Effects of 12-O-Tetradecanoylphorbol 13-Acetate on the Production of mRNAs for Human Tissue-Type Plasminogen Activator

Abstract

The mRNA for human (tissue type) plasminogen activator from a human melanoma cell line (Bowes) was investigated in different translation systems. After translation of poly(A)-rich RNA in Xenopus oocytes a biologically active plasminogen activator was obtained. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the secreted translation products revealed a protein band precipitable with affinospecific antibody and migrating at the same position (apparent molecular mass of approximately 70 000 Da) as the native melanoma cell product. Translation in rabbit reticulocyte lysate yielded an immunoprecipitable band migrating at position corresponding to a molecular mass of 52 000 Da. Addition of 12-O-tetradecanoylphorbol 13-acetate to the cell cultures resulted in increased production of plasminogen activator. Concomittantly more poly(A)-rich RNA could be extracted from the cells and this RNA was more effectively translated by oocytes into biologically active plasminogen activator. Translation of poly(A)-rich RNA from phorbol-ester-treated cells in the reticulocyte lysate system yielded the 52000-Da protein also seen with RNA from untreated cells. However, in addition a prominent protein band of apparent molecular mass of 48 000 Da was detectable. Its intensity increased with increasing doses of tetradecanoylphorbol acetate. This phorbol-ester-induced protein was not precipitable with the affinospecific antibody against plasminogen activator.