Galactosyltransferase activity in metastasizing and nonmetastasizing rat mammary carcinomas and its possible relationship with tumor cell surface antigen shedding.

In order to study the mechanism of tumor cell surface antigen shedding, galactosyltransferase levels were compared in 5 spontaneously metastasizing and 3 nonmetastasizing rat mammary tumors. The enzyme activity both with or without exogenous acceptors was higher in the metastasizing group. This difference did not seem to be due to the variation in levels of degrading enzymes such as pyrophosphatase or beta-galactosidase found in these tumors. Little difference in the biochemical properties of the enzyme was found between the two groups. Most of the enzyme activity (60-70%) was recivered in the microsomal frctosyltransferase was assayed in "purified" plasma membrane fractions, 70% of the activity was associated with the plasma membrane vesicles, in which the enzyme was enriched by factors of 10-40. The number of galactose acceptor sites on the plasma membranes increased in parallel to the metastasizing capacity, indicating the presence of larger numbers of incomplete glycopeptides on their cell surfaces. These findings seemed to indicate that the greater turnover of glycoprotein in the spontaneously metastasizing tumor cell surface was caused by the shedding of surface antigens into the systemic circulation of the host.

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.

Fucosyltransferase activity in metastasizing and nonmetastasizing rat mammary carcinomas.

Fucosyltransferase levels in 6 established strains of spontaneously metastasizing rat mammary tumors (STMT-058, MT-449, DMBA-4, SMT-077, TMT-081, and SMT-2A) were compared with 4 nonmetastasizing strains (MT-W9B, MT-W9A, MT-100, and MT-66) as controls. Two acceptors were prepared from fetuin for the assay, one by acid hydrolysis of N-acetylneuraminic acid and the other by the stepwise removal of N-acetylneuraminic acid and penultimate galactose by Smith degradation. The enzyme that transfers fucose to the first acceptor was designated fucosyltransferase A, whereas the one that uses the second acceptor was designated fucosyltransferase B. Both types of fucosyltransferases were found in this rat mammary tumor system. Whereas the levels of fucosyltransferase A in the 2 tumor groups were comparable, those of fucosyltransferase B were sixfold to sevenfold higher in the metastasizing tumors. This difference in the level of fucosyltransferase B was not caused either by differential hydrolysis of GDP-fucose by pyrophosphatase in the 2 groups or by hydrolysis of the product by fucosidases. Presence of any other inhibitor(s) or activator(s) of fucosyltransferase was excluded by mixing experiments. Optimal conditions for the assay of this enzyme were determined in a representative strain from each group. Under all circumstances, the activity of fucosyltransferase B was higher in the metastasizing tumors. The enzyme was inhibited by nucleoside diphosphates and triphosphates, and guanosine nucleotides were the most efficient inhibitors. Subcellular distributions of the two fucosyltransferases were similar, 35-50% of the enzyme activity being present in the crude microsomes. When plasma membrane factions were prepared from the microsomes, the major part (50-70%) of the enzyme was associated with the light and heavy plasma membrane fractions. Increased activity of fucosyltransferase B in the group of metastasizing tumors may have reflected faster synthesis and shedding of fucose-containing glycoprotein antigens. Similar molecules possibly were also synthesized in the nonmetastasizing cells but at a much slower rate, because the antigen is not easily lost from the cell surface. Any alteration of the specificity of this focosyltransferase in the metastasizing tumors, in addition, may have caused antigen modulation.

Biochemical properties of cyclic nucleotide phosphodiesterase in metastasizing and nonmetastasizing rat mammary carcinomas.

The biochemical properties of cyclic nucleotide phosphodiesterases in a nonmetastasizing and a spontaneously metastasizing rat mammary carcinoma were compared. The phosphooiesterases in both tumors had a pH optimum of around 8.0 and preferentially hydrolysed cyclic purine nucleotides. The rate of hydrolysis of purine nucleotides in the nonmetastasizing tumor was two times higher than in the metastasizing tumor, but the rate of pyrimidine nucleotide hydrolysis was equal in both tumors. Theophylline, caffeine, and D,L-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro20-1724) inhibited the enzyme activity in both tumors; the percent inhibition was the same by each inhibitor. The cyclic nucleotie phosphodiesterase activity in either tumor was stimulated by Mg++, Mn++, and Co++ and suppressed by Ca++, Zn,++, and Ni++. EDTA inhibited the activity below the basal level (activity in the absence of added cation), an this inhibition could be recovered up to the basal level by an equimolar quantity of either Mn++ or Mg++. Further stimulation of the enzyme activity with increasing concentrations of divalent cations was observed only with Mn++. Similar effects were observe with ethylene glycol bis(beta-aminoethyl ether)-tn,n-tetraacetic acid. The stimulatory cations affected both the low and high Michaelis constant (tkm) enzymes in these tumors by increasing the maximum velocity. In the low Km enzyme, the Km was also slightly increased. Neither guanosine 3',5'-cyclic monophosphate nor adenosine 3',5'-cyclic monophosphate had any effect on the hydrolysis of the other at physiologic levels.

