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).

Polptides characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from tumor cells growing both in culture and in the animal, by using an acid/ethanol extraction procedure. Assay of these polypeptides is based on their ability to induce normal rat kidney fibroblasts to form colonies in soft agar. Peptides from murine sarcoma virus-transformed mouse 3T3 cells grown in culture had the highest specific activity in this assay; peptides from sarcomas produced from these cells or from chemically induced transplantable bladder carcinomas of mice were on d as active; and peptides from a chemically induced rat tracheal carcinoma had only one-tenth the activity. Treatment with either trypsin or dithiothreitol destroyed the activity of all of these materials. The properties of these intracellular polypeptides from both virally and chemically transformed cells are similar to those described for the sarcoma growth factors (SGFs) previously isolated from the conditioned medium of sarcoma virus-transformed mouse 3T3 For the purposes of purification of these polypeptides, it was desired to extract them directly from tumor cells, rather than from conditioned medium. It was also important to ascertain whether the SGFs were unique to MuSV transformation, or whether they were representative of a class of proteins with similar properties, which we shall call transforming growth factors (TGFs) (2). We now report finding TGFs in cultured MuSV-transformed 3T3 cells and in sarcomas produced by inoculation of these cells into athymic mice. Moreover, TGFs of specific activity approximately equal to that of the sarcomas have been isolated from chemically induced bladder carcinomas. These TGFs have been extracted from the cells with acid/ethanol, a procedure that has previously been applied to the extraction from organs and from blood of such biologically active polypeptides as insulin (3, 4), glucagon (5), insulin-like growth factor (6), the somatomedins (7), and secretin (8). The demonstrated application of this method to the finding of a class of acid-stable TGFs from cells transformed by either a virus or chemicals brings a unifying concept to the mechanism of carcinogenesis. MATERIALS AND METHODSMuSV-Transformed 3T3 Cells. The Moloney MuSV-transformed 3T3 cell line 3B11-1C was grown in roller bottles as described (1). After the removal of the "sarcoma-conditioned medium," cells were scraped from the bottles into phosphatebuffered saline. After centrifugation at 500 X g for 10 min, the supernatant was discarded and the remaining cell pellet was frozen in the gas phase of a liquid nitrogen freezer.Sarcomas Derived from MuSV-Transformed 3T3 Cells. Cells of the Moloney MuSV-transformed 3T3 cell line 3197-3 (9), deficient in leukemia helper virus, were scraped into sterile phosphate-buffered saline, and 1 X 106 cells were inoculated subcutaneously into nude mice. Tumors were harvested at 2 weeks to 2 months and immediately frozen and stored above liquid nitrog...

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