Purified growth inhibitor from BSC-1 cells and type beta transforming growth factor from human platelets are shown to have nearly identical biological activity and to compete for binding to the same cell membrane receptor. These findings suggest that the growth inhibitor and the type beta transforming growth factor are similar molecules. The data also show that the same purified polypeptide can either stimulate or inhibit cell proliferation depending on the experimental conditions.

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.Embryonic bone development begins with migration of mesenchymal cells to a predetermined site where they either condense, proliferate, and differentiate directly into boneforming cells or pass through an intermediate cartilage stage before they are replaced with bone. In adult life, bone has a remarkable potential to repair itself upon fracture through a process that recapitulates embryonic bone development. Urist (1) and Reddi and Huggins (2) have shown that the cellular events involved in embryonic bone development are reproduced in predictable intervals in subcutaneous implants of demineralized bone matrix in rats. By employing a reconstitution assay in the rat subcutaneous bone induction model (3, 4) and molecular cloning approaches, several osteogenic proteins (OPs), also called bone morphogenetic proteins [BMPs; BMP-2 through BMP-6, OP-1 (also called BMP-7), and OP-2] have been identified (5-8). The predicted amino acid sequences of these proteins indicate that they are all members of the transforming growth factor f8 (TGF-f3) superfamily, sharing a high degree of homology within the C-terminal seven-cysteine domain (9).The TGF-,p superfamily members are signaling molecules thought to be responsible for specific morphogenic events during development (9, 10). For example, increasing concentrations of Xenopus activins can cause animal cap cells to differentiate into various cell types (11) while BMP-4 (closely related to BMP-2) can instruct a ventral posterior positional cell fate on developing mesoderm in the Xenopus blastula (12, 13). In the mouse, localized expression of BMPs has been reported in skin, heart, nervous system, and developing limbs (14). A recent study demonstrates that mutation of BMP-5The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 so...

High concentrations of fetal bovine serum induced colony formation in soft agar by anchorage-dependent, nontransformed mouse AKR-2B and rat NRK cells. The colony-stimulating activity in fetal bovine serum was precipitated by 45% saturated ammonium sulfate and migrated in molecular sieve chromatography as a single peak of activity in the 10,000-15,000 molecular weight range. The colony-stimulating activity was heat and acid stable and was destroyed by trypsin and dithiothreitol, indicating the activity is due to a polypeptide that requires disulfide bonds for biologicalactivity. No competition for binding to the epidermal growth factor receptor was associated with the colonystimulating activity. Isoelectric focusing revealed activity in the pI 4-5 range. The colony-stimulating activity in serum appeared to be of platelet origin because platelet-poor plasma and plateletpoorplasma-derived serum containedlittle activity, whereas acid/ ethanol extracts ofbovine and human platelets had potent colonystimulating activity. Chromatography of platelet extracts on BioGel P-60 revealed peaks of AKR-2B colony-stimulating activity in the 12,000 and 20,000 molecular weight ranges. The other biological and chemical properties of the platelet colony-stimulating activity were the same as those for the serum activity. The data indicate the presence in serum of a platelet-derived growth factor(s) with properties similar to those of the transforming growth factors.Cells that are normally anchorage dependent can be stimulated to form colonies when suspended in semisolid medium in the presence of higher than usual concentrations of serum. O'Neill et al. (1) reported that anchorage-dependent NIL-8 hamster fibroblasts exhibited a serum concentration-dependent growth in soft agarose and that the rate of growth of attached and suspended cells was approximately the same when the concentration of fetal bovine serum was raised to 66%. Peehl and Stanbridge (2) found that normal human fibroblasts would grow suspended in methylcellulose in the presence of 20% fetal bovine serum but not in medium with 10% fetal bovine serum; this anchorage-independent growth was enhanced by the addition of hydrocortisone.

Purified type f3 transforming growth factor from human platelets (TGFI3) radioiodinated with 1251-labeled (17), who also described a simplified scheme for obtaining pure TGFf3 in reasonable quantities from human platelets.In the present study we show that radioiodinated TGFf3 isolated from human platelets by a modification of the procedure of Assoian et al. (17) grown in serum-free MCDB 153 medium supplemented with bovine pituitary extract, EGF, insulin, hydrocortisone, phosphoethanolamine, and ethanolamine as described by Boyce and Ham (30). Stock cultures of all of the cells except the keratinocytes were maintained in McCoy's 5a medium supplemented with 5% (vol/vol) fetal bovine serum. All experiments were performed within 10 passages of the frozen stocks from which the cells were recovered periodically.Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; TGFa and TGF,3, type a and p transforming growth factors.*To whom reprint requests should be addressed. 6757The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad Sci. USA 81 (1984) These stock cells have been shown to be free of mycoplasma contamination by staining with Hoechst no. 33258 stain (31).Soft Agar Colony Stimulation Assay. The assay for the stimulation of colony formation of anchorage-dependent cells suspended in soft agar was performed as described (9) by using AKR-2B (clone 84A) cells as indicators and using the Quantimet 800 image analyzer (Cambridge Instruments, Monsey, NY) to quantitate the colony formation.Preparation of Growth Factors. TGF, from human platelets was purified by the method of Assoian et al. (17) with the addition of a final purification step consisting of reversed-phase C18 HPLC eluted with a 45-60% acetonitrile gradient in H20 with 0.1% trifluoroacetic acid. The platelet TGF,8 was shown to be homogenous on silver-stained 12.5% polyacrylamide/NaDodSO4 gels (32) under both reducing and nonreducing conditions. The protein content of the TGFO preparation was determined by weighing a thoroughly dried sample of the purified protein. EGF was purified from adult male mouse submaxillary glands by the method of Savage and Cohen (33). Crystalline bovine insulin was purchased from Sigma. Purified human platelet-derived growth factor (PDGF) was kindly provided by R. Ross (34). Fibroblast growth factor (FGF) was partially purified from bovine pituitary glands in our laboratory. This FGF preparation, =50% pure, caused one-half maximal stimulation of [3H]thymidine incorporation into AKR-2B cells in the presence of insulin at 150 pg/ml (13). Mouse embryo factor was partially purified from acid/ethanol-extracted 17-day mouse embryos (8) by using ion-exchange and gel exclusion chromatography. Serum-free conditioned medium from AKR-MCA cells was chromatographed on a Bio-Gel P-60 column in 1 M acetic acid...

Type fi transforming growth factor/growth inhibitor (TGF-13/GI) is demonstrated to be a potent stimulator of DNA synthesis in AKR-2B mouse embryo cells with a prolonged (>24 hr) prereplicative phase when compared with other growth factors (epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor) that induce DNA synthesis 12-14 hr after stimulation. In addition, TGF-,B/GI inhibits the early peak of DNA synthesis produced by EGF and insulin before the later stimulatory effects of TGF-13/GI become manifest. TGF-fi/GI induces a marked morphologic transformation in these cells prior to their entry into S phase. Like the other growth factors, TGF-13/GI stimulates an early increase in the rate of protein synthesis in AKR-2B cells and its stimulatory effect on DNA synthesis is enhanced by insulin. The data show that this molecule is a growth factor for certain mesenchymal cells in monolayer culture but only after a prereplicative phase that is significantly longer than that of other growth factors.