Alterations in serum glycosyltransferases and 5'-nucleotidase in breast cancer patients.

We have measured sialyltransferase, galactosyltransferase, and fucosyltransferase as sell as 5'-nucleotidase in the serum of breast cancer patients. Serum sialyltransferase values in 65 normal healthy females ranged from 2.6 to 8.5 units, with a mean of 5.4. In 25 women with operable primary breast cancer, serum sialyltransferase levels were found to be between 6.2 and 15.4 units. Marked elevation of this enzyme level (range, 8.8 to 36 units) was observed in 48 patients with metastatic breast cancer. Galactosyltransferase and fucosyltransferase measurements, however, showed considerable overlap between the controls and the cancer patients. On the other hand serum 5'-nucleotidase and sialyltransferase in breast cancer patients showed very similar patterns. Thus, serum 5'-nucleotidase values in 44 normal females ranged from 11.4 to 23.2 units, whereas the levels found in 30 patients with metastasis were between 25 and 71.8 units. The tissue origin of abnormal levels of serum glycosyltransferases and 5'-nucleotidase was discussed in relation to their physiological significance as well as their role as markers for diagnosing early malignant breast neoplasm and for monitoring the extent of metastasis.

Experimental model for quantitative study of metastasis.

This paper describes the growth of HEp-3, a human epidermoid carcinoma, in the chick embryo. When inoculated onto the chorioallantoic membrane, the tumor grows locally with a doubling time of 21 to 24 hr and disseminates widely in the embryo proper, metastasis to the lung and heart being especially prominent. The tumor secretes large amounts of plasminogen activator (PA) both in vivo and in vitro. This enzyme of human origin can be easily identified in a mixture containing both human and chicken PA's, since each PA has a marked preference for the homologous plasminogen. Thus the presence of human PA in tissues and body fluids can be used as a quantitative marker for HEp-3 metastasis both in embryos and in newly hatched chicks. Two assays for metastasis are described; their respective sensitivities are less than 4 x 10(4) and less than 500 HEp-3 cells per 17-day chick embryo lung (or approximately 1 HEp-3 cell per 400 and per 3 x 10(4) lung cells), respectively. These assays revealed that tumor growth and metastasis are sensitive to embryo age, inoculation onto the chorioallantoic membrane at 10 days being optimal for both. Tumor size, the length of the latent period that precedes the appearance of lung metastasis, and the number of metastatic cells in the lungs are all influenced by inoculum size. Generally, but not uniformly, an increased level of lung metastasis is correlated with tumor size on the chorioallantoic membrane. The attractive features of this system for quantitative study of metastasis are reproducibility rapidity, sensitivity, convenience, and cost.

Serum tyrosinase in malignant disease, its activity, and the electrophoretic patterns of the enzyme as carried by immunoglobulins'.

Serum tyrosinase activity in many persons with metastatic diseases was found to be significantly higher than activity in normal persons. The highest activity was observed in melanoma and breast carcinoma. The electrophoretic patterns of serum tyrosinase, resolved by electrophoresis of a serum tyrosinase fraction followed by incubation of the gel sample with L-dopa, and represented as sets of RF's of melanin bands, were characteristically different in melanoma, breast carcinoma, and certain other diseases. The RF's of melanin and protein bands in the serum enzyme preparations from melanoma patients were concisely defined. Further, some potent serum fractions inhibiting tyrosinase melanogenic activity have been obtained, and the presence of tyrosinase inhibitors in the serum enzyme preparation has also been demonstrated. More detailed exploration of these serum tyrosinase parameters may provide more specific and sensitive detection for certain malignant diseases.

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.

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.

The fibrinolytic enzyme system in hepatic cirrhosis and malignant metastases.

Components of the blood fibrinolytic system were measured in 18 patients with hepatic cirrhosis, in two patients with acute hepatic necrosis, and in 10 patients with hepatic metastases. The frequency of an elevation of plasminogen activator and a reduction in plasminogen in hepatic cirrhosis has been confirmed. Patients with compensated cirrhosis had low levels of the serum inhibitor of plasminogen activation while those with severe hepatic insufficiency or coma due to cirrhosis or hepatic necrosis had elevated levels. The presence of hepatic metastases was associated with reduced plasminogen activator levels and an increase in the fibrinogen concentration.

[Clinical significance of the determination of serum phosphohexose isomerase in malignant neoplasms]. / Significato clinico del dosaggio della fosfoesosoisomerasi serica nelle neoplasie maligne.

The results of a preliminary study on the serous level of PHI in patients undergoing treatment at the Catania Oncology Institute are reported. Of 53 subjects with ongoing cancer, higher than normal values were encountered in 70% of cases. This percentage rose to 82.3% in cancer patients with metastasis affecting the skeleton. Of 12 subjects who underwent mastectomy with no clinical signs of recurrence only three showed abnormal values while in no case of fibrocystic mastopathy were levels higher than normal observed. A constant correlation was identified between effectiveness of surgical, radiotherapeutical or medical treatment and reduction in serous level of PHI. The usefulness of this investigation in oncology is thus confirmed.

[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.