluble factors that caused NRK cells to grow in soft agar. The soluble growth-promoting factors from these three cell lines were acid- and heat-stable (60°C for 30 minutes) but were inactivated by trypsin (100 µm/ml) and dithiothreitol (50 µM). The growth factors from SF-210 and U-343 MG-A were further purified by molecular-sieve chromatography. The partially purified growth factor from U-343 MG-A retained transforming growth factor (TGF)-like activity, had a molecular weight of 9 kD, was potentiated by TGF-β in the soft agar assay, competed effectively with 125I-epidermal growth factor (EGF) radiolabeled for the EGF receptor on A 431 epidermoid carcinoma cells, and was completely inhibited by monoclonal antibodies to TGF-α. The partially purified growth factor from SF-210 had a molecular weight of 17 kD, was not inhibited by monoclonal antibodies to platelet-derived growth factor (PDGF) or TGF-α, and did not bind to a heparin-Sepharose column. These results imply that U-343 MG-A secretes a growth factor with TGF-α-like activity, and SF-210 secretes a TGF with neither TGF-α nor TGF-β activity.

A factor present in fetal calf serum enhances oncogene-induced transformation of rodent fibroblasts

1987 ◽  
Vol 7 (10) ◽  
pp. 3380-3385
Author(s):  
W L Hsiao ◽  
C A Lopez ◽  
T Wu ◽  
I B Weinstein
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Time Course ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fold Increase ◽  
Fibroblast Cell ◽  
Focus Formation ◽  
Heat Stable

Our previous studies indicated that addition of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin to Dulbecco modified Eagle medium supplemented with calf serum enhanced T24-induced focus formation in both the murine C3H 10T1/2 and rat 6 embryo fibroblast cell lines. In the present studies we have found that fetal calf serum (FCS) is more potent than 12-O-tetradecanoylphorbol-13-acetate in enhancing T24-induced focus formation, in terms of the number and size of the foci, in both C3H 10T1/2 and rat 6 cells. Time course studies indicate that FCS can exert this enhancing effect when it is added several days after the transfection with T24 DNA. In rat 6 cells, an 11-fold increase in T24-induced focus formation occurred when the transfected cultures were maintained for only 1 day in 5% FCS, starting 4 days after the transfection. Several known growth factors, including epidermal growth factor, transforming growth factors alpha and beta, insulin, and platelet-derived growth factor, did not enhance T24-induced transformation in these cell systems. Fractionation studies indicate that the factor present in FCS has a molecular weight of about 1,300, is not lipid soluble, and is acid, base, and heat stable. These findings suggest that a factor(s) normally present in serum may enhance the emergence of tumor cells in vivo, by acting in concert with an activated oncogene, during the multistage carcinogenic process.

Proliferation Versus Differentiation: The Effect of Growth Factor Stimulation Detected by HALO.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4231-4231
Author(s):  
Ivan N. Rich ◽  
Karren Hall ◽  
Stephen H. Bartelmez
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Mirror Image ◽  
Target Cells ◽  
Proliferative Potential ◽  
Stage Of Development ◽  
Differentiation Factor ◽  
Colony Forming Assay

