Growth inhibitors in serum, platelets, and normal and malignant tissues

The non-malignant epithelial cell line from Buffalo rat liver (BRL) and its malignant transformant obtained by Rous sarcoma virus infection (RSV-BRL) were used as the indicators. A survey was made in animals for growth inhibitors which could inhibit the growth of BRL, but not the growth of RSV-BRL in culture, under the consideration that the loss of the sensitivity to such inhibitors might be the most dominant cause for acquisition of tumorigenicity. From human platelets, a growth inhibitor was extracted with acid-ethanol and prepared to a homogeneous purity, separated from the major TGF-beta activity, which stimulated the anchorage-independent growth of NRK cells in the presence of EGF. The purified inhibitor (Mr 25-27k; pI 8.7-9.1) showed 50% inhibition on the growth of BRL at 20 pg/ml (0.7 pM), but hardly influenced the growth of RSV-BRL. Its inhibition was specific to epithelial cell lines, thus called epithelial growth inhibitor (EGI). It consisted of two subunits with practically identical Mr, which were cross-linked with disulfide bonds. Its sequence of N-terminal 10 amino acids was consistent with that of TGF-beta. In rats, serum contained a growth inhibitor with Mm 220k. The inhibitor, when treated with high concentrations of urea or guanidine-HCl, liberated an active component with Mr 30k and pI 5.2. When the growth inhibitor with Mm 220k was treated with ethanol-acetone or 1 M acetic acid, its activity increased more than 10-fold. Growth inhibitors were extracted with acid-ethanol from various tissues of rats. The activities of the extracts to inhibit the growth of BRL cells were high in spleen, lung and tumors with relatively high mitotic activities, but low in muscle, heart and brain. Acid-ethanol extract from BRL cells showed growth inhibitory activity toward BRL cells themselves significantly higher than that from RSV-BRL cells. The serum-free media conditioned with BRL and RSV-BRL cells showed a little growth inhibitory activity toward BRL cells. On treatment with acetic acid, their activities increased significantly, comparable for both types of cells. The media thus activated inhibited the growth of BRL cells, but not that of RSV-BRL cells. All the growth inhibitors described above including human platelet inhibitor, whether present in extracts or purified, resembled one another in stability against acid, heat and reducing reagents.