Xenopus oocytes synthesize active human plasminogen activator

Induction of synthesis of the protease plasminogen activator (PA) by hormones, oncogenic viruses and tumor promoters occurs at the transcription level. A novel bioassay for PA messenger RNA was developed to study the regulation of PA synthesis and the genetic elements involved in it. Poly(A)containing RNA from HEp-3, a PA-rich tumor of human origin, was found to direct the synthesis of a new proteolytic activity when microinjected into Xenopoi oocytes. Newly synthesized protease can be detected within a few hours after microinjection of minute quantities of unfractionated mRNA. The new enzymatic activity is indistinguishable from human PA: it is absolutely dependent on human plasminogen; it is neutralized by serum raised against urokinase, the human urinary PA; and it comigrates with urokinase and HEp-3 PA in gel electrophoresis, exhibiting a molecular weight of 60,000. INTRODUCTION Plasminogen activator (PA) is a highly specific serine protease which converts the inactive zymogen plasminogen to the active trypsin-like protease plasmin (see ref. 1 for review). PA is closely correlated with inflammation, cell migration and tissue remodeling as well as with cellular transformation, tumorogenicity, metastasis and tumor promotion (see refs. 2,3 for review; 4,5). The enzyme can be modulated in a variety of vertebrate cell types by physiolocial inducers such as hormones and/or cyclic AMP (2-6), and also by nonphysiological inducers such as oncogenic viruses (2,3), tumor promoters (7-10) and physical and chemical DNA damaging agents (11). Experiments with inhibitors of macromolecular synthesis indicate that regulation of PA synthesis occurs at the level of transcription (9-13), and that RNA synthesis is also required for the suppression and deinduction of PA synthesis (9,12-14). Thus, a study of PA at the level of messenger RNA (mRNA) is necessary for the understanding of the variety of mechanisms controlling its genetic expression. Such a study has not been carried out so far. A prerequisite for this is an assay to monitor PA mRNA. © IRL Press Limited, 1 Falconberg Court, London W1V 5FG. U.K. a35S Nucleic Acids Research Assays of mRNAs, frequently based on the identification of specific translational products synthesized -in MXXJVO, permit only the detection of relatively abundant species of mRNA. Difficulties in detecting scarce mRNA species thus make difficult the study of genetic elements coding for rare cellular proteins of known functions, and also the investigation of mechanisms which regulate their transcription. Missing, and particularly desirable, are assays for rare mRNA sequences, which direct the synthesis of regulatory proteins such as receptors and enzymes. To this class belongs the mRNA for PA, since PA synthesis is estimated to comprise only a small fraction of the cellular protein synthesis, even upon maximum induction, when enzyme levels increase by two orders of magnitude (8,9). We developed a method to identify and quantify PA mRNA, based on its ability to direct the synthesis of a biologically active polypeptide product. The detection of such a product is feasible due to the unique sensitivity of the assays for PA activity, which compensates for the low level of PA mRNA. This high sensitivity is gained by measuring the proteolytic activity of the reaction product, plasmin, rather than that of PA itself (15). The translation of PA mRNA has been carried out by Xenopo6 oocytes, which have been extensively used as a translation system for a variety of microinjected mRNAs since its first introduction by Gurdon and colleagues (16). Xewopu* oocytes have been shown to efficiently perform translation, processing and various post-transiational modifications; in the case of mRNAs for secretory proteins, oocytes carry out also the secretion of the correct translational products (see refs. 17,18 for review). Moreover, mRNAs which direct the synthesis of human (19) and mouse (20) interferon, of mouse g-glucuronidase (21) and of human oligo-isoadenylate synthetase (22) have been shown to be translated in oocytes into biologically active products. We now report that Xznopui oocytes synthesize and secrete proteolytically active human PA in response to the injection of mRNA from a human carcinoma. MATERIALS AND METHODS Growth of HEp-3 tumors A HEp-3 tumor grown in a chick embryo (23) was further propagated in nude mice (ICR, the Weizmann Institute animal facilities) by subcutaneous injections of tumor pieces. Subcutaneous tumors were removed (0.2-0.8 gr/mouse) 2-3 weeks later and transferred immediately into liquid nitrogen.