A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties

A fragment of rat transforming growth factor alpha (TGF alpha) comprising the third disulfide loop (residues 34-43) was selected as a potential antigenic and receptor binding region. Immunization of rabbits with a peptide conjugate resulted in antibodies which were specific for both the peptide and rat TGF alpha, but not for the homologous epidermal growth factor (EGF). The synthetic decapeptide exhibited low affinity for EGF receptors on human cells. Affinity was increased 100x to 0.2% of EGF or TGF alpha binding by blocking the peptide ends. The blocked decapeptide had no mitogenic activity but prevented the mitogenic effect of EGF and TGF alpha on fibroblasts. This decapeptide is an antagonist and contains an important receptor binding region of TGF alpha.     

Inability of anti-epidermal growth factor receptor monoclonal antibody to block "autocrine" growth stimulation in transforming growth factor-secreting melanoma cells

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.     

Identification and initial characterization of transforming growth factor-like mitogen(s) in human anterior pituitary

Transforming growth factor (TGFs) are a family of peptide(s) defined by their ability to induce anchorage-independent growth of non-neoplastic indicator cells in soft agar. We found that acid-ethanol extracts of human anterior pituitary tissues were able to stimulate colony growth of normal rat kidney fibroblasts in soft agar. When subjected to gel-filtration on a column of Bio-Gel P-60 in 1 M acetic acid, the majority of TGF activity eluted in fractions corresponding to an apparent mol wt 15,000. The activity was heat- and acid-stable, but was inactivated by treatment with trypsin and dithiothreitol. Pituitary TGF-like materials did not compete with epidermal growth factor (EGF) for receptor binding and did not require EGF for colony-forming activity. Thus, human pituitary TGF was not like type alpha or type beta TGF.     

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.     

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.     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.     

Transforming growth factor activity of bovine brain-derived growth factor

Bovine brain-derived growth factor (BDGF), whose biochemical properties resemble those of endothelial cell growth factor (ECGF) and brain-derived acidic fibroblast growth factor (acidic FGF), is able to promote colony formation of normal rat kidney fibroblasts (NRK cells) in soft agar. As in the case of transforming growth factor beta (TGF beta), EGF potentiates the anchorage-independent growth promoting activity of BDGF. In the presence of EGF (5 ng/ml), the optimal concentration of BDGF for stimulation of anchorage-independent of NRK cells is approximately 0.5 ng/ml. At higher concentrations, BDGF becomes inhibitory. The anchorage-independent cell growth promoting activity of BDGF differs from that of TGF beta in acid and reducing agent stability.     

Estrogen stimulates growth of mammary tumor cells ZR-75 without activation of S6 kinase and S6 phosphorylation. Difference from epidermal growth factor and alpha-transforming growth-factor-induced proliferation

Growth of the human mammary tumor cell line ZR-75-1 is stimulated by epidermal growth factor (EGF) and alpha-type transforming growth factor (alpha TGF), as well as by estradiol (E2). The role of activation of S6 kinase and S6 phosphorylation in the EGF(alpha TGF)-induced and E2-induced growth was investigated. Maximal effects on growth are observed at 10 nM EGF or alpha TGF. EGF as well as alpha TGF treatment of serum-starved cells leads to rapid activation of S6 kinase; the activity is increased about tenfold after 30 min of EGF treatment and declines with the time reaching about 25% of the maximal activity after 2 h of EGF treatment. Similar to the growth response, S6 kinase is activated at lower doses of EGF than alpha TGF and shows a maximal response at 10 nM for both growth factors. In contrast to this finding the incubation of serum-starved cells with E2 over a concentration range between 1 pM and 10 nM and times from 30 min to 4 h does not lead to increased S6 kinase activity. On investigating whether this lack of response to E2 is due to desensitization of the system by induction of alpha TGF it was found that preincubation of cells with alpha TGF for 2-6 h desensitizes them to reactivation of S6 kinase by alpha TGF, whereas preincubation with E2 does not. When S6 phosphorylation is monitored over times from 1 h to 6 h, it is observed that EGF leads to increased S6 phosphorylation, whereas E2 does not. The rate of onset of protein synthesis in the first 2 h of stimulation, when EGF-induced S6 phosphorylation is maximal, is more rapid with EGF than with E2. The results suggest that different pathway are involved in E2-induced and EGF(alpha TGF)-induced proliferation.     

Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. II. Identification using a sequence-specific monoclonal antibody

Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human alpha-transforming growth factor (alpha TGF). To further characterize the bovine pituitary alpha TGF, it was compared to a human alpha TGF partially purified from the conditioned medium of a human melanoma cell line. An anti-alpha TGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat alpha TGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate 125I-peptide and then tested for recognition of human alpha TGF. Only 2 of 36 antipeptide antibodies recognized the native alpha TGF. The binding of 125I-peptide to MF9 was displaced by human alpha TGF but not by EGF. Bovine pituitary alpha TGF also displaced the binding of 125I-peptide to MF9 in a similar manner to human alpha TGF. Both iodinated human and bovine pituitary alpha TGF were immunoprecipitated by MF9 whereas 125I-EGF was not. Recognition of alpha TGF by MF9 was strongly dependent on sulfhydryl reduction of the growth factors, suggesting that synthetic peptides representing sulfhydryl-rich protein are not ideal immunogens. Tryptic digests of both 125I-alpha TGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C18 high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both alpha TGFs. The comparisons of the bovine pituitary and human melanoma alpha TGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these alpha TGFs are related and that alpha TGF production is not limited to transformed or fetal sources.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Synthesis of a biological active tumor growth factor from the predicted DNA sequence of Shope fibroma virus

A 55-residue peptide comprising the carboxyl portion (residues 26-80) of the Shope fibroma virus growth factor (SFGF), a predicted 80-residue DNA virus gene product that encoded a homologous sequence with the epidermal growth factor transforming growth factor alpha family, was synthesized by a stepwise solid-phase method. The synthetic SFGF (26-80) purified to homogeneity by reverse-phase HPLC was characterized by fission ionization mass spectrometry and amino acid analysis. The disulfide pairings were established by enzymatic digestion and mass spectrometry and were found to be similar to those of EGF and TGF alpha. Synthetic SFGF (26-80) was found to share about 10% of the activities as EGF in the radioreceptor binding to A431 cells, stimulation of [3H]thymidine uptake in NRK cells, and induction of colony formation in soft-agar assay. Our results therefore confirmed that SFGF contained the putative biological activities of the EGF-TGF alpha family and that production of SFGF by Shope fibroma virus infected cells may account for the proliferative diseases associated with this particular virus.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Regulation of cytochrome P-450 CYPIA1 gene expression and proto-oncogene expression by growth factors in primary hepatocytes

The effect of growth factors on the cytochrome P-450 (CYPIA1) gene expression was studied in primary mouse hepatocytes. Of the three growth factors used, i.e. epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin, only EGF or TGF alpha completely blocked CYPIA1 expression in the presence of the CYPIA1 inducer 3-methylcholanthrene (3-MC). This repression was not linked to cell cycle progression of the hepatocyte because insulin was active to induce 'early immediate genes' and DNA replication as well as EGF/TGF alpha but failed to suppress CYPIA1 expression. A specific EGF/TGF alpha receptor-mediated function may repress CYPIA1 gene expression and contribute to the acquisition of a xenobiotic drug resistance phenotype.     

Purification and characterization of a novel growth factor from human breast cancer cells.

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.     

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice.

Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas.