Amplification of tissue plasminogen activator expression from epithelial cell lines.
Two established non-malignant epithelial cell lines, one derived from human breast tissue (BEB), the other from guinea pig ear keratocytes (GPK), have been shown to secrete a tissue plasminogen activator (tPA). The protein yield of this epithelial enzyme is similar to that from the malignant cell line, Bowes melanoma, but as the specific activity is approximately 10-20 fold lower, means of potentiating the yield are being sought. The enzyme is mainly expressed during the cell growth phase rather than from stationary culture cells. Also, production has to be a 2-step operation with initial growth to about 70% confluency in the presence of serum followed by a change to serum-free conditions for the final period of growth after which the enzyme is harvested. To increase the enzyme yield, means of amplifying enzyme expression, of establishing a serum-free medium so that the enzyme can be continuously harvested, and stimulation of stationary-phase cells have been studied. Of the many regulatory agents studied only the hypomethylating agent 5-azacytidine brought about significant increases (3-5 fold) in enzyme secretion. The effect of a number of mitogenic lectins was also investigated and resulted in further increases of enzyme yield (15-20 fold). Concanavalin A considerably extended the culture period in which enzyme secretion occurred and the reasons for this were investigated using tritiated thymidine.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006168:Guinea Pigs; D006801:Humans; D013197:Stanozolol / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
D003208:Concanavalin A; D013197:Stanozolol; D010959:Tissue Plasminogen Activator; D001374:Azacitidine; D013755:Tetradecanoylphorbol Acetate