Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors

The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11-day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin-C (Sm-C) do not increase cell growth, but a 30-40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 microgram/ml) or Sm-C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor.     

Improved clonal and nonclonal growth of human,rat and bovine adrenocortical cells in culture

Summary This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells. Optimal conditions for stimulating clonal growth were determned by testing the efficacy of horse serum (HS), fetal bovine serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors, UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated dishes and DME/F12 medium with 10% FEBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal human adrenocortical cells without fibroblasts overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal and nonclonal early passage human adrenocortical cells had smilar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland fibroblasts, the tumor promoter 12-O-teradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts. In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of human adrenocortial cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and ncreased the cloning efficiency of cultured bovine adrenocortical cells. This work was supported by Research grants AG-00936 and AG-06108 from the National Institute on Aging, Bethesda, MD.     

Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2-fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose-response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.     

Growth-inhibitory effect of TGF-B on human fetal adrenal cells in primary monolayer culture

We examined the effects of transforming-growth factor-B (TGF-B) on growth ([3H]-thymidine uptake) and function (dehydroepiandrosterone sulfate [DHAS] and cortisol production) of human fetal zone adrenal cells. Results indicate that TGF-B significantly inhibits, in a dose-related manner, both basal and epidermal growth factor (EGF)-stimulated cell growth: IC50 = 0.1-0.25 ng/ml. EGF is ineffective in overcoming the inhibitory effect of TGF-B, suggesting a noncompetitive antagonism between the two factors. Also, the inhibitory effect of TGF-B is additive to that of adrenocorticotropic hormone (ACTH). On the other hand, TGF-B (1 ng/ml) does not significantly change basal or ACTH-stimulated DHAS or cortisol secretion. We conclude that, unlike its effect on other steroid-producing cells, TGF-B inhibits growth of fetal zone cells and does not appear to have a significant inhibitory effect on steroidogenesis.     

Epidermal growth factor-like growth factors prevent apoptosis of alcohol-exposed human placental cytotrophoblast cells

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Placental-derived mitogenic factor for human fetal adrenocortical cell cultures

Summary The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit. The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known. Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied. A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and [³H]thymidine incorporation into DNA 5–8 fold. PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography. PDMF is a heat sensitive protein with disulfide bonds required for activity. The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25–50% and epidermal growth factor by 3–4 fold. The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone. Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells. These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures. This implies a direct role for the placenta in control of this fetal organ’s growth, which would make the human feto-placental unit a bi-directional relationship. This research was supported by Grants HD15882 and HD21798 from the National Institutes of Health, Bethesda, MD. This work was presented in part at the 68th Annual Meeting of The Endocrine Society, Anaheim, CA, June 1986. Editor’s Statement This report describes a potentially new relationship between placenta and the regulation of growth of fetal adrenalcortical cells. Since these cells produce several of the essential hormones influencing fetal development, characterization of this factor(s) could provide important insights into the process of differentiation. David A. Sirbasku     

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10⁻⁹ M) with baFGF (40 μg/ml) and EGF(0.01 μg/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E₂ release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network. © 1996 Wiley-Liss, Inc.     

In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C

The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells.     

Placental keratinocyte growth factor: partial purification and comparison with epidermal growth factor

A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.     

Synergistic effects of epidermal growth factor and thrombin on the growth stimulation of diploid chinese hamster fibroblasts

The serum supplement used in the culture of a variety of mammalian cells can be replaced by known growth factors. Diploid Chinese hamster fibroblasts (CHEF/18) will grow for several days in a medium (4F) supplemented with four growth factors: epidermal growth factor (EGF), insulin, fibroblast growth factor (FGF), and transferrin. The growth rate is only about 50% as fast as when fetal calf serum is added. This difference is eliminated by thrombin (10–100 ng/ml; 0.3–3 nM). The CHEF/18 cell line is unique in that no other cell line responds to thrombin in this concentration range. Thrombin acts synergistically with other growth factors to stimulate CHEF/18 cell growth. By itself, thrombin is only mitogenic at elevated concentrations. Thrombin can largely compensate for the absence of EGF and partly for the absence of insulin in serum-free media. Chemically and “spontaneously” transformed cell lines related to CHEF/18 have lost requirements for both EGF and thrombin, and have retained requirements for insulin and transferrin expressed by CHEF/18. No CHEF cells in this work required FGF. These results suggest that the mechanisms by which EGF and thrombin stimulate cells to grow are related.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk