Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1.

Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which alpha FGF and beta FGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.

Cardiac allograft vasculopathy. Preferential regulation of endothelial cell-derived mesenchymal growth factors in response to a donor-specific cell-mediated allogeneic response.

We have previously reported that cell-mediated immunity to vascular endothelium is associated with the development of cardiac allograft vasculopathy (CAV). The mechanism by which a cell-mediated immune response to the coronary vascular is translated into the development of CAV is, however unknown. Peripheral blood mononuclear cells (PBMCs) obtained serially following cardiac transplantation were cocultured with donor-specific human aortic endothelial cells (HAECs) in 47 allograft recipients, 9 of whom had CAV (CAV+) at 1 year by angiography. At 20 hr following coculture, HAEC poly (A+) RNA was isolated, reverse-transcribed, and the cDNA-amplified (PCR) for a panel of growth factors (GFs) known to alter smooth muscle cell proliferation or migration. Relative quantitation of PCR product was performed using high-pressure liquid chromatography (HPLC). Three patterns of GF regulation were observed depending on the GF, the time posttransplant, and whether the patient had CAV: (1) no regulation (TGF-beta, PDGF-A early post-tx); (2) upregulation irrespective of CAV (bFGF, PDGF-B, TGF-alpha early post-tx); and (3) preferential or exclusive upregulation by CAV+ patients (PDGF-A and TGF-alpha late post-tx, HB-EGF early and late post-tx). For example, using PBMCs as stimulators, obtained 6 months posttransplant from CAV+ patients, increases in HAEC-derived PDGF-A chain (31 +/- 7 to 69 +/- 11), TGF-alpha (97 +/- 27 to 201 +/- 23), and HB-EGF (78 +/- 16 to 173 +/- 27) mRNA were demonstrated (all P<0.05 or greater using HPLC peak area as units). These data demonstrate that cell-mediated activation of vascular endothelial cells in patients with CAV results in preferential upregulation of certain endothelial-derived mesenchymal growth factors capable of stimulating smooth muscle cell proliferation and migration.

Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin, basic fibroblast growth factor, and heparin-binding EGF-like growth factor activity.

Amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF) are two recently identified members of the EGF family. Both AR and HB-EGF share with EGF the ability to interact with the type-1 EGF receptor; however, AR and HB-EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin-like molecules on the cell surface influence binding of AR and HB-EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR-2B and Balb/MK-2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of 125I-EGF on AKR-2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with 125I-EGF for cell surface binding and also attenuated AR-mediated DNA synthesis. Thus, as has been suggested for other heparin-binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF-binding receptor appears to be facilitated by interaction with cell-associated sulfated glycosaminoglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB-EGF-induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB-EGF to compete with 125I-EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB-EGF to compete for 125I-EGF binding. These results suggest that, in AKR-2B cells, HB-EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB-EGF and permits HB-EGF-mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin-binding mitogens other than AR.

Time course and contact dependence of allogeneic lymphocyte-induced human aortic endothelial cell-derived interleukin-6.

Vascular endothelial cells secrete the pluripotent cytokine interleukin-6, and the induction of this secretion can be regulated by a number of other immune-related cytokines. To determine whether a cellular alloimmunologic response to vascular endothelial cells alters the expression of interleukin-6 production by endothelial cells, we cocultured peripheral blood lymphocytes with a pool of human aortic endothelial cells. In response to the pool of allogeneic human aortic endothelial cells, lymphocytes from 10 separate donors proliferated to varying degrees after 5 days of coculturing. After 20 hours, human aortic endothelial cell-derived messenger RNA coding for interleukin-6 increased an average of 96% after exposure to allogeneic lymphocytes and the amount of biologically active interleukin-6 released into the media increased 69%. The kinetics of human aortic endothelial cell interleukin-6 messenger RNA expression in response to lymphocytes from an additional three donors was determined over a 48-hour period. Human aortic endothelial cell interleukin-6 messenger RNA increased approximately threefold over control, as early as 2 hours after exposure to allogeneic lymphocytes and returned toward control levels by 48 hours. Activation of six additional isolates of lymphocytes with phorbol myristate acetate before exposure to human aortic endothelial cells resulted in an increase in human aortic endothelial cell-derived interleukin-6 bioactivity regardless of whether the cells were in direct contact with the human aortic endothelial cells, but the interleukin-6 level increase was approximately twofold higher in those cocultures where there was direct contact. These data show that allogeneic lymphocytes have the potential of regulating vascular endothelial cell-derived interleukin-6, and direct lymphocyte-endothelial cell contact appears to be required for optimal interleukin-6 induction in this in vitro system.

Phenotypic variations in resting and activated levels of ICAM-1 expression by cultured human aortic endothelial cells.

ICAM-1 is an inducible glycoprotein important in the adhesion, activation, and transmigration of circulating leukocytes across the vascular endothelial monolayer, and it likely plays a key role in the allogeneic response. To determine the reproducibility and significance of variations in resting levels of cell surface ICAM-1, 3 individual measurements of ICAM-1 levels were performed on 26 individual isolates of human aortic endothelial cells (HAECs) both at rest and following activation by allogeneic lymphocytes, using flow cytometry. Resting HAEC ICAM-1 levels varied 10-fold (range 6-60 mean fluorescence channels) depending on the isolate studied. There were strong correlations (r = 0.71 to 0.77, P < 0.0001) between the three measurements (performed no closer than weekly intervals on separate cultures), attesting to the consistency of the phenotypic expression. Constitutive expression of ICAM-1 was not affected by cell age, based upon comparing a subset of these isolates across 3 population doublings. Levels of HAEC ICAM-1 following allogeneic lymphocyte activation varied 15-fold (range 20-300 mean fluorescent channels) and, more important, correlated with resting ICAM-1 levels (r = 0.58, P = 0.002). Finally, constitutive ICAM-1 expression was related to TNF-alpha-induced ICAM-1 levels based upon a subset of the isolates studied. These data suggest that phenotypic, and likely genetic, differences in quiescent endothelial cell adhesion molecule expression can influence inflammatory responses including alloresponsiveness to the vasculature.

Human keratinocytes are a major source of cutaneous platelet-derived growth factor.

PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy.

In the studies reported here, we demonstrate that transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) is required for the establishment of small colonies of human keratinocytes at clonal densities, but once small (10-15 cells) colonies have formed, the continued growth of these colonies can proceed in the absence of exogenous TGF-alpha or EGF. Equivalent receptor-binding concentrations of TGF-alpha and EGF were equipotent in stimulating colony formation. We also demonstrate that the growth of keratinocytes at high densities proceeds in the absence of exogenous peptide growth factors or hormones. The expression of TGF-alpha mRNA and protein is regulated by both cell density and the presence of exogenous growth factors. The addition of an antibody which blocks the mitogenic effect of mature TGF-alpha had no effect on the autocrine/paracrine growth of these cells at either density. However, monoclonal antibodies which antagonize ligand activation of the EGF receptor inhibit the autonomous proliferation of keratinocytes at high density and abrogate the exogenous TGF-alpha/EGF-independent expansion of colonies at clonal density. The results of these experiments are among the first evidence to demonstrate that normal human epithelial cells in culture exhibit autocrine/paracrine-mediated proliferation. Exogenous growth factors initiate colonies of human keratinocytes that become self-perpetuating in culture. Keratinocytes regulate production of the mitogenic ligand, TGF-alpha, through a density-dependent mechanism, and cell density stringently controls proliferation.

The pattern of cytokine messenger RNA expression in human aortic endothelial cells is different from that of human umbilical vein endothelial cells.

