Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication-stimulating activity (MSA).

A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.     

The autocrine regulation of proliferation in cell cultures. I. A study of the heparin-binding factors with growth-stimulating activity contained in media conditioned with transformed rat fibroblasts]

A study was made of the medium conditioned by spontaneously transformed rat embryo fibroblasts of line Rec1-sf, which are capable of unlimited reproduction in medium free of serum and of other endogenous growth factors (c-medium). Addition of c-medium to stationary cultures of nontransformed rat embryo fibroblasts (REF), spontaneously transformed REF (line Rec1), and cells of Rec1-sf stimulated the incorporation of 3H-thymidine by 1.5-6 times. SDS-polyacrilamide-gel electrophoresis of proteins, marked by 35S-metionine of c-medium of the cell line Rec 1-sf, demonstrated that this medium had proteins with molecular mass from 10 to 110 kDa. The fractionating divisible by 100 ultra-concentrates of c-medium with utilization of heparin-sepharose allowed to isolate two types of heparin-binding proteins. The proteins of the first type took about 5% of all the proteins of c-medium; they were eluted with 1.1 M NaCl and stimulated the incorporation of 3H-thymidine in REF, Rec1 and Rec1-sf cultures by 1.3-1.9 times. The second type proteins took about 1% of all the proteins of c-medium and were eluted with 2M NaCl, and, like the main endogenic basic growth factor of fibroblasts, stimulated the incorporation of 3H-thymidine into REF and Rec1-sf, but not into the culture of Rec1 line cells. The results obtained are discussed in terms of a hypothesis of autocrine regulation of cell proliferation.     

Effects of withdrawal of a mitogenic stimulus on progression of fibroblasts into S phase: differences between serum and purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a rat liver cell line. A comparison between calf serum and purified MSA was made regarding the regulation of the fibroblast cell cycle. Addition of serum or MSA to stationary, quiescent cells stimulates them to enter the DNA synthetic phase after a characteristic lag period. Exposure to serum for shorter periods of time will irreverisbly commit cells to continue through the cell cycle and initiate DNA replication in the absence of serum. In contrast, the withdrawal of purified MSA from the medium results in an abrupt halt in the progression of cells towards S phase. The results of labeled thymidine incorporation and autoradiographic experiments clearly indicate that the point at which cells become irreversibly committed to enter the DNA synthetic period is at or near the G1-S boundary. The abrupt decay of the stimulation upon withdrawal of purified MSA provides a unique opportunity to investigate the biochemistry of this discrete phase of the cell cycle.     

Stimulation of DNA synthesis,cell multiplication,and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [³H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [³H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [³H] thymidine incorporation into DNA. MSA causes a 2–10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [³H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [³H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.     

Reversible regulation by magnesium of chick embryo fibroblast proliferation

The rate of 3H-thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100-fold, but unlike the case of Mg2+-deprivation this can occur without significant effect on the rate of 3H-thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H-thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.     

Dextran T-500 improves survival and spreading of chick embryo cells in serum-free medium

The supplementation of serum-free Eagle's minimal essential medium (EMEM) with Dextran T-500 significantly improves attachment, spreading and survival of chick embryo cells in primary (myoblasts) and secondary (fibroblasts) cultures. These effects were observed in quiescent cultures incubated at 4 degrees C as well as at 37 degrees C. The results suggest that dextran can be used to simplify conditions for research on factors influencing cell proliferation and differentiation in serum-free media.     

Cultivation of mammalian cells in serum-free media]

P3 cell lines can be grown in protein- and lipid-free synthetic medium. Using the P3 cell culture, we have shown that these cells produce autocrine growth factors and cell-substrate attachment factors. Because the cultured cells produce proteinase-inhibitor, spent medium is applicable for inactivating the action of trypsin at the time of cell passage. In addition, we have tried to cultivate various types of cells in serum-free media on the market (ASF103, ASF104 and GIT). Many cell lines can grow in these media, but inoculum dependency is observed in some cell lines. Production of monoclonal antibody by a hybridoma cell line is rather enhanced in these media. These media can be preserved at 4 degrees C or -20 degrees C for a relatively long period. These media added with EGF support the growth of Syrian hamster embryo cells at an early passage. The growth of human diploid fibroblasts in GIT medium added with EGF is a little less compared in a serum-containing medium.     

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.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Studies on carcinogenesis by avian sarcoma viruses. VI. Differential multiplication of uninfected and of converted cells in response to insulin

Insulin can replace the factor(s) in calf serum whose amount is limiting for multiplication in cell culture of chicken embryo fibroblasts and of chicken embryo fibroblasts infected and converted by avian sarcoma virus. In serum-free, insulin-containing medium, converted cells multiply more than do uninfected cells. It appears, therefore, that the increased multiplication in cell culture of converted cells as compared with uninfected cells results from a decreased requirement by the converted cells for an insulin-like activity found in serum.     

