Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are structurally related peptides. Purified human TGF-alpha produced in Escherichia coli and pure natural mouse EGF were compared for their ability to bind to target cells in vitro and to promote angiogenesis in the hamster cheek pouch bioassay. Both polypeptides were found to bind in vitro to several target cells, including endothelial cells, and to stimulate their DNA synthesis in an equipotent fashion. In vivo, however, TGF-alpha was more potent than EGF in promoting angiogenesis and, because TGF-alpha is known to be secreted by a variety of human tumors, it is suggested that this growth factor may contribute to tumor-induced angiogenesis.

ABSTRACT

Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A. 431 cells and selected according to their ability to inhibit the binding of radiolabled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerfil tool to study the structure, processing, and mode of action of EGF receptors.Epidermal growth factor (EGF), a single-chain polypeptide hormone isolated from the mouse submaxillary gland (1), is a potent mitogen for a variety of epidermal and epithelial cells both in vivo and in vitro (2). The initial interaction of EGF with target cells occurs at specific membrane receptors, which were identified as 150,000-to 170,000-dalton glycoproteins (3)(4)(5). Various methods were used to visualize and follow the cellular locations ofEGF receptors in culture cells (6-8). The general pattern that emerges from these studies is that EGF binds to diffusely distributed, laterally mobile (9) membrane receptors, which cluster rapidly, over coated regions of the plasma membrane, in a temperature-sensitive process (2, 8, 10). The EGF-receptor complexes then pinch off, by an energy-dependent process, and form vesicles inside the cell. The endocytic vesicles interact with other intracellular organelles, where the hormone and presumably also the receptor are degraded (2, 11). Concomitantly with this now well-established sequence of events, EGF induces a diverse repertoire of physiological processes. These include early responses such as stimulation of nutrient uptake (for review see ref.2), enhanced phosphorylation ofendogenous membrane proteins (5,(12)(13)(14), and changes in cell structure (15) and cytoskeletal organization (16). Delayed responses to EGF include the activation of cytoplasmic enzymes and stimulation of DNA synthesis (2). The relationship between the biological activity of EGF and the topographical processing of the EGF-membrane receptor complexes is still unresolved.

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