New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues.

Abstract

Proteins potentiated by epidermal growth. factor (EGF) to induce a transformed phenotype in non-neoplastic rat kidney fibroblasts in cell culture have been isolated from many non-neoplastic tissues of the adult mouse, including submaxillary gland, kidney, liver, muscle, heart, and brain. They resemble previously described transforming growth factors (TGFs) isolated from neoplastic cells as follows: they are extractable by acid/ ethanol and are acid-stable, low molecular weight (6000-10,000) polypeptides requiring disulfide bonds for activity, and they cause anchorage-independent growth of non-neoplastic indicator cells that will not grow in soft agar in their absence. Partial purification of these TGFs from submaxillary glands of male mice shows that they are distinct from EGF. Unlike previously described extracellular TGFs, but like certain cellular TGFs from neoplastic cells, they are potentiated by EGF in their ability to promote anchorageindependent growth. The isoelectric point of the submaxillary gland TGF protein is near neutrality. Chromatography on Bio-Gel P-30 followed by high-pressure liquid chromatography has resulted in a 22,000-fold overall purification. The most purified protein is active in inducing growth in soft agar at 1 ng/ml when assayed in the presence of EGF. The data add further evidence to the concept that neoplasia may result from a quantitative, rather than qualitative, alteration in non-neoplastic biochemical processes. We have recently described (1) the isolation and characterization of a set of low molecular weight, acid-stable polypeptides, called transforming growth factors (TGFs), from several neoplastic mouse tissues including fibroblasts transformed by Moloney sarcoma virus (MSV) and a transplantable bladder carcinoma originally induced by a chemical carcinogen. These polypeptides are intracellular proteins that are extractable by acid/ethanol. Similar extracellular transforming polypeptides, called sarcoma growth factors (SGFs), were first isolated by De Larco and Todaro (2) from the conditioned medium of MSVtransformed mouse fibroblasts grown in culture. Several other extracellular transforming polypeptides derived from neoplastic cells ofboth human (3) and animal (4) origin have been reported recently.All of these polypeptides cause the following set of changes when applied to untransformed, non-neoplastic indicator cells growing in culture, and these changes provide an operational definition of TGFs: (i) loss of density-dependent inhibition of growth in monolayer; (ii) overgrowth of cells in monolayer; (iii) change in cellular shape, with the result that the indicator cells assume the neoplastic phenotype; and (iv) acquisition of anchorage independence, with the resultant ability to grow in soft agar. Untransformed, non-neoplastic cells will not form progressively growing colonies in soft agar, and this property of anchorage-independent growth of cells in culture has a particularly high correlation with neoplastic growth in vivo (5-7).

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

“…Three of these clones, (all derived from exposure to 10 mM EMS) scored positive in the secondary and tertiary screens and were named NRK/EMS clones B, F, and J. Consistent with previous studies (Roberts et al, 1981;Assoian et al, 1983), we observed that a few colonies occasionally formed when the non-mutagenized NRK cell parent was cultured with serum and EGF alone. However, the colonies recovered from this culture condition never yielded clones that maintained high efficiency colony formation (>75 % of cells seeded; refer to Materials and Methods) in secondary or tertiary screens.…”

“…For example, TGF-/$1 does not directly stimulate DNA synthesis in adherent, G0-synchmnized fibroblasts, and it can even inhibit the proliferation mediated by mitogens such as serum and EGF (Massagu6, 1990;Moses et al, 1990;Sporn and Roberts, 1990). TGF-~I is also unusual in its ability to stimulate anchorage-independent growth of certain fibroblastic cell lines (Roberts et al, 1981;Assoian et al, 1983;Moses et al, 1985;Tucker et al, 1984). These distinct cellular effects of TGF-/$1 are consistent with recent studies showing that the TGF-/31 receptor has a serine/threonine kinase activity rather than the tyrosine kinase activity typically associated with peptlde growth factor receptors (Linet al, 1992;Massagu6, 1992).…”

Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth.

“…The collected supernatants were assayed for TGF-/3 biologic activity using a soft agar assay. This assay relies upon the ability of TGF-/3 to enhance the growth of NRK cells in agar in the presence of EGF (10). Supernatants conditioned by unactivated T cells had low levels of TGF-/3-1ike biologic activity (0.6-1.6 pM), while supernarants from PHA-activated T cells had levels 10-50-fold higher (20-74 pM, Table lI).…”

“…The bioassay for TGF-/3 depends upon the ability of TGF-t3 to induce anchorage-independent growth of normal rat kidney (NRK) fibroblasts grown in the presence of 5 ng/ml EGF in soft agar. The soft agar assay was performed as previously described by measuring colony formation in 0.3 % agar (10). Colonies were stained after 7 d of incubation, and those >62 #M in diameter were counted using an Omnicon image analysis system programmed to count a 5-cm 2 area ofa 35-mm-diam plate.…”

Synthesis and secretion of transforming growth factor beta isoforms by primary cultures of human breast tumour fibroblasts in vitro and their modulation by tamoxifen

“…TGF-P is a member of a superfamily of regulatory peptides, existing as three isoforms TGF-fl,, TGF-f2 and TGF-#3 in mammalian species. These are multifunctional peptides that are usually stimulatory to cells of mesenchymal origin (Roberts et al, 1981), but inhibitory to certain epithelia . This growth factor is involved in cellular proliferation and differentiation during development, and defective TGF-P signalling may be implicated in carcinogenesis (Roberts et al, 1988).…”