[Macro-creatine kinase: not all increased CK-MB activity signifies a heart infarct]. / Macro-creatine-kinase: niet iedere verhoogde CK-MB-activiteit duidt op een hartinfarct.

Assay of creatine kinase MB isoenzyme plays an important role in the diagnosis of acute myocardial infarction. An increase in CK-MB is frequently interpreted by the clinician as objective evidence of myocardial cell damage. However, increases of CK-MB may be found in several circumstances in which patients have not sustained an acute myocardial infarction. An important cause of elevated CK-MB values unrelated to acute MI is the presence of macro-creatine kinases in the patient's plasma. With immuno-inhibition procedures macro-CK is often measured as CK-MB, leading to falsely elevated CK-MB. In this paper macro-CKs, their clinical importance and their interference with CK-MB determination are discussed.

Mechanisms of cellular invasiveness: a comparison of amnion invasion in vitro and metastatic behavior in vivo.

We employed a sensitive in vitro amnion invasion assay to examine the relationship of the invasive ability of numerous mouse and human tumor cell lines and their variants to their ability to spontaneously or artificially metastasize; we also studied possible enzymatic activities involved in the in vitro invasion process. In vitro invasive ability of tumor cells was strongly correlated with spontaneous metastatic ability from the subcutaneous site, regardless of the ability of tumor cells to form artificial metastases when introduced intravenously. However, normal nontumorigenic human trophoblast cells were also highly invasive. Various collagenase inhibitors totally abrogated amnion penetration by all invasive cells; various inhibitors of plasmin, plasminogen, and plasminogen activators prevented invasion in most, but not all, cases. Thus, amnion penetration provides a rigorous test for tumor cell invasiveness required for spontaneous metastasis in vivo, and invasiveness is strongly dependent on metalloproteinase activity, which usually follows plasmin activation.

Gelatinases of murine metastatic tumor cells.

The properties of gelatinases secreted in culture medium of murine fibroblasts, macrophages, colonic carcinoma, FBJ virus-induced osteosarcoma, Lewis lung carcinoma and mammary tumor cells were compared. Normal fibroblasts and macrophages secreted gelatinases of 60,000 and 95,000 molecular weights, respectively. Tumor cells secreted both of these gelatinases, although the relative amounts of the 60 kDa and 95 kDa gelatinases differed among the cell lines. The cell lines that had the greatest metastatic potential to the lung secreted the highest amount of 95 kDa gelatinase. The 95 kDa gelatinase produced by tumor cells had properties similar to that of macrophages.

Activity of porphobilinogen deaminase in peripheral blood mononuclear cells of patients with metastatic cancer.

Porphobilinogen deaminase (PBGD), one of the enzymes in the pathway of heme synthesis, was found to be elevated in peripheral mononuclear cells of 60% of patients with epithelial tumors and metastatic spread, but only in 14% of patients with tumor and no evidence of metastases. The combination of both high lactic dehydrogenase and high PBGD afforded a sensitivity of 40%, but a specificity of 96% in diagnosing metastatic spread.

Involvement of heparanase in tumor metastasis and angiogenesis.

The capacity of various blood-borne cells, whether normal or malignant, to extravasate was found to correlate with heparanase-mediated degradation of HS in subendothelial ECM. This degradation was stimulated by proteases or plasminogen and inhibited by native heparin and by various modified nonanticoagulant species of heparin. These heparins also induced a marked reduction in tumor cell metastasis and autoimmune diseases in experimental animals. Heparanase-mediated degradation of HS in ECM also released EC growth factors that are stored in ECM, most likely by high affinity binding to HS. Such growth factors were extracted from subendothelial ECM synthesized in vitro and from basement membranes of the cornea in vivo, and are structurally and functionally related to bFGF;bFGF binds to ECM and is readily released by incubation with either HS, heparin or low MW heparin fragments as well as by various normal and malignant cells and by heparanase-mediated degradation of ECM HS. In contrast, there was little or no release of growth-promoting activity upon incubation of ECM with hyaluronic acid, chondroitin sulfate or chondroitinase ABC. A model is proposed suggesting that regulation of capillary growth and neovascular response may result from displacement of an angiogenic protein (bFGF) from its storage sites within basement membranes.

