Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

Tumor interactions with the vasculature: angiogenesis and tumor metastasis.

Antibodies to plasminogen activator inhibit human tumor metastasis

Proteolytic degradation of extracellular matrix in tumor invasion.

Plasminogen activation and regulation of pericellular proteolysis.

The plasminogen activator activity of human breast tumors was determined quantitatively in extracts of 33 primary tumors, 11 lymph node metastases, and 11 normal tissues. Activities varied more widely in the neoplastic tissues than in the normal ones. The mean activator content of the tumors (primary and metastasis) was significantly higher ( p < 0.01) than that of normal tissues: 4.4-fold on a g wet tissue basis and 2.1-fold when the activities were referred to the protein content of the extracts. Primary and metastatic tumors were not significantly different from one another in their plasminogen activator content. In the six cases where tumors and normal tissue pairs from the same patients could be compared, the mean of the individual tumor:normal ratios was 10.0 (wet weight) or 3.6 (extract protein). In addition to the activator content, the molecular type of the enzymes was also determined, using goat antibody formed against human urinary urokinase as an inhibitor of activator activity. On the average, 80% of the activity of the tumor extracts could be inhibited by the antibody; extracts of normal breast tissue contained only 62% urokinase-like activator. The activators were also characterized by sodium dodecyl sulfate gel electrophoresis using two different methods for the zymographic visualization of activator bands. The fraction of urokinase-like enzyme determined by the fibrinogen copolymerization method agreed well with the results obtained by immunoinhibition. No correlation was found between activator activity or activator composition, on the one hand, and the degree of differentiation of the tumors, the fraction of positive lymph nodes found, or the amount of estrogen-binding protein (or their molecular size) in the cytosol of the tumors, on the other. This apparent lack of correlation may reflect variations in local physiological conditions which necessitate the presence of widely differing amounts of activator in order to ensure a critical amount of the product of the reaction catalyzed by this enzyme, i.e. , plasmin.

Plasminogen Activator Activity and Composition in Human Breast Cancer

The secretion of plasminogen activator by explants of 92 human malignant tumors was studied in short-term organ culture. Where possible, adjacent normal tissue of the presumed origin of the tumors was also studied. The study included adenocarcinomas of the lung, colon, prostate, breast, and stomach and different types of sarcomas. In addition to the measurement of secretion rates, all tissues were quantitatively extracted to determine the amount of cell-bound enzyme. Both culture fluids and extracts were analyzed with respect to the type of plasminogen activator they contained by immunoinhibition with goat immunoglobulin G formed against purified human urinary urokinase sodium dodecyl sulfate:gel electrophoresis followed by zymography. The study yielded the following conclusions: ( a ) the measurement of plasminogen activator secretion rates gives a much sharper differentiation between malignant and normal tissues than does the amount of extractable enzyme; ( b ) the enzymes secreted in short-term organ culture are, in the great majority of the cases studied, of the urokinase type, even when a large fraction of the activator contained in the tissue is of the vascular type; ( c ) the secretion rates of metastatic tumors of the colon are much lower than those of the primary ones; ( d ) immunoperoxidase staining of tissue sections reveals that urokinase is localized predominantly in the tumor cells. The low secretion rates of metastatic tumors, probably a reflection of this property in the original cell that gave rise to the metastatic focus, could be of advantage to circulating cancer cells. Such cells would not dissolve the microthrombus thought to be essential for the arrest of cancer cells in the capillaries of target organs.

https://cancerres.aacrjournals.org/content/canres/43/11/5517.full.pdf

Plasminogen Activator Secretion of Human Tumors in Short-Term Organ Culture, Including a Comparison of Primary and Metastatic Colon Tumors

Metastatic malignant melanomas from 16 patients, extracted with Triton X-100, were analyzed for plasminogen activator activity by azocaseinolysis . In 6 cases tumor explants were set up also in short-term organ culture, and the rate of plasminogen activator secretion into the culture medium was determined. Both the extractable activator content [8.66 +/- 7.8 "Committee on Thrombolytic Agents" (CTA) U/g tissue] and the activator secretion rates (0.90 +/- 1.6 CTA U/g/hr) were low in comparison with values for other human tumors. In addition to the activity, the type of plasminogen activator also was determined by immunoinhibition with goat antihuman urokinase antibody in the azocaseinolytic assay, as well as by sodium dodecyl sulfate (SDS) gel electrophoresis followed by zymography on fibrin-agar, in the presence and absence of antibody. On the average, 77% of the activator activity was of the urokinase type in the extracts, and 90% in the culture fluids. Immunoperoxidase reaction for the detection of urokinase showed this enzyme to be localized mainly in the cell membrane of the melanoma cells; stromal elements showed no specific staining. These results are of interest in view of the findings made recently by investigators in several laboratories that in all but one of the melanoma cell cultures derived from metastatic human tumors, only the vascular type ("tissue activator") was cell associated or was secreted into the culture medium. The possible reasons for this discrepancy are discussed.

Plasminogen Activators in Human Malignant Melanoma

Conditioned media from explants of human colorectal and gastric tumors in short-term organ culture were analysed for plasminogen activator activity, activity toward the synthetic urokinase substrate, Spectrozyme-UK, and for the presence of urokinase antigen using monospecific goat antibody, by enzymelinked immunosorbent assay. Comparisons were made between primary tumors, adjacent normal mucosa and metastatic lesions. These analyses were carried out on unfractionated culture fluids and on fractions obtained by fast protein liquid chromatography separation using Superose 6 gels. Plasminogen activator activity, tested by azocaseinolysis in the presence of added plasminogen, was restricted to peaks of 55 kD and 155 kD. These were of the urokinase type as shown by specific immunoinhibition and by absorption by an antiurokinase antibody Affigel 10 column. Spectrozyme-UK, in addition to these peaks, detected a series of higher molecular weight activities, the largest of which appeared in the void volume, and were therefore of >106 molecular weight. These activities were greatly increased by inclusion of trace plasmin indicating that these components were mostly in their proenzyme forms. The characteristics of these very large enzymes were similar to those isolated earlier from a human lung cancer cell line [10]. Comparison of the primary and metastatic tumors confirmed earlier observations showing that urokinase secretion by the metastatic tumors was greatly reduced in comparison with the primary tumors: in the colon carcinomas it was 10 per cent of the value for the primary, in the gastric tumors 3 per cent, whether means or medians were compared (P<0·0001). This large difference was characteristic only of plasminogen activator secretion assayable by azocaseinolysis; activities toward Spectrozyme-UK, and antigen reacting with anti-urokinase antibody, were considerably less different in the two groups. In individual tissues, no correlation was found between the amount of extractable plasminogen activator and amounts secreted, or between the latter and the amount of lactic acid released. It is postulated that the greatly reduced plasminogen activator secretion by explants of metastatic tumors may be a phenotypic characteristic of distinct advantage for cancer cells destined to initiate metastatic foci, and may contribute to the ability of circulating cancer cells to lodge in the blood vessels of the target organ.

Judith M. Madeja

Papers

Plasminogen Activator Activity and Composition in Human Breast Cancer

Plasminogen Activator Secretion of Human Tumors in Short-Term Organ Culture, Including a Comparison of Primary and Metastatic Colon Tumors

Plasminogen Activators in Human Malignant Melanoma

Secretion of plasminogen activators by human colorectal and gastric tumor explants.