Purification and Characterization of a Novel Transforming Growth Factor1

Previous studies have indicated that an autostimulatory transforming growth factor was required for the optimal growth of SW-13 adrenal carcinoma cells in soft agar. The production of SW-13 colony-stimulating activity by other human malignant cell lines of both epithelial and mesenchymal origin has been demonstrated. Evidence was presented indicating that the stimulating activity detected in crude acid-ethanol extracts was an acidand heat-stable polypeptide requiring disulfide bonds for full activity. This activity was detected more frequently in tumors and human cancer cells in culture of epithelial origin than of mesenchymal origin and in a variety of nonneoplastic tissues. In the present study, this activity, termed epithelial transforming growth factor (TGFe) because of its ability to stimulate soft agar growth of certain epithelial cells, was partially purified from bovine kidney. Fourfold purification of the kidney acid-ethanol extract with 50% maximal growthstimulatory activity of 10 «ig was achieved using molecular sieve chromatography where TGFe eluted with an apparent molecular weight of 20,000-25,000. The next purification step, molecular sieve high perform ance liquid chromatography, yielded a 50% maximal growth-stimulatory activity of 50 ng and an 800-fold purification from the initial acid-ethanol extract. TGFe eluted in the M, 11,000 range. Reversed phase high performance liquid chromatography with a Ci«column was then used, yielding a single or double peak of SW-13 colony-stimulating activity at 30-35% acetonitrile. The degree of purification was 11,000-fold with a 50% maximal growth-stimulatory activity of 3.5 ng. Analysis of the peak on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major and sometimes single band with a molecular weight of 23,000-25,000. Extraction of protein from the polyacrylamide gel dem onstrated that only the M, 23,000-25,000 band stimulated soft agar growth of SW-13 cells. The biological activity of the partially purified TGFe was found to differ from other known growth factors with regard to its ability to stimulate soft agar growth of SW-13 cells with the exception of basic fibroblast growth factor (FGF). The acid lability of FGF, the different molecular weights of these two growth factors, the lack of stimulation of soft agar growth of A431 cells, and the lack of binding of TGFe to FGF receptors indicated that TGFe was not related to basic FGF. Partially purified TGFe was also found to stimulate soft agar growth of two squamous cell carcinoma lines, A431 and 1)562. and the mouse embryo-derived AKR-2B cells. These data suggest that TGFe is a unique factor distinct from those previously described and it is speculated that TGFe may play a role in normal and neoplastic growth of epithelial cells. INTRODUCTION TGFs4 are operationally defined as polypeptides stimulating anchorage-independent growth of nontransformed anchoragedependent cells through high affinity binding to membrane receptors. TGFa and TGF/3 are the two major representatives. TGFa is a A/r 5600 single chain polypeptide with 3 intrachain Received 12/16/86; revised 5/22/87; accepted 6/5/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation was supported by USPHS Grant CA 27217 awarded by the National Cancer Institute, Department of Health and Human Services. 2To whom requests for reprints should be addressed. Present address: Depart ment of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. 3 Present address: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232. 'The abbreviations used are: TGF, transforming growth factor; EGF, epider mal growth factor; FGF, fibroblast growth factor, RP-HPLC, reverse phase high pressure liquid chromatography; BSA, bovine serum albumin; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; PDGF, platelet-derived growth factor; ODS, octadecylsilane; !• I)>„. 50% maximal growth-stimulatory activity. disiiltidc bonds. It shares 30 to 35% sequence homology with human and rodent EGF (1). TGFa competes with 125I-labeled EGF for binding to the EGF receptor completely and equivalently (2, 3). TGFa activity is similar if not identical to that of EGF. It is a potent mitogen for mesenchymal and epithelial cells in monolayer but only a weak stimulator of anchorageindependent growth in soft agar of mesenchymal cells (4). TGFa acts, like EGF, in synergism with TGF/3 in promoting soft agar growth of nontransformed NRK cells (4, 5). Unlike EGF, TGFa has been identified only in embryonic (6) and tumor tissue and transformed cells (1, 4, 5, 7, 8) but not in adult nonneoplastic tissues and organs. TGF/3 is a M, 25,000 polypeptide consisting of two identical 112-amino acid chains held together with disulfide bonds (912). Platelets are the major source of TGF/3 (9, 13). TGF/3 is a ubiquitous growth factor. It is produced by transformed and nontransformed cell lines (14-16), solid human neoplasms (17, 18), nonneoplastic tissues (10-13, 17-19), and mouse embryos (20). The ubiquity and high degree of conservation among species of TGF0 (21) suggest an important role in the control of cell growth and proliferation (22). It acts on many cells, both mesenchymal and epithelial, and its effect differs, depending on the cell type tested (22-24). It stimulates anchorage-inde pendent growth of anchorage-dependent mouse embryo AKR2B cells (14,16) and confers transformed morphology on them in a reversible manner (13, 25). TGF/3 is very similar to the growth inhibitor purified from African green monkey BSC-1 cells by Holley et al. (26, 27). They both inhibit the growth of epithelial cell lines, bind to the same receptor with the same affinity, and have the same molecular weight of 25,000 (23). Previous studies have indicated the presence of an apparently novel TGF (18) in a variety of tissues, both neoplastic and nonneoplastic. It stimulated soft agar growth of SW-13 cells, derived from a human small cell carcinoma of the adrenal. Evidence was presented suggesting this TGF was responsible for autocrine stimulation exhibited by these cells. This SW-13stimulating activity appeared to be an acidand heat-stable polypeptide requiring disulfide bonds for its activity. It could be separated from TGF/3 by molecular sieve chromatography and RP-HPLC. In this paper we report partial purification and further characterization of this novel TGF, called TGFe because of its ability to stimulate soft agar growth of certain epithelial cells. MATERIALS AND METHODS Cell Culture. SW-13 (28), HT-1080 (29), JEG-3 (30, 31), AN3 CA (32), and BS-C-1 (33) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A431, A549, A204, A375 (34), and NRK (clone 49F) (35) cell lines were a gift from Drs. G. J. Todaro and J. E. De Larco. The D562 cell line was provided by Dr. R. E. Scott who purchased it from the ATCC (36). SW620 and SW480 cell lines were given by Dr. R. J. Coffey, Jr., who obtained them from the ATCC (37). Drs. W. Rowe and N. Teich provided the original stock of AK.R-2B cells (38, 39); clone 84A was obtained from J. A. Proper (20). C3H/10TV2 cells and their transformed counterpart C3H/MCA 58 cells were obtained from Dr. C. Heidelberger (40). All cell lines were maintained in McCoy's Medium 5a (Grand Island Biological Co., Grand Island, NY) supplemented with 5% (v/v) fetal