No abstract

We have analyzed the role of plasminogenactivator inhibitor type 1 (PAI-1) in the regulation of tumor cell-mediated extracellular matrix degradation. Immunocytochemical analysis revealed PAM-i associated with microgranular and fibrillar material of the extracellular matrix and demonstrated the presence of PAM-i as a cell surface-associated antigen. Transforming growth factor j3 significantly reduced matrix degradation mediated by HT-1080 human fibrosarcoma cells. This inhibition was correlated with an increase in PAM-i antigen expression, whereas urinary-type plasminogen activator (u-PA) secretion was unaffected. In this experimental system, PAT-i regulated extracellular matrix breakdown, as added PAM-i inhibited matrix solubilization, whereas monoclonal antibodies to PAM-increased it. A cell line (LPAI) producing high levels of biologically active PAM-was estab- Plasminogen-activator inhibitor type 1 (PAI-i) is a member of the serpin family of protease inhibitors and reacts specifically and rapidly with both tissue-type (t-PA) and urinary-type

We have analyzed the CM of 20 human tumor cell lines for the presence of PA, PA-I and PC. Most of the cell lines expressed PA activity as measured by a radioiodinated fibrin plate assay. The urinary type and tissue-type PA activities were specifically quantified by means of purified inhibitory antibodies. U-PA and/or t-PA antigen, as measured by radioimmunoassays, were detected in all but 4 of the CM and were generally 10 times more concentrated than PA activity, indicating the presence of specific PA-Is. Analysis of CM by electrophoresis followed by fibrin-agarose zymography demonstrated the presence not only of free but also of inhibitor-complexed PA. Affinity purification demonstrated that 8/20 cell lines expressed detectable PA-I activity. The PA-I1 and PA-I2 inhibitors were most frequently observed, while PN was recovered only from CM of the HT1080 fibrosarcoma cell line. PC activity, as measured by the plasma recalcification time method, was found in 9/20 CM. It was of the thromboplastin tissue factor type since most of its activity was lost when assayed with a Factor VII-deficient plasma.

Abstract. A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (L~) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two-to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. I~r~ cells and reference HT-1080 fibrosarcoma cells, in contrast to control L~o cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using antiu-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alon~ is sufficient to confer to a cell an experimental invasive phenotype.

Urokinase (u-PA) and the urokinase receptor (u-PAR), are thought to play a critical role in the invasive and metastatic properties of cancer cells. The HT29 human colon-carcinoma cell line was selected to evaluate these aspects. HT29 cells express u-PA receptors (100,000 sites/cell, KD = 1.5 nM), but no PA activity and therefore are unable to generate plasmin in the presence of plasminogen. These cells have been transfected with a human u-PA cDNA to investigate whether secreted u-PA would enhance in vitro extracellular matrix degradation, and whether the binding of u-PA to the cell surface is determinant. Five clones were selected for stable expression of high PA activity. These clones were capable of marked plasminogen-dependent degradation of R22 smooth-muscle-cell-derived extracellular matrix, whereas the parental cell line contributed to an insignificant breakdown only. Aprotinin, polyclonal anti-u-PA IgG, recombinant PAI-2, and co-culture with human PAI-I-producing mouse L cells significantly inhibited this degradation. Furthermore, a peptide displacing u-PA from its receptor as well as 2 different polyclonal anti-u-PA receptor IgGs decreased the breakdown after 24 hr by as much as 70% and 81%, respectively. These results show that the binding of u-PA to its receptor plays an important role in in vitro matrix breakdown by HT29 u-PA transfectants.