Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Modulation of growth of human carcinoma SW-13 cells by heparin and growth factors

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.     

Comparison of the ability of basic and acidic fibroblast growth factor to stimulate the proliferation of an established keratinocyte cell line: modulation of their biological effects by heparin, transforming growth factor beta (TGF beta), and epidermal growth factor (EGF)

The bioactivity of both bFGF and aFGF in the BALB/MK-1 cell line has been compared to that of EGF. Our results indicate that, for that cell type, aFGF was far more potent than bFGF in inducing cell proliferation. In the presence of heparin, aFGF was as potent as EGF. In addition, excess bFGF has an inhibitory effect on the proliferation of MK cells exposed to a saturating concentration of aFGF, therefore acting as a partial agonist of aFGF. Surprisingly, bFGF, although it had low biological activity, was capable of synergizing the effect of EGF. In its presence, cultures exposed to saturating concentration of EGF have a final cell density 3- to 4-fold higher than that of counterpart cultures exposed to EGF alone. TGF beta, which in previous studies has been shown to inhibit the growth of keratinocytes, also inhibited the growth of BALB/MK-1 cells in response to either bFGF or aFGF. These studies suggest a role for FGF in regulating BALB/MK proliferation. aFGF provides positive growth signals which can be negatively modulated by excess bFGF or TGF beta, while bFGF, although a poor mitogen, could act by potentiating the effect of subsaturating concentrations of EGF.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

Internalization of growth factor-receptor complexes under the influence of antibodies initiates cell apoptosis in vitro

Antibodies against growth factors like IGF1, IGF2, aFGF, bFGF and, to a certain extent, TGF alpha and EGF were shown to cause apoptosis of normal and tumorigenic cells while apoptotic cell death could be prevented neither by single growth factors nor by serum. Antibodies to growth factors caused apoptosis by interacting with growth factors bound to their receptors on the cell surface. The phenomenon is likely to be associated with active internalization of growth factor receptors loaded with ligands. Apparently these activated receptors are essential for cell survival and their disappearance from the cell surface initiates apoptosis.     

Responses of keratinocytes to substrate-bound vitronectin: growth factor complexes

Insulin-like growth factor-1 (IGF-I) can associate with the extracellular matrix protein vitronectin (VN) via select IGF-binding proteins, and the resulting complex stimulates responses in a variety of cell types. As VN can also associate with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we hypothesized that the multimeric nature of VN could be exploited to deliver multiple growth factors to the cell surface. We report here that VN enhances bFGF but not EGF stimulated [(3)H]-leucine incorporation in the HaCAT keratinocyte cell line, with VN synergistically enhancing cell migration in response to both EGF and bFGF when presented as a VN-bound complex. Furthermore, the addition of EGF and/or bFGF to IGF-I:IGFBP-5:VN complexes significantly enhances both [(3)H]-leucine incorporation and migration of HaCAT cells above that induced by IGF:IGFBP-5:VN complexes alone. Indeed, similar responses are observed in primary cultures of human skin keratinocytes, highlighting the potential use of these novel complexes for a wide range of tissue repair applications.     

Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity.

Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Purification and characterization of heparin-binding growth factors from porcine uterus.

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.     

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.     

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha-transforming growth factors in ras-transformed mouse mammary epithelial cells

A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c-Harvey ras proto-oncogene, NMuMG/rasH, or by the polyoma middle T-transforming gene, NMuMG/pyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha-transforming growth factor (alpha TGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage-independent growth of the NMuMGrasH cells. Likewise, human EGF or human alpha TGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum-free medium supplemented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, whereas these same growth factors under comparable culture conditions have little or no effect upon the anchorage-dependent growth of the ras-transformed NMuMG-rasH cells. The biological refractoriness of the NMuMG/rasH cells to human EGF or human alpha TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high-affinity population of binding sites for mouse [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage-independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive alpha TGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of alpha TGF mRNA in the NMuMG/rasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto-oncogene, but not by the polyoma middle T-transforming gene, become unresponsive to exogenous EGF or alpha TGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells

Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK-21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.     

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.     

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

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.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.