Faculty Bibliography

Incorporation of the serine protease active site reagent diisopropyl fluorophosphate (DFP) into a plasminogen activator with an Mr of approximately 52000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (NaDodSO4), having one stainable band under nonreducing as well as reducing conditions with an Mr of approximately 52000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After NaDodSO4-polyacrylamide gel electrophoresis, the active enzyme showed one band under nonreducing conditions, but after reduction, two bands with Mr values of approximately 20000 and 32000 were observed. The active enzyme incorporated [3H]DFP into the approximately Mr 32000 band, while no incorporation was observed into the inactive form. These findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher molecular weight form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence

Two melanoma cell lines, each derived from a different patient with metastatic disease, were very similar in their appearance, their growth characteristics, and their tendency to differentiate and to pigment in culture as they become confluent. These lines, UCT-Mel 1 and UCT-Mel 2, were used to study the effects of retinoic acid and other derivatives of vitamin A. When added to UCT-Mel 1 cells, retinoids had only a modest effect on plasminogen activator release and were without measurable effect on morphology, growth, or tyrosinase synthesis. In contrast, when added to UCT-Mel 2 cells, retinoids appeared to induce a more differentiated state evident as an inhibition of cell proliferation and the assumption of a dendritic morphology. Paradoxically, however, retinoids caused a striking inhibition of the density-dependent intracellular accumulation of tyrosinase and melanin that was taken to represent spontaneous in vitro differentiation. Culture of UCT-Mel 2 cells in the presence of retinoic acid resulted in initial inhibition followed by marked stimulation of cellular plasminogen activator release. The data suggest that the manner in which retinoids exert their effects on cells in vitro does not depend on the histological origin of the tumor cells being studied but on the innate responsiveness of that particular cell line to the retinoid or compound in question

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types

We describe a method for 5-hydroxy-3-indoleacetic acid in urine based on its reaction with nitrosonaphthol in the presence of mercaptoethanolamine, an odorless compound. Mercaptoethanolamine shifts the color maximum from 540 nm to 640 nm, intensifies the color and discharges the color of interfering substances. The method is rapid and free from interferences

The effects of retinoic acid on cultured human cells derived from normal and neoplastic tissues were studied. Retinoic acid consistently induced plasminogen activator synthesis by cells of mesenchymal origin (with the exception of adult skin fibroblasts) but not by cells of epithelial origin. The effect of retinoic acid was more pronounced than that of equimolar concentrations of retinol or retinyl acetate. Dexamethasone inhibited the retinoid-induced increase in plasminogen activator in lung- and foreskin-derived fibroblasts. Cells derived from normal or neoplastic tissues showed no consistent differences either in baseline rates of plasminogen activator release or in the magnitude of the retinoid effect

A survey of 56 normal and neoplastic tissues has shown that plasminogen activators were released by cultured human cells in several molecular weights and their susceptibility to inhibition by antibodies to the normal urinary enzyme, urokinase. Melanoma cells characteristically secreted plasminogen activators which were immunochemically distinct from urokinase and which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a prominent, closely spaced doublet with an apparent molecular weight of approximately 70,000 and a minor molecular weight component of approximately 60,000. Enzymes with similar characteristics have been observed in serum-free harvest fluids collected from other neoplastic tissue (a breast carcinoma, a glioblastoma, a malignant teratoma, a uterine sarcoma, and a carcinoma of the renal pelvis) and from normal tissue (8-week embryo fibroblasts, normal esophageal fibroblasts, and one culture of normal adult bladder epithelium). Plasminogen activators released by cells derived from most normal adult tissues, or from a 26-week-old embryo, and from other tumors of ectodermal or mesenchymal origin were inhibited by anti-urokinase antibody and showed a closely spaced doublet with a molecular weight of 60,000 as the most abundant molecular species with no evidence of the enzyme with a molecular weight of 70,000

To explore the interaction of tumor promoters and sarcoma virus transformation with cellular regulatory mechanisms, we have studied induction of plasminogen activator synthesis by these agents in a background of changing cyclic nucleotide concentrations. We have confirmed the original report of Wigler and Weinstein (Nature, 259: 232, 1976) that phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, induces high levels of plasminogen activator production by chick embryo fibroblasts. Sarcoma virus transformation sensitizes the fibroblasts by lowering the threshold concentration for response to the action of PMA, and the effects of transformation and PMA on plasminogen activator synthesis are synergistic rather than additive. The plasminogen activators produced in the PMA-, virus-induced, or synergistically stimulated cultures are indistinguishable. Enzyme production in all three conditions is strongly but reversibly inhibited when cyclic nucleotide levels are raised by exposure to cyclic adenosine-3':5'-monophosphate or cholera toxin. A substantial fraction of the morphological effect that accompanies transformation is not affected by concentrations of cyclic nucleotides that suppress plasminogen activator production, and the two phenomena are therefore at least partially independent expressions of transformation in this system

Low concentrations of Vitamin A stimulated plasminogen activator synthesis (PA) in chick embryo fibroblasts (CEF). It caused a dose dependent and reversible increase in PA synthesis in both normal CEF and CEF infected with a temperature sensitive mutant of Rous Sarcoma virus (RSV-Ts68). Both induction and deinduction of PA could be inhibited by Actinomycin D. Vitamin A also accentuated the morphological changes associated with transformation in the Rous Sarcoma virus infected cells. The effects of Vitamin A on PA synthesis were essentially similar to those of the known tumour promoter, phorbol myristate acetate (PMA). Both Vitamin A and PMA were found to act synergistically with sarcoma gene expression as far as PA synthesis was concerned