Endothelial cells most readily available and most frequently used in investigations of alloimmunity and cytokine expression and function are derived from human umbilical veins. It is unclear whether cells derived from fetal venous tissue are relevant to phenomena related to the adult allograft, especially in areas such as cardiac allograft vasculopathy, a chronic rejection process directed against the coronary arteries. Human aortic endothelial cells (HAECs) were compared to human umbilical vein endothelial cells (HUVECs) for their constitutive expression of poly (A)+ RNA coding for a group of cytokines known to stimulate smooth muscle cell proliferation, including acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, transforming growth factor beta, platelet-derived growth factor A-chain, platelet-derived growth factor B-chain and amphiregulin. Poly (A)+ RNA coding for basic fibroblast growth factor and transforming factor-beta was consistently expressed by all nine isolates of HAECs, but platelet-derived growth factor A- and B-chain were expressed in only six of the nine isolates. In most cases this was related to the presence of transforming growth factor alpha expression. In contrast, HUVECs consistently expressed basic fibroblast growth factor, transforming growth factor-beta, and both platelet-derived growth factor chains. Transforming growth factor alpha expression was never seen in the HUVEC isolates. No endothelial cell isolate expressed mRNA coding for acidic fibroblast growth factor or amphiregulin. There appear to be differences between cytokine gene expression patterns by endothelial cells from different vascular beds.

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.

Amphiregulin messenger RNA is elevated in psoriatic epidermis and gastrointestinal carcinomas.

Amphiregulin (AR) is a heparin-regulated, epidermal growth factor-like growth factor capable of stimulating the proliferation of non-tumorigenic cells while inhibiting cell proliferation in some human tumor cell lines in vitro. In the present study, we have investigated AR mRNA expression in normal, hyperproliferative, and neoplastic human epithelium. Our results demonstrate that, compared with the adjacent uninvolved epithelium, AR mRNA expression is markedly elevated in epidermal biopsies derived from three human psoriatic lesions as well as in biopsies derived from five human colon carcinomas and three human stomach carcinomas. Moreover, analysis of a colon carcinoma by in situ hybridization revealed that AR mRNA is localized to the epithelium.

Inhibition of autonomous human keratinocyte proliferation and amphiregulin mitogenic activity by sulfated polysaccharides.

We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Expression of multiple species of basic fibroblast growth factor mRNA and protein in normal and tumor-derived mammary epithelial cells in culture.

We examined the expression of the basic fibroblast growth factor (bFGF) gene in cultured normal and tumor-derived human mammary epithelial cells at both the transcriptional and translational level. Northern blot analysis revealed three bFGF mRNA transcripts of 7.5, 4.4, and 2.2 kilobases in all four strains (donors) of normal cells (HMECs) we examined and in the immortal mammary cell line HBL-100. Of the four mammary tumor-derived cell lines we examined (MCF-7, BT-474, T-47D, and Hs578T), only the Hs578T cells produced detectable levels of bFGF mRNA. Western blot analysis of cell lysates using an anti-bFGF monoclonal antibody revealed corresponding results. bFGF protein was detected in normal HMEC strains 161 and 48 (other normal strains not tested), in HBL-100 cells, and in Hs578T cells, but not in the other tumor cell lines. In each case, three distinct molecular weight species of bFGF protein were detected which migrated in sodium dodecyl sulfate-polyacrylamide gel at 18, 24, and 27 kDa. We also investigated the ability of bFGF to stimulate the proliferation of normal and tumor-derived mammary epithelial cells. Addition of bFGF to serum-free cultures of these cells had no effect on the proliferation of HMECs under a variety of conditions and was weakly mitogenic for Hs578T cells. Our results indicate that normal HMECs produce bFGF mRNA and protein(s), whereas only some mammary tumor-derived cells express this gene. Thus, our results do not support a general role for expression/overexpression of bFGF in the development of mammary tumors. However, bFGF could play a role in the normal development and homeostasis of the mammary gland.

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.

Synoviocytes synthesize, bind, and respond to basic fibroblast growth factor.