Purified multiplication-stimulating activity from rat liver cell conditioned medium: comparison of biological activities with calf serum, insulin, and somatomedin

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a line of rat liver cells (CRL), The biological activities of purified CRL MSA for chicken embryo fibroblasts were compared with those of calf serum to determine which activities are important for the stimulation of DNA synthesis and mitosis. In a balanced salt solution, only glucose and amino acids were needed in addition to purified CRL MSA to stimulate DNA synthesis maximally. Purified CRL MSA stimulated the rates of uptake of glucose and α-aminoisobutyric acid. Only the stimulation of the rate of glucose uptake appeared to be a primary response to purified CRL MSA since the stimulation was not inhibited by actinomycin D or cycloheximide. The stimulation of the rate of uptake of α-aminoisobutyric acid was inhibited by actinomycin D. CRL MSA differed from calf serum in its inability to commit cells irreversibly to synthesize DNA after the removal of CRL MSA and in its lack of the ability to stimulate the migration or prolong the survival of chicken embryo fibroblasts. Comparative studies indicated that purified CRL MSA had functional similarities to insulin and somatomedin. CRL MSA may be representative of a family of small polypeptide hormones having insulin-like activity which are involved in the control of cell multiplication.     

Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts.

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ or K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate. Addition of 1.8 mEGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

Sulfation and mitogenic activities of growth factors in human serum: importance of compounds of molecular weight lower than 1,000 in the global expression of activity of this serum on 3 different cellular types

A human serum ultrafiltrate contains compounds needed for a maximal expression of sulfation and mitogenic activities of the corresponding retentate. These low molecular weight (less than 1,000) molecules have no effect, by their own, on 35SO4(2-) uptake in chick embryo cartilage, but show a significant synergistic effect with serum growth polypeptides. Tested alone, they display a slight mitogenic activity as measured by 3H-thymidine incorporation in chick embryo fibroblasts or in lymphocytes activated by phytohemagglutinin. Here they also act in a synergistic way on mitogenic activities of growth factors from human retentate. A fraction (P2B), partially purified from human plasma ultrafiltrates, produces the same synergistic response with human retentate in the three cellular systems (cartilages, fibroblasts, activated lymphocytes). However, concentrations which lead to optimum responses are very different according to the cell type studied. These results suggest the existence of several compounds which can each act specifically on a cellular system.     

Cumulative population doublings as the determinant of chick cell lifespan in vitro

Chick embryo fibroblasts were maintained at confluency for up to 35 days in medium containing 0.5% or 0.75% fetal bovine serum or 2.5% or 5.0% horse serum. At weekly intervals cells were subcultured and serially propagated in medium containing 10% FBS until their replicative lifespans were completed. The results showed that the replicative lifespan of embryonic chick fibroblasts was dependent on the cumulative number of population doublings undergone by the culture and was not related to the calendar time cells were in culture. Further characterization of 0.75% FBS maintained chick cells returned to 10% FBS medium showed that cells had protein contents and incorporated ³H-thymidine into DNA at a rate that resembled that of young cells, despite an advanced chronological age.     

The insulin signal initiating cellular differentiation is preserved by chick embryo myoblasts incubated at 2 degrees C

Insulin pulse treatment, lasting for 1 to 3 min, stimulates differentiation of chick embryo myoblasts to myotubes and myofibers in a serum-free medium. The use of serum-free medium supplemented with 2,2'-thiodiethanol permits terminal differentiation of chick embryo myoblasts to cross-striated, spontaneously contracting myofibers. The experiments carried out showed that incubation of chick embryo myoblasts after the insulin pulse treatment for 10 days at 2 degrees C does not inhibit the progress of their differentiation. The data demonstrate that differentiation can be interrupted at any time by transferring cells to 2 degrees C and resumed without delay after returning to 37 degrees C.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast

The effect of tunicamycin (TM) on the synthesis and secretion of sulfated proteoglycans and hyaluronate was examined in chick embryo fibroblasts and chondrocytes. The incorporation of the precursors [³H]glucosamine, [³H]mannose and [³⁵S]sulfate into glycoconjugates in both the cell layer and medium of cultures was determined. In the chick embryo fibroblast, but not in the chondrocyte, synthesis of sulfated proteoglycan was inhibited 60–75% by TM (5 × 10⁻⁸ M), while synthesis of hyaluronate and protein was only inhibited slightly. The inhibition of sulfate incorporation into glycosaminoglycans of the chick embryo fibroblast was overcome to a great extent by addition of β-xyloside, which provides an exogenous initiator for chondroitin sulfate synthesis. TM treatment also altered cell shape and surface morphology in chick embryo fibroblasts, as observed by phase contrast and scanning electron microscopy (SEM). Cells treated with TM became rounded, and increased numbers of microvilli and blebs appeared on the cell surface. These alterations in cell morphology were reversed by removal of TM, but not by exogenous addition of xyloside, chondroitin sulfate or the adhesive cell surface glycoprotein fibronectin. These results demonstrate that TM inhibits synthesis of sulfated proteoglycans in the chick embryo fibroblast and causes a dramatic alteration in cell shape and surface morphology.     

Cytoskeletal and DNA synthesis modification by concanavalin A in embryonic fibroblasts maintained in serum-free and serum-added medium

The administration of lectin concanavalin A (Con A) to in vitro cultures of chick embryo fibroblasts caused dose-dependent changes in cell shape, cytoskeleton network and DNA synthesis. After 6 hrs. even a low doses of Con A produced evident effects in serum-free cultures, whereas higher doses were required to cause alterations in cells cultured in serum-added medium. As the sugar competitor mannoside abolishes the effects, it would seem that the lectin acts by binding to transmembrane receptors and that the fibronectin present in the serum engages the receptors so that they are not available to Con A.