Ornithine decarboxylase activity in hepatic and extrahepatic metastases from colorectal cancer.

The values of ornithine decarboxylase (ODC) activity in hepatic and extrahepatic metastases from primary colorectal cancer were studied. Adjacent noninvolved tissue was used as a control. Liver metastases had significant elevated ODC levels over surrounding liver (0.271 vs. 0.065, respectively, P less than .008). Results similar to those found in liver metastases were noted in extrahepatic metastases (median value, 0.271). This study discusses the possible reasons for these elevations and emphasizes that these differences may have potential roles in the areas of diagnosis, staging, monitoring of the disease, and therapeutic interventions in colorectal cancer and its hepatic and extrahepatic metastases.

Heparanases and tumor metastasis.

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.

Glycolytic enzyme activities in breast cancer metastases.

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases.

Recent studies in this laboratory demonstrated that several sulphated polysaccharides can inhibit metastasis of the rat mammary adenocarcinoma 13762 MAT, probably by preventing the passage of tumour cells through the walls of blood vessels. In order to directly test this possibility, 13762 MAT cells were cultured with (35S)O4(=)-labelled subendothelial extracellular matrices (ECM) and ECM degradation was monitored in either the presence or absence of different sulphated polysaccharides. Degradation products were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent autoradiography. The 5 sulphated polysaccharides that had previously been shown to possess anti-metastatic activity were potent inhibitors of the degradation of subendothelial ECM by 13762 MAT cells. In contrast, of the 4 polysaccharides tested that failed to inhibit metastasis, 3 had no effect on ECM breakdown and one (carrageenan-kappa) was substantially less effective at inhibiting ECM degradation than the anti-metastatic preparations. It was also shown that 13762 MAT cells produce a heparan sulphate-specific glycosidase (heparanase) that degrades the heparan sulphate side-chains of the ECM, the action of this enzyme rather than that of other ECM-solubilizing enzymes being inhibited by the antimetastatic sulphated polysaccharides. Additional experiments indicated that the anti-coagulant activity of the polysaccharides probably plays a minor role in their anti-metastatic effects since heparin, almost completely depleted (98-99.5%) of heparin molecules with anti-coagulant activity by passage over an anti-thrombin III column, retained its ability to inhibit 13762 MAT heparanases and was almost as effective as unfractionated heparin at inhibiting tumour-cell metastasis. Collectively, these data suggest that sulphated polysaccharides inhibit the metastasis of 13762 MAT cells by inhibiting tumour-cell-derived heparanases involved in the penetration of the vascular endothelium and its underlying basement membrane by tumour cells.

Do proteases play a role in cancer invasion and metastasis?

The main difference between a benign and malignant tumor is the ability of the malignant form to invade normal tissue and spread or metastasize to distant sites throughout the body. It is the ability to form metastasis which makes cancer such a difficult disease to treat. Evidence suggesting that proteolytic enzymes are involved in cancer spread is as follows: proteases are involved in normal destructive events and tissue remodelling, correlations exist between different protease activities and metastatic potential in model tumor systems, inhibitors and antibodies against proteases inhibit metastasis in model systems and the finding of highest levels of protease activity at the invading front in tumors. The most likely mechanism by which proteases could mediate metastasis is by catalyzing degradation of the extracellular matrix and basement membranes. It is concluded that if proteases could be proved to play a role in the spread of human cancers, inhibition of these enzymes could open up new therapeutic approaches for the control of malignancy.

Heterogeneity of isozyme expression in tumor cells does not correlate with metastatic potential.