Abstract In order for stem and progenitor cells to initiate the proliferation process to grow in vitro and form colonies of differentiated cells, growth factors are required. Some factors are specific for proliferation, some for differentiation. Others exhibit both a proliferative and differentiation function. To date, the classical colony-forming assay (CFA) has been used to ascertain the effectiveness of growth factors either individually or in combination. However, the CFA cannot differentiate between a proliferation or differentiation factor because the assay itself is a differentiation assay. We have now developed a unique in vitro system which measures the proliferative capacity of cells to respond to different growth factors. Based on the colony-forming assay, HALO (Hematopoietic Assays via Luminescence Output) measures cell proliferation as a function of the concentration of intracellular ATP which drives a luciferin/luciferase reaction. The end-point of the reaction is bioluminescence detected in a plate reader luminometer. Since the assay does not require cells to differentiate into colonies, no manual enumeration is needed. Furthermore, HALO is a rapid, non-subjective and standardized proliferation assay performed in 96-well plates with high-throughput capability. At least 14 different cell populations with differing proliferative potential from 5 different species can be detected simultaneously. For the present study, both mouse and human bone marrow target cells were used. Eight replicate microcultures of 100ul each were performed to investigate the dose-dependent effect of single growth factors on cell proliferation. The results are summarized as follows. Proliferation of mouse and human hematopoietic cells can be detected between 1 and 2 days and is optimal between 4 and 7 days respectively. The growth factors, SCF, IL-3, IL-6, Flt3-ligand, EPO, TPO and GM-CSF alone, all induce proliferation at less than 1ng/ml. At growth factor concentrations typically used in the colony-forming differentiation assay, many are inhibitory. The EPO dose response for murine CFU-E proliferation measured at 21h produced a flat response and inhibition at doses above 10mU/ml. The mirror image profile was obtained for CFU-E differentiation at 48 h. These results demonstrate that at the CFU-E level, EPO is a differentiation factor and not a proliferation factor. However, EPO alone does stimulate proliferation at the BFU-E stage of development. In conclusion, it is now possible to distinguish between a proliferation and differentiation factor at different hierarchical levels in the hematopoietic system.

scholarly journals GROWTH OF THE FOWL CORYZA BODIES IN TISSUE CULTURE AND IN BLOOD AGAR

1939 ◽  
Vol 69 (2) ◽  
pp. 199-209 ◽  
Author(s):  
John B. Nelson
Keyword(s):  
Tissue Culture ◽  
Growth Factor ◽  
Chick Embryo ◽  
Agar Medium ◽  
Normal Growth ◽  
Cellular Component ◽  
Nutrient Media ◽  
Embryo Tissue ◽  
Cultural Requirements ◽  
Culture Supernatants

Evidence is presented that the growth capacity of chick embryo tissue for the fowl coryza bodies is conditioned by a diffusible cellular component which is essential for their multiplication. This growth factor is inactivated at pH 6, but withstands a temperature of 100°C. for 60 minutes. An amount sufficient to promote a normal growth of the specific bodies may be present in tissue culture supernatants long after its content in the tissue is exhausted. Postembryonic tissue (liver and spleen) contains a variable amount of growth factor and is not a satisfactory substitute for the chick embryo. Multiplication of recently isolated fowl coryza bodies is not demonstrable in nutrient media enriched with blood. Experiments with one strain, however, indicate that an adaptation to fluid blood in an agar medium may take place after many generations in tissue culture. The probable bacterial nature of the fowl coryza bodies is discussed on the basis of their cultural requirements.

scholarly journals STUDIES ON THE FACTORS ESSENTIAL TO THE INITIATION AND MAINTENANCE OF MULTIPLICATION OF PSITTACOSIS VIRUS (6BC STRAIN) IN DEFICIENT CELLS IN TISSUE CULTURE

1954 ◽  
Vol 99 (5) ◽  
pp. 461-479 ◽  
Author(s):  
John D. Hare ◽  
Herbert R. Morgan
Keyword(s):  
Chick Embryo ◽  
Viral Infections ◽  
Defined Medium ◽  
Virus Multiplication ◽  
Virus Growth ◽  
Molecular Weights ◽  
Heat Stable ◽  
Time Of Infection ◽  
Embryo Tissue ◽  
Tissue Tropisms

