Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EGF) receptor, the type I insulin-like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti-EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell-cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin- and IGF-I-induced growth stimulation of melanoma cells was inhibited by MAb alpha IR-3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF-I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.     

Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

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.     

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.     

The purification of fully active recombinant transforming growth factor alpha produced in Escherichia coli

Recombinant human transforming growth factor alpha (TGF alpha), which is active as assessed by competition with epidermal growth factor (EGF) for binding to the EGF receptor, has been produced in Escherichia coli and separated from misfolded and inactive forms of recombinant TGF alpha using reverse-phase high performance liquid chromatography. The purified recombinant TGF alpha was used to produce a monoclonal antibody that binds to active TGF alpha specifically. The antibody was coupled to Sepharose and used as an independent method for purifying active TGF alpha. The EGF receptor binding activity of antibody affinity purified TGF alpha is comparable to that of high performance liquid chromatography-purified active TGF alpha, and is 0.55 mg of EGF eq/mg of TGF alpha. The disulfide arrangement of the active TGF alpha was determined after digestion with thermolysin, and found to be analogous to the disulfide arrangement previously determined for EGF (Savage, C. R., Hash, J. H., and Cohen, S. (1973) J. Biol. Chem. 248, 7666-7672).     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

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.     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

Human epidermal growth factor (EGF) receptor sequence recognized by EGF competitive monoclonal antibodies. Evidence for the localization of the EGF-binding site

Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

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.     

Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.     

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.     

A novel ganglioside, de-N-acetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor receptor kinase and as a stimulator for cell growth

A novel ganglioside, de-N-acetyl-GM3 (neuraminyllactosylceramide, II3NeuNH2LacCer), was found in the monosialoganglioside fraction of A431 cells and B16 melanoma cells by high-performance liquid chromatography, thin-layer chromatography, and immunoblotting with its specific monoclonal antibody DH5. This novel type of membrane ganglioside strongly enhanced the kinase activity associated with the epidermal growth factor (EGF) receptor, and it showed 32, 35, and 12% growth stimulation as compared with control cultures of A431, Swiss 3T3, and B16 melanoma cells, respectively. Exogenously added de-N-acetyl-GM3 did not alter the affinity of EGF binding to its receptor. These properties of de-N-acetyl-GM3 are in striking contrast to those of GM3 and its lyso derivative (lyso-GM3) which were previously shown to inhibit EGF receptor kinase activity and to inhibit growth in the same cells. These data indicate that de-N-acetylation at the sialic acid moiety of GM3 ganglioside is an important mechanism for modulation of EGF-dependent cell growth. The mechanism is antagonistic to that of GM3-dependent modulation of receptor function.