The major purpose of these studies was to determine whether the expression of isozymes by tumor cells was heterogeneous among tumor cell subpopulations within a neoplasm and whether expression of one or another isozyme correlated with metastatic potential of tumor cells. The expression levels of 40 isozymes were determined in 56 cell lines, many of them clonal, from nine different murine and human tumors. The enzymes chosen for study are involved in nucleotide, carbohydrate and pentose phosphate metabolism, and as such are indicators of the general metabolic and differentiational status of the cell. The tumors studied included two murine and two human malignant melanomas, four murine fibrosarcomas, and one human prostatic adenocarcinoma. The lines isolated from these tumors consisted of cells that are tumorigenic non-metastatic, tumorigenic low metastatic and tumorigenic highly metastatic. Clonally derived cell lines from a given tumor differed in their expression of a number of different isozymes, including adenosine deaminase, creatine phosphokinase-B and lactate dehydrogenase. Different patterns of isozyme expression were observed among different tumor types as well as between tumors of the same type; however, there were no differences in isozyme expression for any enzyme tested that correlated with metastatic ability of tumor cells.

Effects of retinoids on type IV collagenolytic activity in melanoma cells.

The effects of retinol, all-trans-retinoic acid, isotretinoin and etretinate on the activity of basement membrane collagen degrading enzyme was studied in melanoma cells. The results indicated that retinoids at concentrations of up to 10(-6) M did not significantly affect type IV collagenolytic activity in these cells in vitro. Since type IV collagenolytic enzyme may be involved in the metastatic potential of tumour cells, it appears that retinoids do not affect the metastatic potential of melanoma cells by affecting type IV collagenolytic activity.

Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line.

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80-90 kD as determined by affinity-labelling with [3H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5-8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.

Cathepsin B-like proteinase as a marker for metastatic tumor cell variants.

Serial transplantation of a spontaneous BDX rat tumor, classified as an anaplastic sarcoma, gives rise to two variants; a rapidly growing nonmetastatic line (AS) and a slowly growing, invasive, and highly metastatic variant (ASML). The availability of two cell lines of the same origin but with markedly differing metastatic potential offers an ideal model for the identification of the cellular properties involved in invasive and/or metastatic behavior. The present work focuses on the pattern of various proteinases in the two tumor cell variants. The findings disclosed one major consistent difference which relates to a cathepsin B-like cysteine proteinase. The metastatic ASML variant manifests exceedingly high intracellular cathepsin B-like activity; in the nonmetastatic AS variant, the activity of this proteinase is significantly lower. Other proteinases, in particular elastase-like, chymotrypsin-like, collagenase-like enzymes and plasminogen activator, showed low, essentially comparable activity patterns. Thus, cathepsin B-like proteinase is a marker enzyme of the metastatic ASML tumor cell variant.

Ectopic creatine kinase MB production in metastatic cancer.

This case concerns the presence of high serum levels of creatine kinase MB isoenzyme in a patient with metastatic cancer. This patient did not have a myocardial infarction, so the source of the CK-MB was investigated. Because of the observation of macro-creatine kinase in patients with cancer, it was necessary to rule out the presence of this form of the enzyme. Extensive laboratory analysis demonstrated that the isoenzyme was true CK-MB, not an atypical or macro CK. Results of the study showed that ectopic production of the isoenzyme was the apparent source of the high serum activity. Homogenization of the cancer tissue demonstrated the presence of a high percentage of CK-MB activity. The implications of CK-MB production in cancer are discussed, including the use of various technics to rule out interfering activities when situations such as this occur.

Secretion of basement membrane collagen degrading enzyme and plasminogen activator by transformed cells--role in metastasis.

The relationship of a basement membrane collagen degrading enzyme (BM collagenase) and plasminogen activator (PA) was studied in a number of non-malignant and malignant human and murine cell lines. Several non-malignant cell lines secreted significant amounts of PA but not detectable BM collagenase activity whereas the malignant cell lines, with one exception, secreted both enzymes. Therefore, the secretion of BM collagenase appears to be a characteristic of many malignant cells whereas PA is synthesized also by normal cells. The BM collagenase needed proteolytic activation for maximal activity indicating that it is secreted in a latent form. The addition of plasminogen to the culture medium of human fibrosarcoma cells (HT-1080) resulted in maximal activation of the enzyme. Plasmin, but not plasminogen, increased the activity of partially purified enzyme protein. Accordingly, the activation of latent BM collagenase in vivo may be facilitated by PA through the conversion of plasminogen to plasmin. It is suggested that the secretion of BM collagenase concomitantly with PA is a prerequisite for metastasis.