The growth of psittacosis virus (6BC) was studied in cultures of minced whole chick embryo tissue maintained in either Hanks-Simms solution or Hanks's balanced salt solution (BSS), and in neither medium could sustained, long-term virus growth take place. Addition of beef embryo extract (BEE) to cultures at a time when virus multiplication was declining reversed this general trend and resulted in greater virus growth. This virus-stimulating action of BEE was only partially diminished by colchicine, a mitotic inhibitor, indicating that the action of BEE was not due entirely to the development of a larger population of cells as a result of its enhancement of cell proliferation. Chick embryo tissue cultivated for 13 days in BSS prior to infection lost its ability to support the growth of psittacosis virus, but this capacity could be restored by the addition of BEE, alone or with colchicine, at the time of infection. A significant amount of virus was adsorbed to tissue in BSS alone, indicating that the failure of virus to grow in depleted tissue maintained only in BSS after infection was not due entirely to failure of virus to attach to and invade the cells. It was found that an ultrafiltrate and a dialysate of BEE contained the major part of the stimulating capacity of the whole extract, indicating that the active materials were substances of low molecular weights. Autoclaved lactalbumin hydrolysate was an active stimulator, suggesting that the materials responsible for its activity were relatively heat-stable. Since a chemically defined medium (Parker 199) was equally effective in stimulating viral growth, it should be possible eventually to define the chemical nature of the virus stimulators. The implications of the findings are discussed with special reference to their application in the study of tissue tropisms and of latency in viral infections of cells.

Labelling of basement membrane constituents in the living chick embryo during gastrulation

Development ◽  
1984 ◽  
Vol 79 (1) ◽  
pp. 113-123
Author(s):  
Esmond J. Sanders
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Concanavalin A ◽  
Primitive Streak ◽  
Basal Surface ◽  
Stage Of Development ◽  
Pass Through ◽  
The Relationship

The basement membrane of the living chick embryo epiblast has been labelled with ultrastructural markers in order to study the movement and turnover of this structure during gastrulation. Two problems were addressed in these experiments. Firstly, to what extent does the basement membrane move medially with the epiblast during morphogenesis? Secondly, what is the relationship to the basement membrane of the so-called interstitial bodies? The ultrastructural markers used were concanavalin A conjugated to ferritin and fibronectin antibodies conjugated to peroxidase. Embryos were cultured using the technique of New, and the label was applied to the periphery of the basal surface of the epiblast through a hole in the endoblast at the early primitive streak stage of development. The embryos were then allowed to develop to the full primitive streak stage in the presence of the label. When the position of the label was determined after incubation, it was found to have accumulated in large amounts at the edge of the primitive streak at the point where the basement membrane is disrupted. This indicates that constituents of the basement membrane are transported medially with the epiblast cells and are sloughed off as the latter pass through the primitive streak. This movement of basement membrane constituents is counter to the direction of migration of the underlying mesoderm cells. When embryos are exposed to label for only 1 h, then washed and incubated for a further three hours, the marker was found in the interstitial bodies and not distributed throughout the basement membrane itself. This suggests that the interstitial bodies, which have been implicated in influencing the migration of the mesoderm cells, are turnover products of the basement membrane to which they are attached.

Ultrastructural immunocytochemical localization of fibronectin in the early chick embryo

Development ◽  
1982 ◽  
Vol 71 (1) ◽  
pp. 155-170
Author(s):  
E. J. Sanders
Keyword(s):  
Chick Embryo ◽  
Basal Lamina ◽  
Regional Differences ◽  
Developmental Stages ◽  
Primitive Streak ◽  
Extracellular Material ◽  
Cell Surfaces ◽  
Mesoderm Cell ◽  
Stage Of Development ◽  
Primary Location

Fibronectin was localized using peroxidase and biotin-avidin-ferritin techniques in developmental stages of the chick embryo from laying to primitive streak formation. The primary location of fibronectin at all stages examined was the basal lamina of the epiblast and its associated extracellular material. The remaining tissues showed little or no immunochemical deposit. At the primitive streak stage of development there were some regional differences in the density of the deposit on the basal lamina. Closely adjacent to the primitive streak, where the basal lamina is fragmentary, deposit was sparse or absent. In this, and other regions, mesoderm cell surfaces did not stain except where they closely approached the stained basal lamina or interstitial bodies. Staining was variable in the basal lamina anterior to the primitive streak, but in a number of cases particularly heavy deposit was noted overlying the crescent of late hypoblast. This area seemed to correspond with the anterior fibronectin- rich band reported by others using immunofluorescence localization.

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