Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction.

Abstract

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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