Rheumatoid arthritis (RA) is a systemic disease characterized by the destructive proliferation of synovial tissue. It has been suggested that this proliferative lesion resembles a malignancy. Although polypeptide growth factors have been implicated in malignant cell growth, their role in the pathogenesis of proliferative but non-neoplastic diseases such as RA has not been extensively studied. We tested the hypothesis that the synoviocyte itself may be a source of growth factor activity. We demonstrated that culture supernatants from synoviocytes obtained from patients with RA, osteoarthritis, and traumatic joint disease contain mitogenic activity. This activity has biologic properties identical to those of basic fibroblast growth factor (bFGF). Specifically, the mitogenic activity is synergistic with insulin and binds to heparin-agarose, but elutes with 2.0M NaCl. In addition, synoviocyte extracts contain a peptide with a molecular weight of approximately 16,000, which reacts with antibody specific for bFGF. Cultured synoviocytes express the bFGF gene, express receptors for bFGF, and proliferate in response to bFGF. We conclude that bFGF derived from the synoviocytes themselves may play a role in stimulating their proliferation in an autocrine manner in disease states such as RA.

Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements.

Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell. Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.

Differential expression of mRNA coding for heparin-binding growth factor type 2 in human cells.

The proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin-binding growth factor type 2/basic fibroblast growth factor (HBGF-2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF-2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type-beta also increased HBGF-2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF-2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum-free medium and at higher levels in cells rapidly growing in serum-containing medium. In contrast to fibroblasts, mRNA coding for HBGF-2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF-2/bFGF, our results suggest that HBGF-2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC-25) expresses mRNA coding for HBGF-2/bFGF, suggesting that the gene may become activated in some carcinomas.

Transforming growth factor alpha and beta expression in human colon cancer lines: implications for an autocrine model.

Three human colon cancer lines (SW480, SW620, WIDR) secrete different levels of transforming growth factor beta (TGF beta)-like and transforming growth factor alpha (TGF alpha)/epidermal growth factor (EGF)-like molecules into serum-free conditioned media as measured by competing activity in TGF beta and EGF radioreceptor assays. SW480 cells, the highest producers of TGF beta-like activity, lack detectable TGF beta receptors while SW620 cells, the highest producers of TGF alpha/EGF-like activity, lack EGF receptors. This study investigated the production of these growth factors at the mRNA level and examined the mechanism of loss of detectable receptors. Using complementary DNA probes for TGF beta and TGF alpha, it was demonstrated that mRNA levels correlated with the amounts of TGF beta and TGF alpha produced; TGF beta gene expression was highest in SW480 cells and TGF alpha gene expression was highest in SW620 cells. Acid washing of the SW480 cells prior to performing the TGF beta binding assay resulted in the unmasking of substantial levels of TGF beta receptors. Neither acid washing nor preincubation with suramin uncovered EGF receptors in SW620 cells. Also, and in contrast to the other two lines, EGF receptor expression could not be detected in SW620 cells by Northern gel analysis of receptor messenger RNA or by immunological analysis of receptor protein. Thus two distinct mechanisms (occupation of TGF beta receptor in SW480 cells, or absence of EGF receptor in SW620 cells) explain the lack of detectable TGF beta and EGF receptors in the binding assays. The autocrine hypothesis remains viable for TGF beta in SW480 cells but not for TGF alpha in SW620 cells; this would not discount a paracrine role in this latter case.

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.

Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium.

Type beta transforming growth factor-growth inhibitor (TGF beta/GI) causes normal human prokeratinocytes to arrest growth predominantly in the G1 phase of the cell cycle within 48 h after log phase cultures are exposed to the factor in serum-free medium. The growth arrest induced by TGF beta/GI is reversible because the cells from treated cultures can be replated into fresh medium and grown into large colonies. Normal prokeratinocytes are demonstrated to secrete TGF beta/GI-like molecules into the culture medium and to have specific cell surface receptors for this molecule. In contrast, a human squamous cell carcinoma, SCC-25, does not arrest growth when exposed to TGF beta/GI. These cells, unlike the normal prokeratinocytes, do not exhibit detectable cell surface receptors for the factor.