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.

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal prokeratinocytes (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor (3 resulted in an early induction of c-sis mRNA. The increase in c-sis mRNA was followed by a corresponding increase in protein similar to platelet-derived growth factor (PDGF) in the culture medium. In addition, PDGF-regulated genes (c-fos and c-myc) were stimulated by transforming growth factor 13 with delayed kinetics relative to that seen in other cell systems with direct PDGF stimulation. A model is proposed in which the monolayer mitogenicity of transforming growth factor 13 is mediated by the induction of c-sis and PDGF and the subsequent autocrine stimulation of c-fos, c-myc, and other PDGFinducible genes.The traverse of G, proceeds by an ordered sequence ofevents under growth factor control (1, 2). The cellular responses subsequent to growth factor binding are of great interest. A strategy, or at least an accepted paradigm, has evolved for regulating growth at the level of protooncogene transcription (3, 4). For instance, platelet-derived growth factor (PDGF), the polypeptide believed to be responsible for initiating cell cycle traverse in many mesenchymal cells (5), stimulates a rapid and transient induction of both c-fos and c-myc mRNA in quiescent cells (6)(7)(8). The product of the c-sis protooncogene is the B chain of PDGF while the c-erbB gene product is homologous to the cytoplasmic domain of the epidermal growth factor (EGF) receptor (9-12). Moreover, it has been shown that the receptor for colony stimulating factor 1, a macrophage growth factor, is similar or identical to the product of the c-fms protooncogene (13 MATERIALS AND METHODSCell Culture. Mouse embryo-derived AKR-2B cells (14-16) were grown in McCoys 5A medium as described (16). To prepare cultures for RNA purification, 300,000 cells/100-mm Petri dish were cultured in serum-supplemented McCoys SA medium and allowed to grow to confluence. On day 5 after plating the spent medium was removed and fresh serum-frfe MCDB 402 (Kansas City Biologicals, Lenexa, KS) was added for an additional 2 days to assure quiescence and removal'of residual serum factors (16). Cultures were then stimulated for the indicated period of time with fresh MCDB 402 alone (containing 10 ,g of Pentex bovine serum albumin/ml) (Miles) or supplemented with the indicated reagents (also containing 10 ,ug of bovine serum albumin/ml). TGF-,8 from human platelets was purified by a modification of the method of Assoian et al. (17) as described by Tucker et al. (18). The platelet TGF-,B was shown to be homogenous by silver staining of NaDodSO4/polyacrylamide gels under reducing and nonreducing conditions and autoradiography of gels of reduced and nonreduced 1251-labeled . RNA Isolation. Cytoplasmic poly(A)+-containing RNA was prepared by a modification of published procedures (19). Subsequent to stimulation cultures were scraped with a rubber policeman and collected by centrifugation (235 x g for 5 min). The cell pellet was resuspended in lysing buffer (140 mM NaCl/1...

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR).Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.