Identification and characterization of polypeptide growth factors secreted by murine embryonal carcinoma cells

Undifferentiated P19 and PC13 murine embryonal carcinoma (EC) cells have been analyzed for their ability to secrete polypeptide growth factors. This has been carried out by a combination of specific bioassays and the use of biochemical and immunological detection methods. Both P19 and PC13 EC cells secrete a platelet-derived growth factor (PDGF)-like growth factor, a type beta transforming growth factor, and insulin-like growth factors. In addition, PC13 EC cells secrete a heparin-binding growth factor functionally related to fibroblast growth factor, while P19 EC cells secrete transforming growth factor-alpha. This is the first demonstration for secretion of transforming growth factor-alpha by an equivalent of early embryonic cells. The possible paracrine growth stimulating effects of these growth factors have been tested on differentiated derivatives of P19 EC cells, corresponding to all three germ layers. The differences in growth factor production by various embryonal carcinoma cells are discussed in relation to the developmental origin of these cell lines.     

A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.     

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

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

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells

The regulation of gelsolin levels during differentiation of the murine embryonal carcinoma cell line, PC-13, was investigated using nucleic acid and immunological probes. A cDNA clone, Mu-319, which contained the entire coding sequence for the cytoplasmic form of murine gelsolin was isolated using a polyclonal antibody. Gelsolin was detected in several cell lines but was not detectable in three undifferentiated embryonal carcinoma cell lines. Levels of gelsolin mRNA increased 10-fold during the differentiation of the murine embryonal carcinoma cell line, PC-13. Differentiation of PC-13 was accompanied by changes in cell shape, from small indistinct cells to large flat cells. The accumulation of gelsolin mRNA in PC-13 cells began 12-24 h after addition of the differentiation-inducing agents. In comparison, 2-5A-dependent RNase activity showed a 40-fold increase beginning after 24 to 36 h and c-fos mRNA were shown to increase about 9-fold beginning 36 to 60 h after induction of differentiation. The levels of gelsolin per se, as determined by immunoreactivity were also shown to increase with differentiation of PC-13 cells. These results suggest that gelsolin may play a role in the restructuring of actin filaments which accompanies the dramatic changes in cell shape during differentiation.     

PC13 embryonal carcinoma cells produce a heparin-binding growth factor

A polypeptide growth factor has been isolated from serum-free medium conditioned by mouse PC13 embryonal carcinoma cells, which is strongly mitogenic for Swiss 3T3 fibroblasts. On a CM-2-SW high-performance liquid chromatography cation-exchange column at low pH, this growth factor elutes at a salt concentration very close to that of basic fibroblast growth factor (FGF). The growth factor is mitogenic for a mesodermal derivative of embryonal carcinoma (EC) cells, but not for differentiated derivatives with endodermal or ectodermal characteristics, again similar to FGF. The PC13-derived growth factor binds to heparin-Sepharose, and elutes from this column at similar salt concentrations as FGF. These data demonstrate that PC13 embryonal carcinoma cells produce a basic heparin-binding growth factor (HBGF). Since the initial purification steps are similar to those used by Heath & Isacke (EMBO j 3 (1984) 2957 [7]) for isolation of a PC13 embryonal carcinoma-derived growth factor (ECDGF), which is cationic with a molecular weight (MW) close to that of FGF, the present heparin-binding growth factor (HBGF) is most likely identical with ECDGF.     

Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The F9-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 pM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenically identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells.     

Somatomedins/insulin-like growth factors, their receptors and binding proteins are present during mouse embryogenesis

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3-4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30-45 X 10(3) Mr. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.     

Differentiation-dependent production of a platelet-derived growth factor-like mitoattractant by endoderm cells derived from embryonal carcinoma cells

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA-5E (visceral endodermlike) cells produced little of this factor. However, PYS-2 (parietal endodermlike) and Dif 5 endoderm cells were found to produce significant amounts of endoderm-derived mitoattractant (EDM) activity. The activity secreted by the Dif 5 cells was partially purified using gel filtration chromatography using chemotaxis and mitogenic assays as markers for biological activity. The partially purified activity competes with [125I]iodo-platelet-derived growth factor (PDGF) for binding to target cells, and the biological activity is neutralized with anti-PDGF IgG, suggesting shared domains in the two molecules. However, the factor appears to be different from PDGF, based on its thermal stability, molecular weight, and charge. The differentiated endoderm cells including retinoic acid (RA)-treated F9, Dif 5, PSA-5E, and PYS-2 cells also exhibit specific [125I]iodo-PDGF binding, and the PSA-5E cells respond to PDGF as a chemoattractant. Conceivably, such a PDGF-like factor may contribute to the regulation of cell growth and migration during the early stages of embryogenesis.     

PC13 embryonal carcinoma-derived growth factor.

A potent growth factor, PC13 embryonal carcinoma-derived growth factor (ECDGF), has been isolated from serum-free medium conditioned by PC13 murine embryonal carcinoma cells. ECDGF is a single chain, cationic hydrophobic molecule of 17 500 daltons. ECDGF will induce DNA synthesis in established fibroblast cell lines and the immediate differentiated progeny of PC13 EC cells in vitro, and consequently appears to differ from other well characterised growth factors both in structure and action.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Early mouse embryos produce and release factors with transforming growth factor activity

Summary Previous studies have shown that extracts from mouse embryos at mid and late stages of development contain factors that exhibit transforming growth factor activity. The work reported here demonstrates that cultured mouse embryos at significantly earlier stages of development produce and release factors that exhibit the characteristic property of transforming growth factors. Specifically, the data demonstrate that embryos cultured from the blastocyst stage in serum-containing medium or in serum-free medium release factors that promote the anchorage-independent growth of normal rat kidney fibroblasts. It is shown that these factors are produced and released by cells derived from the inner cell mass and by trophoblasts. The precise developmental stage when production of these factors first begins has not been determined but our findings suggest that these factors are produced by cell types associated with early postimplatation embryos. This work was supported by the Laboratory of Viral Carcinogenesis at the National Cancer Institute and by grants from the National Cancer Institute (CA-36727) and the University of Nebraska Medical Center (22-271-732). Editor's Statement This paper presents evidence that, in an in vitro assay system, early embryonic cells are capable of both synthesizing and secreting TGF-like growth factors, implicating the production of these factors in the events of early development. David W. Barnes     

Inhibitory effects of transforming growth factor-β on laminin production and growth exhibited by endoderm-like cells derived from embryonal carcinoma cells

Previous studies have shown that two mouse embryonal carcinoma (EC) cell lines do not express cell surface receptors for transforming growth factor type-beta (TGF-beta) until they are induced to differentiate. To understand the effects of TGF-beta in this model system, we have examined the effects of TGF-beta on parietal endoderm-like cells derived from EC cells. We have determined that TGF-beta exerts three effects on these cells. TGF-beta inhibits proliferation of the parietal endoderm-like cells, and this occurs even in the presence of growth factors that stimulate their proliferation. TGF-beta also alters the morphology of the parietal endoderm-like cells by increasing their spreading. Moreover, the morphological effect of TGF-beta is observed in the presence of dibutyryl cyclic AMP (dbcAMP), which reduces the spreading of these cells. Lastly, TGF-beta, but not other growth factors, decreases the production of laminin by the parietal endoderm-like cells. This was unexpected since TGF-beta has been shown to increase the production of extracellular matrices in other systems. Thus, our findings indicate that parietal endoderm-like cells provide a useful system for broadening the study of TGF-beta. Furthermore, our findings provide additional support for the possibility that TGF-beta plays important roles during the early stages of mammalian development.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.