Effects of growth factors and kinase inhibitors on the properties of human adipose-stromal cells in different culture conditions

The best-fit environments for the proliferation and differentiation of human adipose tissue-derived stromal cells (hADSCs) may require specific media and stimuli. The characteristics of hADSCs cultured in different media might be different. We evaluated the effects of growth factors on the proliferation and differentiation of hADSCs and compared the effects of these growth factors on hADSCs cultured in different serum-contained media. The effects of kinase inhibitors on the proliferation of hADSCs were also examined. The combination of insulin-transferrin-selenium (ITS) and fibroblast growth factor (FGF) induced the greatest increase in proliferation, while the combination of ITS and epidermal growth factor (EGF) showed the second-greatest effect in the human serum (HS)-contained media. By contrast, in the fetal bovine serum (FBS)-contained media, ITS had little effect while FGF had better results than the combination of ITS and EGF. The inhibitory potency of the kinase inhibitors was different in the two different media. These results suggest that hADSCs may have different characteristics according to the culture conditions, and that extrapolations of results from animal studies, using FBS media, may not correlate to findings in human studies using HS.     

Effect of placental factors on growth and function of the human fetal adrenal in vitro

Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.     

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary log-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-β1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified. © 1995 Wiley-Liss, Inc.     

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.     

Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Decreased heme content and cessation of cell growth in cultured chick embryo fibroblasts in the presence of horse serum: Stimulation of heme synthesis and cell growth by iron

A new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO₄ to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme synthesis.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

Human blood-derived fibrin releasates: Composition and use for the culture of cell lines and human primary cells

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-β1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1-10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Human dermal fibroblasts express multiple bFGF and aFGF proteins

Summary We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.     

Impaired proliferation response after PDGF induction in fibroblasts from Hutchinson-Guilford Progeria syndrome

Fibroblasts from a Hutchinson-Guilford Progeria Syndrome (HGPS) patient were compared to normal human fibroblasts to determine if differences existed in growth factor mediated cell proliferation. Cultures of progeric fibroblasts were exposed individually to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), platelet poor plasma (PPP) and fetal bovine serum (FBS). Autoradiographic studies using 3H thymidine showed that progeric fibroblasts had similar labeling indices relative to controls after exposure to FBS and EGF. In contrast, progeric cells made competent with PDGF and later treated with 5% PPP had a significantly lower labeling index. This and preliminary observations on fos RNA accumulation suggests the possible existence of a genetic defect in HGPS fibroblasts.     

Induction of DNA synthesis by fibroblast growth factor in temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts arrested at restrictive temperature.

Four temperature-sensitive cell-cycle mutants of rat 3Y1 clonal fibroblasts representing separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125 and 3Y1tsH203) are arrested at restrictive temperature, primarily with a G1-phase DNA content (temperature arrest). We examined various factors affecting signal transduction for activity which induces DNA synthesis at the restrictive temperature when added to the temperature-arrested cultures of these mutants. The factors examined were theophylline, dibutyryl cyclic AMP, cholera toxin (CT), dibutyryl cyclic GMP, sodium nitroprusside, phorbol 12-myristate 13-acetate, 1-oleoyl 2-acetylglycerol, bombesin, vasopressin, basic fibroblast growth factor (FGF), platelet-derived growth factor, A23187, monensin, epidermal growth factor (EGF), insulin and fetal calf serum (FCS). None of these factors induced DNA synthesis in 3Y1tsH203. In one mutant (3Y1ts121), FGF, EGF and FCS individually induced DNA synthesis. In the other 2 mutants (3Y1tsD123 and 3Y1tsG125), FGF and CT individually induced DNA synthesis. The FGF-induced DNA synthesis was suppressed by islet-activating protein (IAP) in 3Y1tsD123 and 3Y1tsG125, but not in 3Y1tsF121. The CT-induced DNA synthesis was also suppressed by IAP, as previously shown. When temperature-arrested cultures were shifted to a permissive temperature, all 4 mutants initiated DNA synthesis in the presence of IAP. These results suggest that (1) a cell can prepare for the initiation of DNA synthesis by using several independent signal transduction pathways, and (2) in a given situation, the cell uses a particular pathway because of its availability, which depends on the culture conditions.     

Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.     

Umasking large and persistent reductions in proliferation rate of aging cells

Summary We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.     

A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors.

The effects of fibroblast and epidermal growth factors on proliferation and differentiation of cultured myoblasts and chondrocytes have been compared. FGF stimulated myoblast proliferation, as determined by monitoring levels of DNA synthesis during seven days growth in vitro and by the morphology of the cultures after myotube formation. EGF has relatively little effect on myoblast proliferation. With chondrocytes, both FGF and EGF are mitogenic and FGF's, but not EGF's effect is potentiated by dexamethasone. One implication of these results is that in the course of differentiation cell types which develop from the same embryonic origin as fibroblasts are controlled by different sets of mitogenic factors. Myoblasts become primarily dependent on mitogenic agents such as FGF while chondrocytes can respond to both FGF and EGF.     

Bypass of the ts block of tsJT60, a G0-specific ts mutant from rat fibroblasts, by fetal bovine serum and epidermal growth factor

tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.