A Transforming Growth Factor From MCG-T14 Mouse Mammary Carcinoma Cells

Abstract

Mitogenic activity was assayed in the harvest fluid concentrates (HFC) from. human· mammary cell.lines ·(T47P, HSO 578T, MDA-157, HBL-100 and BT-20) and a mouse mamamry carcinOinii (MCG-T14) cell line. Data showed that the HFC from four of the human cell lines (T47D,.HSO 578T, MDA-157, and HBL-fOO) and the mouse cell line (MCG-Tl4) were sour.ces of mitogenic activity. The mitogenic activity from the HFC was not due to the action of the serine protease, plasminogen activator. The mitogenic substance was also not a cell degradation product. The HFC from the human mammary carcinoma line BT-20 contained a non-dialyzable inhibitory activity which su~pressed the activities of the mitogerts in fetal bovine serum. Attempts to isolated the mitogenic activity from HFCs proved impractical due to proteolytic breakdown. However, a transforming growth factor (TGF) from acid-ethanol extracts of MCG-T14'mouse mammary carcinoma ' ' cells was isolate~ and·partially characterized. This factor stimulated thymidine uptake in BALB/c 3T3 cells and promoted anchorage-independent growth of NRK-49F cells in soft agar. The mitogenic activity, designated T14-TGF, stimulted thymidine uptake in conflue.nt-quiescent BALB/c 3T3 cells to the same extent as tha,t exhibited by 5% calf serum. T14-TGF was potentiated by EGF in its ability to promote colonial growth of NRK-49F cells. In competition binding assays, T14-TGF and EGF competed for binding to BALB/c 3T3 and A431 cells. However, EGF was a more efficien~ competitor than T14-TGF in all experiments and T14-TGF exhibited only partial inhibition of EGF-binding to A431 cells. This suggests that T14-TGF may have contained subfractions which did not compete for EGF binding sites. Nerve growth factor, fibroblast growth factor, and multiplication stimulating activity did not compete with T14-TGF for binding .. 1 sites .on BALB/c 3T3 cells. In addition, iodinated T14-TGF did not bind to NR6/6 3T3 cells which lack EGF receptors. It was concluded that Tl4-TGF must interact with EGF receptors specifically~ 2 T14-TGF was purified to apparent electrophoretic homogeneity by electroelution from 15% SDS-urea polyacrylamide gelso The factor was basic (pi 8.3), had anapparent moleculaJ; weight of 14,000 and was selectively inactivated by trypsin. In addition, T14-TGF exhibited no proteolytic activity and contained no carbohydrate residues. The biological activity of T14-TGF was resistant to inactivation by acid or heat and· denaturation by guanidine HCl. T14-TGF was completely inactivated by treatment with dithiothreitol which indicated that it required one or more intact disulfide .bridges for activ~ty. A unique characteristic of T14 ... TGF was that this factor bound very strongly to. both DEAE or carboxymethyl f:on excha'Iigers. T14-TGF contained high proportions of basic amino acid residues lys~ne and arginine.