Publisher Summary This chapter discusses the cellular adhesiveness and extracellular substrata. Cell adhesiveness is a fundamental cell property. It plays a role in developmental processes such as cell migration during embryogenesis and morphogenesis in response to particular extracellular matrices; it plays a role in homeostatic processes such as tissue and organ stability, thrombosis, inflammation, and wound healing; and it plays a role in the pathology of various disease states, for instance, in the invasive and metastatic behavior of malignant cells, in disorders of platelet function, and in disorders of leukocyte function. Adhesiveness is common to many different cell types under a variety of conditions, and the underlying mechanisms are analogous for active cell-substratum adhesion occurring in vitro to artificial and to model physiological substrata and in vivo to fibrinogen or fibrin deposits, to the basement membrane, and to other acellular components of the connective tissue.

Cellular adhesiveness and extracellular substrata.

Trans-membrane control of the receptors on normal and tumor cells. II. Surface changes associated with transformation and malignancy.

Cysteine proteinases are a subclass of endopeptidases which require activation by thiol reagents. A tumor cysteine proteinase which appears to be related to lysosomal cathepsin B has been implicated in the ability of tumor cells to invade the extracellular matrix and to metastasize to secondary sites. Lysosomal cathepsin B can degrade such components of the extracellular matrix as collagen, fibronectin and proteoglycans. Activity of this cathepsin B-like cysteine proteinase (CB) has been correlated with tumor malignancy in a number of tumor lines yet not in all tumor lines studied. CB activity in tumors seems to be associated with the viable tumor cells, probably with the plasma membrane of these tumor cells. CB activity has been measured in the sera, urine, ascites fluid and pancreatic fluid of tumor-bearing patients. CB is released from tumor explants and tumor cells in vitro as well as from normal subcutaneous tissue exposed to tumor-conditioned medium. Cathepsin B from normal tissues is rapidly inactivated above pH 7.0. Therefore, CB in tumor cell membranes or released from tumor cells (or from host cells in response to tumor cells) may not possess proteolytic activity at neutral pH and thus may not facilitate tumor cell invasion. However, CB exhibits enhanced stability at neutral or slightly alkaline pH's. There is not yet definitive proof that CB plays a role in tumor invasion and metastasis. There is, however, an increasing body of correlative evidence relating CB activity and tumor malignancy. This correlative evidence plus preliminary evidence that tumor CB can degrade components of the extracellular matrix in vitro suggests that CB may be one proteinase active in a proteolytic cascade resulting in tumor invasion and metastasis.

Cysteine proteinases and metastasis.

The intracellular distribution of specific protease, plasminogen activator (PA), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution. A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na+,K+-ATPase, 5'-nucleotidase, and [3H]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like" elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.

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Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells.

Anticoagulants in the treatment of cancer

Beef pancreas trypsin inhibitor has been shown to promote parallel alignment of hamster tumor cells in culture and to increase the adhesiveness of rounded cells. It has a slightly depressing effect upon cell proliferation. Soybean inhibitor depressed cell proliferation but had no effect upon cell alignment. Egg white inhibitor had no effect upon proliferation or cell alignment. The synthetic protease inhibitor N -α- p -tosyl-l-lysine chloromethyl ketone HCl had no effect upon cell alignment but depressed cell proliferation. It was toxic in concentrations in excess of 20 µm. Beef pancreas trypsin inhibitor had no effect upon cell proliferation or cell interaction of nontumorigenic hamster embryo cells in culture. The results suggest that some aspects of the morphology and social behavior of the tumor cells studied may be associated with protease activity that can be inhibited by beef pancreas trypsin inhibitor.

https://cancerres.aacrjournals.org/content/canres/32/11/2469.full.pdf

Effects of Protease Inhibitors on Growth of Hamster Tumor Cells in Culture

Hamster tumor cells requiring glutamine for survival and growth were used in this study. By gradually reducing the glutamine concentration over a period of 3 weeks, it was possible to establish a line which grew in the absence of glutamine. These cells, in contast to the glutamine-dependent cells, showed parallel alignment which is typical of normal hamster embryo fibroblasts. They were, however, readily distinguishable from normal cells because they grew to higher maximum density, were more refractile when examined by phase-contrast microscopy, and were more readily dispersed with trypsin. Injection of these cells into hamsters resulted in the development of slow growing solid tumors. When cells from these tumors were explanted into tissue culture, they were found to have reverted to glutamine dependence and to display the random crisscross growth pattern which was typical of the tumor cell line from which they were originally derived.

Properties of a hamster tumor cell line grown in a glutamine-free medium

Summary Free amino acids were measured in mouse ascites tumor cells taken from susceptible and resistant mice on the 5th and 7th days after inoculation. Large numbers of samples were analyzed by the combined use of an automatic analyzer and a computer program designed to calculate statistical significances and to generate multicomparison tables. In comparison with cells taken from the resistant strain on the 5th day, cells from the susceptible strain were higher in alanine and slightly higher in tyrosine. Glycine and serine were slightly lower in cells from the susceptible strain. On the 7th day arginine was detected in only very small traces in cells from the resistant strain, whereas arginine levels in the susceptible strain were not significantly different from those found on the 5th day. Comparison of the levels of the other amino acids in cells taken from the two strains on the 7th day revealed no significant differences. The effects of administering i.p. a large dose of histidine are also described.

Free amino acids in E.L.4 mouse ascites tumor cells taken from susceptible (C57BL) and resistant (B10.D2) mice.

Pyrrolidone carboxylic acid, which is formed spontaneously when glutamine is incubated at 37°C in a phosphate or bicarbonate medium, was found to inhibit growth of a line of hamster tumor cells which required glutamine for survival and growth.

Constance Weinstein

Papers

Effects of Protease Inhibitors on Growth of Hamster Tumor Cells in Culture

Properties of a hamster tumor cell line grown in a glutamine-free medium

Free amino acids in E.L.4 mouse ascites tumor cells taken from susceptible (C57BL) and resistant (B10.D2) mice.

Inhibition of growth of hamster tumor cells in vitro by pyrrolidone carboxylic acid in a glutamine-dependent system.